Dual-responsive smart nano-platform targeting peptide modi�cations synergistically enhances multimodal therapy for liver cancer

The success of clinical therapies against liver cancer is largely determined the accuracy rate of treatment. Herein, we designed a dual-responsive smart nano-platform (HMCuS@DOX@9R-P201) could realize multimodal synergistic therapy. The nano-platform could precisely recognize the protein marker FOXM1c-DBD on the surface of HepG2 cells. The apoptosis rate of HepG2 cells reached 98.51% under near-infrared (NIR) laser irradiation, and the tumor inhibition rate of HMCD9P NPs + L treatment group was as high as 88.2% in mice. Moreover, it could up-regulate the apoptosis-related protein Bak, down-regulate PARP-1, Bcl-2, and Caspase 8, and inhibit the pathway protein FOXM1, thus down-regulating Skp2, up-regulate p27Kip1, and precise induction of multimodal synergistic therapy based on chemotherapy, PTT, and PDT to improve anti-HCC e�cacy and reduce side effects. Overall, we report a liver cancer-targeted smart nano-platform with promising anti-liver cancer effects and multiple synergistic therapeutic mechanisms.


Introduction
Primary liver cancer is one of the most aggressive and deadly solid malignant tumors.Hepatocellular carcinoma (HCC) accounts for approximately 90% of all primary liver cancer cases [1].Currently, the main treatment strategy for liver cancer involves a combination of surgical treatment (including liver resection and liver transplantation) and chemotherapeutic drugs.However, surgery is limited to patients with earlystage disease and is associated with a high recurrence rate.Conventional chemotherapeutic drugs cannot recognize cancer cells and can kill both cancer cells and normal cells, inducing serious adverse effects [2][3][4].Therefore, a nano-drug delivery system that can improve the treatment of liver cancer is urgently required [5,6].
In the last decade, two non-invasive light-activated treatment modalities, photothermal therapy (PTT) and photodynamic therapy (PDT), have proven to be promising treatment techniques for patients with cancer [7][8][9].PTT and PDT have potential advantages over chemotherapy in terms of high site-selectivity, controllability, and fewer systemic effects.In PTT, the photothermal material rapidly generates heat in the tumor area following laser irradiation to induce irreversible damage (40-45°C) or thermal ablation (> 45°C) in cancer cells [10,11].In PDT, a laser with a speci c wavelength is used to irradiate the tumor.The photosensitizer internalized by tumor cells is excited by the laser, and this energy is transferred to the surrounding oxygen, leading to the generation of highly reactive singlet oxygen species.The singlet oxygen oxidizes cellular biomolecules to produce cytotoxic effects, leading to cell damage or even death, thus achieving anti-tumor effects [12,13].However, both PTT and PDT have some inherent drawbacks.
Single-mode therapy is insu cient for completely eradicating tumors and lacks the ability to target cancer cells.Therefore, a targeted synergistic therapeutic nano-medicine system should be constructed for late-stage tumor research and clinical treatment [14,15].Among the various photothermal materials available, hollow mesoporous copper sul de nanoparticles (HMCuS NPs)-a new type of NIR nano-responsive material-have good photothermal stability and biocompatibility [16][17][18].Copper sul de, as a p-type semiconductor, has a strong local surface plasmon resonance (LSPR) effect on NIR light, leading to an improved photothermal effect [19][20][21].And copper ions leaking from copper sul de under NIR light can produce hydroxyl radicals (⋅OH) in a redox reaction with the buffer matrix surrounding the tumor environment, enabling PDT [22,23].More importantly, the hollow porous shell structure and small size of HMCuS NPs allow the NPs to be used as multifunctional nano-carriers for co-loading various types of chemotherapeutic drugs [24][25][26].Owing to this versatility, the designed drug delivery system can be used for the synergistic application of chemotherapy, PTT, PDT, and bio-imaging.However, most bare mesoporous materials have problems such as poor targeting and premature drug release [27,28].Therefore, it is highly desirable to design a smart nano-platform by combining both tumor targeting and a controlled on-demand drug release strategy.
9R-P201 peptide is a molecularly targeted lead drug.Screening studies show that it has a high a nity for FOXM1, which is a transcription factor highly expressed in HCC cells [29][30][31].Interestingly, HMCuS NPs modi ed with 9R-P201 peptide could confer several advantages.Act as a tumor-targeting fraction, 9R-P201 peptide could bind to the speci c marker FOXM1, which is highly expressed on the surface of HepG2 cells.It allows nanodrug enter the nucleus and bind to the FOXM1c-DBD in the nucleus and exert its inhibitory effect [32,33].Moreover, 9R-P201 peptide could react with the amino group added to the surface of HMCuS to form an amide bond, which breaks following NIR irradiation in the tumor microenvironment (TME), realizing the effects of an intelligent slow-release drug platform.In addition, the 9R-P201 peptide is biocompatible and biodegradable in vivo.Given these advantages, a 9R-P201 peptidemodi ed smart nano-platform drug delivery system could provide a new direction for liver cancer treatment.
In this study, a 9R-P201 peptide-modi ed HMCuS-NH 2 @DOX TME-responsive smart nano-platform was designed to synergistic enhance multimodal therapy for HCC as a drug delivery system.Doxorubicin (DOX) was chosen as the model drug because of its dual role as a chemotherapeutic and uorescent agent.Materials used for preparing HMCuS@DOX@9R-P201 NPs were systematically evaluated based on a series of physicochemical and biological properties, including morphology, particle size, surface morphology, drug delivery e ciency, in vitro photothermal effects, TME and NIR laser response release pro les, intracellular ROS generation properties, in vitro cellular uptake, uorescence imaging properties, cytotoxicity, intracellular drug release, apoptosis, in vivo photothermal performance, and NIR thermographic properties.Finally, the in vivo anti-tumor e cacy and histological effects of the developed HMCuS@DOX@9R-P201 NPs were analyzed.The HMCuS@DOX@9R-P201 smart nano-platform for multimodal therapy and drug delivery designed in this study represents a promising nano-therapeutic agent that can effectively synergize chemotherapy, PTT, and PDT while achieving superior antitumor e cacy.

Apparatus
In this study, the following equipment were used for different experiments.

Synthesis of HMCuS
First, 100 µL CuCl 2 (0.5 M) was added to 50 mL of deionized water containing 0.48 g PVP-K30 and stirred magnetically at 25°C.Then, 50 mL of sodium hydroxide solution (pH = 9.0) was added, followed by 26 µL of N 2 H 4 •H 2 O (50%) to obtain a bright yellow suspension of Cu 2 O spheres.After 5 min of incubation at room temperature, 400 µL of aqueous Na 2 S solution (320 mg/mL) was added to the suspension.The solution was heated at 60°C for 2 h.Finally, the CuS nanospheres were centrifuged at 12000 rpm for 10 min and then washed twice with deionized water to neutral.Freeze-drying was performed to obtain HMCuS.

Synthesis of HMCuS-NH 2
HMCuS (50 mg) was dispersed in 200 mL of ultrapure water under ultrasonication, and subsequently, 100 mg of mercaptoethylamine was added and stirred for 24 h.The precipitate was obtained by centrifugation and washed twice with ultrapure water until neutral.It was then freeze-dried to obtain the aminated HMCuS complex (HMCuS-NH 2 ).
Then, 10 mL of PBS containing 20 mg DOX was added, stirred at room temperature for 24 h, and washed using centrifugation to obtain HMCuS-NH2@DOX.

Calculation of encapsulation rate and drug loading capacity
A mixture of DOX in PBS (pH = 7.4) was prepared to obtain aqueous DOX solutions (2, 4, 8, 10, 20, and 40 µg/mL), and the absorbance values at 488 nm were measured using a UV spectrophotometer under lightproof conditions.The amount of drug remaining in the supernatant was calculated based on the absorbance value of the drug in the supernatant at the maximum absorption wavelength using the standard curve.The encapsulation rate and drug loading capacity were calculated according to the input amount.
Drug loading rate (%) = (mass of drug input -mass of drug in supernatant)/mass of carrier × 100% Encapsulation rate (%) = (mass of drug input -mass of drug in supernatant)/mass of drug input × 100%

Evaluation of in vitro photothermal performance
In vitro photothermal conversion ability of HMCuS and HMCD9P NPs was tested to determine their NIR photothermal conversion effects.At a certain power, 1 mL of the sample solution (different concentrations) was placed in a uorescence spectrophotometer quartz dish and irradiated with an 808nm laser for a certain time.The temperature value was recorded every 30 s. Next, 1 mL of a certain sample concentration was added to a uorescence spectrophotometer quartz dish.The solution was irradiated with an 808-nm laser for a certain time at various power settings, and the temperature value was recorded every 30 s. Next, the temperature changes in the samples at different concentrations under 808-nm laser irradiation were monitored and imaged using an infrared thermal imaging camera.In addition, the photothermal stability of the nanomedicines was evaluated following multiple laser on/off treatments.In each cycle, the nanomedicine solution was irradiated with the laser for 10 min, which was then turned off and then cooled naturally to room temperature.The on/off cycle was repeated ve times, and the temperature change pro le over these cycles was recorded by plotting the change in temperature against exposure time.
In vitro stimulation-triggered drug release studies HMCD9P NPs samples (2 mg) were packed into dialysis bags (MWCO = 3500) and placed in 30 mL of phosphate buffer (pH = 7.4/6.8/5.4)within a constant temperature water bath shaker for drug release experiments (37°C, 100 rpm).In the laser group, an 808-nm laser (1 W/cm 2 ) was used for 10 min.Then, 1 mL of sample was removed after 1, 2, 4, 6, 8, 10, 12, 24, and 36 h and supplemented with the same volume of drug release medium.The absorbance values of different concentrations of DOX solutions were measured at 488 nm using a UV spectrophotometer to analyze the drug release data.

Hemolysis test
Retroorbital mouse blood samples (0.5 mL) were added to a tube containing sodium heparin, and the whole blood was centrifuged at 2000 rpm for 5 min.The cells were washed with saline until the supernatant became free of red colour.The lower erythrocyte layer was prepared into a 4% erythrocyte suspension based on the volume ratio and set aside.Then, 0.5 mL of the 4% erythrocyte suspension was added to a set of numbered 1.5-mL centrifuge tubes.The suspension was treated with 0.5 mL of the NPs in physiological saline (25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, 400 µg/mL, and 800 µg/mL).Meanwhile, 0.5 mL of saline was added for the negative control, and 0.5 mL of water was added for the positive control group.The tubes were incubated at 37°C for 3 h and centrifuged at 2000 rpm for 5 min.Then, the supernatant was added to a 96-well culture plate, and the OD of hemoglobin at 570 nm was measured using an enzyme marker.

In vitro cytotoxicity evaluation
In vitro tumor activity assays for DOX, HMCuS, HMCuS-NH 2 @DOX, and HMCD9P NPs were performed using the MTT method.HepG2 cells, SNU-739 cells, A549 cells, and HL-7702 cells were added to 96-well plates and cultured for 24 h.DOX, HMCuS, HMCuS-NH 2 @DOX, and HMCD9P NPs were diluted to different concentrations with the culture medium and placed in test tubes for use, and a blank control group without inoculated cells and a negative control group with no drug treatment were set up.The IC 50 values for the different cells were measured 48 h after treatment.The laser control group was divided into 8 subgroups: DOX group, DOX + L group, HMCuS group, HMCuS + L group, HMCuS-NH2@DOX group, HMCuS-NH 2 @DOX + L group, HMCD9P NPs group, and HMCD9P NPs + L group.The non-laser groups were incubated until 24 h after dosing, while the laser groups were irradiated with an 808-nm laser (1 W/cm 2 , 5 min) after 4 h of dosing and then incubated until 24 h.At the 24 h mark, 50 µL of MTT solution (1 mg/mL) was added to each well for 4 h.The plate was shaken, and 100 µL of DMSO solution was added per well.The plate was shaken for 10 min and the absorbance value at 570 nm was measured using the multifunctional enzyme marker A. The cell inhibition rate was calculated by the formula: cell inhibition rate × 100% = (average OD of negative group -average OD of experimental group)/(average OD of negative group -average OD of blank group), and the corresponding IC 50 value was calculated by the IC 50 value calculation software.

Apoptosis assay
The Annexin V-FITC/PI Apoptosis Assay Kit was used to assay the level of apoptosis.HepG2 cells in the logarithmic growth stage were inoculated in 24-well plates.The original medium was discarded after 24 h of incubation and replaced with drug-containing medium.The cells were divided into 14 groups: blank group, blank + L group, HMCuS group, HMCuS + L group, DOX group, DOX + L group, HMCuS-NH 2 @DOX group, HMCuS-NH 2 @DOX + L group, HMCD9P NPs 1 µg/mL group, HMCD9P NPs 1 µg/mL + L group, HMCD9P NPs 5 µg/mL group, HMCD9P NPs 5 µg/mL + L group, HMCD9P NPs 10 µg/mL group, and HMCD9P NPs 10 µg/mL + L group.HMCuS, DOX, and HMCuS-NH 2 @DOX were all administered at a dose of 5 µg/mL.The laser groups were irradiated with an 808-nm laser (1 W/cm 2 , 5 min) 4 h after administration and then incubated until 24 h.Cells were collected by trypsin digestion.Then, they were mixed with 5 µL of PBS containing 5 µL of Annexin V-FITC with 5 µL of PI in a centrifuge tube and stained for 15 min at 37°C under dark conditions.The cells were centrifuged to recover the dye and washed twice with 1 mL of PBS.Finally, 600 µL of PBS was added.The cells were aspirated through a pipette and ltered through a 400 mesh lter, and the samples were used for ow cytometric detection.

Assessment of intracellular ROS production
HepG2 cells in the logarithmic growth phase were inoculated in 6-well plates, and different groups of samples were added after 24 h of incubation under normal conditions.A total of 8 groups (blank group, blank + L group, HMCD9P NPs 0.25 µg/mL group, HMCD9P NPs 0.25 µg/mL + L group, HMCD9P NPs 0.5 µg/mL group, HMCD9P NPs 0.5 µg/mL + L group, HMCD9P NPs 1 µg/mL group, and HMCD9P NPs 1 µg/mL + L group) were prepared.The blank group was left untreated and the laser group was irradiated using an 808-nm laser (1 W/cm 2 , 5 min) at 4 h after treatment with the drug-containing medium, followed by the addition of pre-diluted DCFH-DA (concentration 10 µM, solvent PBS) to the wells of each sample group.Subsequently, the 6-well plates were protected from light at 37°C for 30 min and nally observed using an inverted uorescence microscope.Cells with high ROS production appeared green in the eld of view.

Cellular uptake
In order to test the cellular uptake behaviour and uorescence imaging properties of HMCD9P NPs, HepG2 cells in the logarithmic growth phase were inoculated in two 6-well plates.Two groups were prepared, one was treated with the competitive inhibitor 9R-P201 peptide and the other was not.All the cells were placed in an incubator at 37°C and 5% CO 2 for a certain period.After the cells grew to an appropriate density, the original medium was removed, and 2 mL of the HMCD9P nano-loaded system in fresh medium (5 µg/mL) was added to each well and incubated for 1, 2, 4, and 6 h, respectively.Cells in the competitive inhibitor group were incubated for 30 min in advance using medium with an excess of free 9R-P201 peptide (1 mg/mL), allowing the 9R-P201 peptide in the medium to compete with the NPs for binding to the receptor on the surface of HepG2 cells.All cells were collected, centrifuged, washed, and resuspended in 500 µL PBS.They were ltered through a sieve and placed in the ow cyrometer for assay.
Cell uptake was detected using confocal laser scanning microscopy, and cells were inoculated in small laser confocal dishes and incubated.After the cells grew to a suitable density, the original culture medium was removed and replaced with 500 µL of fresh medium containing the HMCD9P nano-loaded system (5 µg/mL).The cells were incubated for 1, 2, 4, and 6 h.They were then washed with PBS, and 500 µL of the nuclear dye Hoechst 33342 was added (1 µg/mL).The cells were stained at 37°C for 10 min and protected from light.After staining, the cells were washed with PBS, and an appropriate amount of PBS was added.The cellular uptake of the HMCD9P nano-loaded system was then observed using confocal laser scanning microscopy, with the nuclear dye and nano-system being excited at 405 and 488 nm, respectively.
In vivo uorescence imaging of the liver tumor targeting ability of HMCD9P NPs All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Henan University and experiments were approved by the Biomedical Research Ethics Sub-Committee, Henan University (No. HUSOM2022-094).
To investigate the uorescence effect of NPs on HCC targeting in vivo, saline-con gured HMCD9P NPs were administered to seven mice with established hepatic tumors (H22 cells) using tail vein injections.After 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h of treatment, uorescence images of the nano-drug in the tumor and in each organ was acquired using a small animal 3D imaging system.(Ex = 488 nm, Em = 600 nm).

Evaluation of in vivo tumor therapeutic effect of NPs
To further study the in vivo antitumor effect, H22 cells were selected to construct a subcutaneous transplantation tumor model in BALB/c mice.The antitumor effect of the nano-drugs was studied following tail vein injection combined with laser irradiation.Two mice were selected for the intravenous injection of different agents (Saline group [saline] and HMCD9P NPs).The tumor area was irradiated with an 808-nm laser (1 W/cm 2 , 5 min) at 6 h after intravenous injection, and the temperature change was detected in real time using in vivo infrared thermography.In addition, mice were randomly divided into 7 groups of 5 mice each.The subcutaneous transplantation tumor model was constructed using H22 cells in BALB/c mice, and the anti-tumor effect of the nano-medicine was studied via tail vein injection combined with laser irradiation.The tumor size was observed daily, and when the tumor volume reached 70 mm 3 , the mice in each of the 7 groups (control group (saline group and HMCuS group), HMCuS + L group, DOX group, DOX + L group, HMCD9P NPs group, and HMCD9P NPs + L group) were treated with the respective drugs.The doses were administered every other day for seven doses, and the body weight and tumor volume of the mice were measured while administering the drug.The tumor area was irradiated with an 808-nm laser (1 W/cm 2 , 5 min) on the second day of administration.The tumor volume of the mice was calculated as V = W 2 ×L/2 (V stands for tumor volume, W stands for short tumor diameter, and L stands for long tumor diameter).

Biochemical testing
In order to evaluate the health status of the mice after treatment with various drugs, blood was collected following treatment in ordinary centrifuge tubes.After 2 h at room temperature or overnight at 4°C, the blood samples were centrifuged at 2-8°C for 15 min at 3000 rpm.The supernatant was taken for biochemical analysis, including tests for ALT, AST, ALB, ALP, BUN, and UA.

Immunoblot analysis of HCC tumor tissues for apoptosisrelated proteins
Tumors obtained from mice of different groups were stored in liquid nitrogen, weighed, and homogenized in lysate liquid (tumor weight = 10 mL:1 g (1 mL of lysate was added to 10 µL of PMSF)) with a homogenizer.Homogenates were poured into EP tubes and lysed on ice for 30 min, vortexed every 5 min, and then centrifuged at 12000 rpm for 10 min in a high-speed cryogenic centrifuge.After centrifugation, the supernatant was carefully removed, protein quanti cation was performed, and protein concentration was calculated.Then 5× sample buffer (1/4 of the sample volume) was added to the sample, and the mixture was boiled for 10 min in a water bath.Samples from different groups were passed through electrophoresis and transmoulding, and then sealed with 5% skimmed milk powder at 25°C for 2 h, followed samples were incubated with the corresponding primary antibody at 4°C for 12 h.The strips were washed 3 times with TBST (Tris-HCl, NaCl, tween20) and incubated with the secondary antibody at 25°C for 2 h.Finally, it was washed with TBST for 3 times and detected the protein expression was detected using immun-imprint chemi-luminescence reagent (ECL) plus reagent.

RESULTS AND DISCUSSION
Design and characterization of HMCuS@DOX@9R-P201 NPs The process for preparing the HMCuS@DOX@9R-P201 smart nano-platform and its application for the multimodal synergistic treatment of liver cancer was shown in Scheme 1. First, HMCuS NPs with a cagelike structure was synthesized via ion exchange.Their unique cage-like structure was used to encapsulate the uorescent chemotherapeutic drug Dox, which greatly improved the drug loading capacity.Then, the HMCuS NPs were surface-modi ed with the 9R-P201 peptide, which had HCC-targeting properties as it could speci cally target the FOXM1 receptor on the surface of HCC cells.The synthesized nano-drug delivery system was predicted to be a promising nanotherapeutic agent for the precision treatment of liver cancer.
Particle size and dispersion were characterized using transmission electron microscopy (TEM) images of hollow mesoporous copper sul de and complexes.The prepared hollow mesoporous copper sul de drug carrier HMCuS was found to have a hollow mesoporous structure, which was clear, small in size, and homogeneous in shape, with a size of about 140 nm (Fig. S1A).Thus, it could passively target tumor cells owing to the enhanced permeability and retention (EPR) effect.After Dox loading, a black substance could be detected inside the cage, indicating that the loading of DOX was successful (Fig. S1B).The unique cage-like structure of HMCuS NPs substantially enhanced the chemotherapeutic effect of DOX.
After the 9R-P201 peptide was added, a transparent layer could be detected surrounding the NPs, indicating the successful surface modi cation of the NPs with the 9R-P201 peptide that could precisely target liver cancer cells (Fig. S1C).Characterization of the HMCuS NPs and the components of the NPs was also performed based on zeta potential measurements (Fig. S1D).The zeta potential of HMCuS was − 13.6 mV, but it changed to -2.5 mV in HMCuS-NH 2 after the addition of the amino group, indicating successful modi cation.The potential increased to 29.7 mV after loading DOX, as DOX is positively charged, indicating successful drug loading.Finally, the zeta potential changed to 40.6 mV when the 9R-P201 peptide was attached.The potential value of the 9R-P201 peptide itself was 14.5 mV.This demonstrated successful modi cation with the 9R-P201 peptide.Fig. S1E shows the X-ray diffraction (XRD) pattern of HMCuS NPs.The crystallographic planes corresponding to the derivative peaks at 2θ = 27.797°,29.339°, 31.829°,47.928°, 52.702°, and 59.372° were 101, 102, 103, 110, 108, and 116, respectively, consistent with the standard data of CuS (JCPDS No. 06-0464).This demonstrated the successful synthesis of HMCuS NPs [34].Fig. S2 depicts an energy-dispersive detector (EDS) plot of HMCuS, in which only the Cu and S elements were detected, further validating the synthesis of HMCuS.To better demonstrate the successful modi cation of the β-mercaptoethylamine ligand, the infrared spectrum was characterized (Fig. S3).A characteristic peak of the S-H stretching vibration at 2558 cm − 1 was observed for mercaptoethylamine.In contrast, this characteristic peak of the S-H stretching vibration was not detected in HMCuS-NH 2 , but the characteristic peak of the C-N stretching vibration of the amino group appeared at 1210 cm − 1 .This demonstrated that mercaptoethylamine had been added to HMCuS.Fig. S1F shows a full wavelength scan of the NPs obtained using a UV-Vis spectrophotometer.Both HMCuS and HMCuS-NH 2 showed strong UV absorption in the NIR region.The UV absorption weakened marginally after the loading of DOX and the 9R-P201 peptide.In addition, the feeding ratios of HMCuS-NH 2 and DOX were screened.When HMCuS-NH 2 :DOX was 1:2, the drug loading rate and encapsulation rate of HMCuS-NH 2 @DOX were 57.4% and 67.5%, respectively.When HMCuS-NH 2 :DOX was 1:1, the drug loading rate and encapsulation rate of HMCuS-NH 2 @DOX were 49.5% and 98.1%, respectively.When HMCuS-NH 2 :DOX was 2:1, the drug-loading rate and encapsulation rate of HMCuS-NH 2 @DOX were 33.2% and 99.4%, respectively.Therefore, HMCuS-NH 2 :DOX = 1:1 was used for the subsequent reactions to avoid drug waste and poor drug loading.

In vitro photothermal properties
The strong absorption of HMCuS in the NIR region encouraged us to evaluate its photothermal effects in vitro.Therefore, a series of photothermal experiments were carried out.Fig. S1G shows the temperature distribution of the HMCD9P NP solution (100 µg/mL) under 808-nm laser irradiation (1 W/cm 2 ) for 5 laser on/off cycles.The photothermal stability of HMCD9P NPs was found to be good.Figure 1A shows the temperature variation in HMCuS NPs of different concentrations under 808-nm laser irradiation (1 W/cm 2 ) for 8 min.Meanwhile, Fig. 1B shows the temperature variation in HMCuS NPs (100 µg/mL) irradiated with the 808-nm laser for 8 min at different power settings.When 100 µg/mL HMCuS NPs were irradiated with the 808-nm laser (1 W/cm 2 ) for 8 min, the temperature rose to 58°C, at which tumor cells could be killed.Therefore, the photothermal conversion properties of HMCuS NPs proved their value as an excellent photothermal material for antitumor therapy.Figure 1D shows the temperature variation in HMCD9P NPs of different concentrations under 808-nm laser irradiation (1 W/cm 2 ) for 8 min.Similarly, Fig. 1E shows the temperature variation in HMCD9P NPs (100 µg/mL) irradiated with the 808-nm laser for 8 min at different power settings.When 100 µg/mL HMCD9P NPs were irradiated with an 808-nm laser (1 W/cm 2 ) for 8 min, they warmed to 53°C.This temperature was lower than that reached by HMCuS NPs (58°C), likely owing to DOX loading and the presence of the 9R-P201 peptide.These ndings were consistent with the UV-Vis absorption results.The photothermal performance of HMCD9P NPs was evaluated by detecting the temperature change in HMCD9P NPs under continuous 808-nm laser irradiation using infrared thermography (Fig. 1C).The temperature of HMCD9P NPs solutions of different concentrations (0, 10, 25, 50, 100, and 200 µg/mL) increased rapidly following 1 W/cm 2 laser irradiation for 5 min.The results showed that HMCD9P NPs have excellent photothermal properties and could be used for anti-tumor photothermal therapy.In conclusion, the excellent photothermal properties of HMCuS NPs could enable their application as a promising photothermal material in the thermal ablation of cancer.

TME-triggered and NIR laser response release curves
Chemotherapeutic drug release from HMCD9P NPs was tested in different mediums.After 36 h, in vitro release from HMCD9P (Fig. 1F) was only 6.1% and 25.5% higher in pH 5.4 phosphate buffer than in pH 6.8 and pH 7.4 phosphate buffer in the absence of 808-nm laser irradiation.In the presence of 808-nm laser irradiation, the corresponding laser groups showed a higher release rate than the non-laser groups.Notably, the release rate in pH 5.4 + L phosphate buffer was as high as 51.3% and 70.7% when compared with pH 6.8 and pH 7.4 phosphate buffer.The results indicated that the HMCD9P nano-drugs could break their amide bonds in a microacidic environment and then open the pores of the hollow mesoporous material to release the drug, achieving dual stimulated drug release properties of acid response and NIR laser response [35][36][37].In particular, the pH-sensitive drug release of DOX was due to the protonation of the amino group in the DOX structure by the acidic environment, which led to hydrogen bond breakage and facilitated drug release.In addition, under NIR laser irradiation, HMCuS generated heat and thus reduced the viscosity of the surrounding medium, facilitating drug diffusion.This indicated that when the nano-medicine targets liver cancer cells, DOX release would be promoted by the acidic environment and external NIR laser irradiation.This dual stimulus-responsive drug release pro le could enhance the antitumor effect of the drug at the tumor site.

Hemolysis test
Hemolysis caused by nonspeci c interaction with haemoglobin is an obstacle that must be overcome in the application of nano-drug carriers in vivo.In order to evaluate the biosafety of the HMCD9P nano-drug, retroorbital blood samples collected from healthy BALB/c mice were used for a hemolysis assay.HMCD9P nano-drugs of different concentrations (25,50,100,200,400, and 800 µg/mL) were added to the red blood cell suspension and incubated for 4 h. Figure 2A and 2B show that the hemolysis rate tended to increase slightly with increasing sample concentration, but no signi cant hemolysis was observed.The hemolysis rate remained lower than 5% at 800 µg/mL, indicating that the designed HMCD9P nano-drug had a good safety pro le and could be used for intravenous administration.

In vitro cytotoxicity evaluation
In order to detect the cytotoxicity of DOX, HMCuS and its complexes were incubated with HepG2, SNU-739, A549, and HL-7702 cells for 48 h.Table S1 shows that HepG2 cells showed better under treatment with HMCD9P NPs than did SNU-739 cells and A549 cells.It was less toxic to normal hepatocytes HL-7702 cells and showed selective effects, indicating that HMCD9P NPs can speci cally target HCC cells.The inhibition rate in the HMCuS NPs group was only about 50% at concentrations up to 500 µg/mL (Fig. 2C), indicating that the agent was almost non-toxic to HepG2 cells.This demonstrated that HMCuS NPs had high biosafety.The biosafety of HMCuS NPs as nano-medicine carriers and photothermal materials was a key concern.Next, the cytotoxicity of DOX, HMCuS, and their complexes against HepG2 cells was examined with and without laser irradiation for 24 h. Figure 2C and 2D show that the cytostatic rate increased in all groups following laser irradiation, with signi cant phototherapeutic (thermal and photodynamic therapy) effects being detected.The higher cytostatic rate of HMCD9P NPs + L when compared with HMCuS-NH 2 @DOX + L was presumably due to the increased cellular uptake of NPs induced by the 9R-P201 peptide modi cation.In addition, although both phototherapy and chemotherapy increased the cytostatic rate, the desired therapeutic effect was not achieved.However, in the HMCD9P NPs + L group, the cytostatic rate increased substantially to 87.65%, revealing its high cytotoxicity.This was mainly due to the combination of phototherapy through NIR laser irradiation and synergistic tumor suppression owing the chemotherapeutic effect of DOX, providing excellent therapeutic results for liver cancer.

Apoptosis assay
Apoptosis refers to the autonomous and orderly death of cells through genetic regulation and is necessary for maintaining a stable intracellular environment.To investigate the effect of HMCD9P NPs loaded with DOX on apoptotic cell death, we performed a ow cytometry assays.As shown in Figs.S4A  and 4B, the apoptosis rate of HepG2 in the HMCuS group increased from 6.23-11.03%after laser irradiation, it indicates that HMCuS photothermal material can induce massive cell death under NIR laser irradiation.The apoptosis rate before and after DOX (5 µg/mL) with and without laser irradiation remained almost unchanged.However, the apoptosis rate in the HMCD9P NPs 10 µg/mL (DOX: 1.65 µg/mL) group was as high as 98.51% after laser irradiation.These results suggest that the combination of chemotherapy and with PTT and PDT using HMCD9P NPs inhibits HCC cell proliferation primarily by inducing apoptosis.

Assessment of intracellular ROS production
The production ROS is important for killing tumor cells.The TME typically has high concentrations of H 2 O 2 , and copper ions can catalyze the decomposition of H 2 O 2 to produce ⋅OH via Fenton-like reactions.
As shown in Fig. S4C, the photodynamic effects of NPs in cells were observed using an inverted uorescence microscope.A relatively weak green uorescence signal could be observed after the cellular uptake of NPs, even without NIR laser irradiation, this may be due to ⋅OH generation from copper ion leakage.The intracellular green uorescence signi cantly enhanced following NIR light irradiation, and the uorescence intensity tended to increase with increasing NP concentrations.It indicates that the release of copper ions accelerated after the irradiation of NIR laser, and the released copper ions reacted with the buffer matrix around the tumor to produce a large amount of ⋅OH, that enhanced the PDT effects of NPs.

Cellular uptake
The uptake of HMCD9P NPs in HepG2 cells and A549 cells was monitored using ow cytometry.The cells treated with HMCD9P NPs at a concentration of 5 µg/mL (DOX concentration 0.825 µg/mL).In comparing cells treated with competitive inhibitors (Fig. 3B) with those without inhibitor treatment (Fig. 3A), the intracellular uptake of HMCD9P NPs was found to decrease signi cantly with the addition of the competitive inhibitor 9R-P201.The results showed that nanoparticles modi ed with 9R-P201 showed higher selectivity to HepG2 cells.In addition, more HMCD9P NPs were taken up in HepG2 cells than that in A549 cells (Fig. S5).This indicated that 9R-P201 peptide on the surface of HMCD9P NPs could drive the active targeting of the drug delivery system into cancer cells via HepG2 cell surfacespeci c receptor-mediated endocytosis then speci cally targeting HepG2 cells.
In order to investigate the effect of nano-drug release in tumor cells, nano-drugs were incubated with HepG2, A549, and HL-7702 cells for different periods and then subjected to confocal microscopy.
Figure 3C shows the uptake of the HMCD9P nano-drug in HepG2 cells at 2, 4, 6, and 8 h of treatment.An increase in drug treatment duration increased the amount of NP entry into the nucleus.This indicated that the 9R-P201 peptide on the surface of HMCD9P NPs could prompt the active targeting of the drug delivery system into HepG2 cells via speci c receptor-mediated endocytosis.Fig. S6 shows the uptake of the HMCuS-NH 2 @DOX nano-drug into HepG2 cells at 2, 4, 6, and 8 h.As the duration of treatment increased, there was an increase in NPs accumulation in the cytoplasm.Unlike HMCD9P NPs, HMCuS-NH 2 @DOX NPs did not enter the nucleus, indicating that the 9R-P201 peptide has a membranepenetrating effect and allows NP entry into the nucleus to exert its FOXM1c-DBD inhibitory effect.Fig. S7 shows the uptake of the HMCD9P nano-drug into A549 cells at 2, 4, 6, and 8 h of action.As the duration of treatment increased, there was an increase in NP accumulation in the cytoplasm.However, uorescence was not evident, indicating that HMCD9P NPs were capable of targeting HCC cells compared to HepG2 cell uptake.Fig. S8 shows the uptake of the HMCD9P nano-drug in HL-7702 cells (human normal stem cells) at 2, 4, 6, and 8 h of action.The uptake in HL-7702 cells was minimal over a long period, indicating that the designed nano-drug delivery system could Speci c targeting HCC cells and had little effect on normal hepatocytes.

In vivo uorescence imaging to study the tumor targeting ability of NPs for HCC
To assess the bio-distribution of HMCD9P NPs in vivo, HMCD9P NPs were injected into H22 tumorbearing mice via the tail vein.As shown in Fig. 4A, the uorescence signal at the tumor site gradually increased after the injection of HMCD9P NPs, the uorescence intensity was brightest and peaked at the tumor site after injection 6 h and started to diminish at 8 h.The time-dependent enhancement of the uorescence signal at the tumor site could be attributed to HMCD9P NP accumulation through the EPR effect and active targeting of HCC.The liver and kidneys also uoresced due to metabolism, but the heart, spleen, lungs, and other organs showed no signi cant uorescence signal.This demonstrated that the multifunctional nano-drug delivery system could speci cally deliver the nano-drugs with low systemic toxicity.

In vivo anti-tumor effect of NPs
To further investigate the anti-tumor effect of the nano-medicines in vivo, we used H22 cells to construct a subcutaneous tumor transplantation model in BALB/c mice.Subsequently, we studied the anti-tumor effect of the nano-medicines using tail vein injection of the nano-drugs combined with laser irradiation.Two groups of mice were selected for the intravenous injection of different doses (Saline, HMCD9P NPs).
The tumor area was irradiated with an 808-nm laser (1 W/cm 2 , 5 min) at 6 h after the intravenous injection.Thermography in vivo was performed with an infrared thermographer to detect temperature changes in real time.The temperature at the tumor site in mice injected with HMCD9P NPs increased rapidly to 57.8°C during 5 min of laser irradiation (Fig. 4B).The rapid increase in temperature attributed to the targeting ability of the NPs and the high photothermal effect of HMCuS.Thermal imaging results showed that HMCD9P NPs had good photothermal properties for anti-tumor thermal therapy.
The anti-tumor effect of this drug delivery system was assessed by comparing the tumor and mice indices in the control, HMCuS, HMCuS + L, DOX, DOX + L, HMCD9P, and HMCD9P + L groups after 7 doses (each group received a dose of 5 mg/kg; laser conditions: 808-nm laser, 1 W/cm 2 , 5 min; 6 cycles of irradiation).After incubation for 14 days, all mice were killed and tumor tissues were removed by dissected.As shown in Fig. 5A and 5B, the tumors were signi cantly smaller and showed excellent antitumor effects in the HMCD9P NPs + L group.As shown in Fig. 5C, the mice in the HMCuS, HMCuS + L, HMCD9P, HMCD9P + L, and Control groups showed a similar increase in body weight.Meanwhile, mice in the positive drug groups DOX and DOX + L exhibited a signi cant decrease in body weight.This was probably because the drug showed greater toxicity and prevented normal growth in mice.As shown in Fig. 5E, compared to the control group, the HMCD9P and HMCD9P + L groups experienced a more obvious inhibitory effect on tumor growth, with the HMCD9P + L group having the best inhibitory effect.
The tumor weight in the HMCD9P + L group was only 0.098 g, much lower than that in the control (0.832 g) and DOX (0.351 g) groups.The tumor volume of mice in the HMCD9P + L group was only 75.77 mm 3 , much lower than that in the control and DOX groups (683.052 and 207.13 mm 3 , respectively) (Fig. 5D).
The tumor inhibition rate reached 88.2%, it shows that the nano-drug HMCD9P can effectively inhibit the growth of tumors through synergistic chemotherapeutic, photothermal and photodynamic multimodal treatment under laser irradiation with minimal toxic effects on mice.

Histological analysis
Sections of tumor tissue and heart, liver, spleen, lung and kidney tissues were stained with hematoxylineosin (H&E) and observed for tissue damage using light microscopy.The hematoxylin dye is basic and gives a purplish-blue colour to chromatin in the nucleus and ribosomes in the cytoplasm.In contrast, eosin is an acidic dye that primarily renders the components of the cytoplasm and extracellular matrix red.As shown in Fig. 6A, there was no signi cant changes in the organs in mice treated with HMCD9P, HMCD9P + L, HMCuS, or HMCuS + L when compared with the control organs, indicating the biosafety of the nanomaterials.However, the liver, spleen, and kidney tissues were slightly damaged by free DOX and DOX + L, indicating that DOX is toxic to mice.The results further con rmed the good in vivo biosafety of the drug nano-carrier system synthesized in this study.After H&E staining, the control group showed a dense arrangement of tumor cells with a large amount of internal stroma (Fig. 6C).Meanwhile, the free DOX group showed some vacuole formation in the tumor tissue, although the tumor cell density was similar to that of the control group.In contrast, tumor tissue sections from the nano-drug HMCD9P + L treatment group exhibited extensive vacuolization and typical morphological features of apoptosis, such as cytoplasmic loss and nuclear chromatin contraction and fragmentation [38,39].Apoptosis analysis based on TUNEL staining of tumor tissue revealed the strongest uorescent signal in the HMCD9P + L group.These morphological changes suggested that the anti-cancer effect of HMCD9P + L was the most pronounced, resulting in the death of a large number of tumor cells.In addition, blood was collected from different groups of mice at the end of tumor treatment for biochemical analysis, including liver function indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), alkaline phosphatase (ALP), the renal function indicators blood urea nitrogen (BUN), and uric acid (UA) [40,41].As shown in Fig. 6B, all indexes were within the normal range in each group, indicating that the treatments had no adverse effects on liver and kidney function in mice.

Effect of different treatment agents apoptosis-related proteins in HCC tumor
The Bcl-2 gene is a potential inhibitor of apoptosis and regulates cell death.The inhibition of Bcl-2 gene expression in malignant cells can promote apoptosis [42,43].Bak is a member of the major pro-apoptotic Bcl-2 family and is required for the death of apoptotic cells.An increase in its expression induces apoptosis [44].The PARP gene is a multifunctional post-translational protein-modifying enzyme present in most eukaryotic cells.It is activated by the identi cation of structurally damaged DNA fragments and is considered a receptor for DNA damage.Furthermore, PARP is a cleavage substrate for caspases, which are core molecules for apoptosis.Therefore, they play an important role in DNA damage repair and apoptosis [45,46].Caspase is a cysteine protease involved in apoptosis, and its family members directly or indirectly induce the structural features of apoptosis and are at the core of the apoptotic transduction pathway.Caspases play a key role in the process of apoptosis by initiating the exogenous apoptotic transduction pathway and indirectly activating the endogenous apoptotic transduction pathway [47,48].
FOXM1, a member of the FOX gene family, is a transcription factor associated with cell proliferation.Its aberrant activation is closely linked to cell proliferation and division and has emerged as an important target [49,50].Skp2, an important regulator of the cell cycle, speci cally recognizes phosphorylated substrates and mediates their ubiquitination, and several cell cycle regulators -such as p27kip1, p21, cyclinA, cyclinD1, and cyclinE -are substrates of the ubiquitin proteasome pathway.Skp2 is also involved in the regulation of cellular signalling and transcriptional regulation as a controller of signal transduction [51,52].p27kip1 is a cell cycle protein-dependent kinase inhibitor that regulates the cell cycle, apoptosis, and cell motility.The overexpression of p27kip1 inhibits the cell cycle and increases apoptosis, and therefore recognized as a marker of senescence [53,54].As shown in Fig. 7A, the expression of apoptosis-related proteins after the treatment of tumor tissues in different groups of mice were analyzed using western blot.The expression of the apoptosis-related protein Bak was upregulated and the expression of PARP-1, Bcl-2, and Caspase 8 was downregulated after treatment.In addition, the induction of apoptotic pathways was observed (Fig. 7B), indicating that the HMCD9P nano-loaded system inhibited FOXM1 expression and thus down-regulated the expression of the downstream target gene Skp2, resulting in the reduced degradation of the p27Kip1 protein and induction of cell death.

Conclusion
In conclusion, a dual-response smart nano-platform consisting of HMCuS@DOX@9R-P201 NPs was designed for multimodal anti-HCC treatment and its multiple synergistic therapeutic mechanisms were evaluated.Compared with other recently reported multifunctional NPs, these NPs offer more precise liver cancer targeting (the apoptosis rate of HMCD9P NPs + L group was as high as 98.51%) and TME-and NIR laser-responsive drug release properties.In addition, the developed NPs have excellent anti-tumor activity and good biocompatibility.More interestingly, the HMCD9P nano-loaded system can up-regulate the expression of the apoptosis-related protein Bak, down-regulate the expression of PARP-1, Bcl-2, and Caspase 8, and inhibit the expression of FOXM1, thereby down-regulating the expression of its downstream target Skp2.As a result, it can prevent the degradation of the p27Kip1 protein and induce cell death.In summary, the designed HMCuS@DOX@9R-P201 NPs could serve as a comprehensive nanotherapeutic agent with a great potential for effective and precise induction of multimodal synergistic therapy based on chemotherapy, PTT, and PDT to improve anti-HCC e cacy and reduce side effects.Based on the above results, this multimodal smart nano-platform for anti-liver cancer therapy could facilitate clinical management of liver cancer in the future. Figures