Prodrug nanosystem equipped with a sonosensitizer for combined chemotherapy and sonodynamic therapy of melanoma

Abstract

Melanoma is a severe eruptive disease caused by melanocyte lesions and characterized by aggressive tumors, which has become one of the fastest evolving cancers worldwide, and its incidence and mortality rates have shown larger increases than other types of cancers.

Finding effective medicines has become an important topic in collaborative research. Many compounds have been removed from clinical application because of poor physicochemical properties, especially low solubility, a high clearance rate and toxic side effects. In this study, a reactive oxygen species (ROS)-responsive camptothecin (CPT) prodrug delivery system (CTS-S-CPT@IR 780 NPs) in which CPT was conjugated to chitosan was developed. The synthesized CTS-S-CPT conjugate self-assembled to form NPs loading IR 780 in solution. Sonodynamic therapy (SDT) at the tumor site can activate the sonosensitizer IR 780 to release large amounts of ROS and heat, inducing apoptosis. ROS can also cleave carbon‒sulfur bonds and release the chemotherapeutic drug CPT. ROS-responsive CTS-S-CPT@IR 780 NPs activated by combination chemotherapy/SDT were successfully prepared for tumor-targeted drug delivery and can effectively inhibit tumor growth in vivo and in vitro with lower toxic side effects, and localized, controllable, and on-demand release drug. This prodrug approach has spawned hope for overcoming such treatment dilemmas.

Graphical abstract

Schematic illustration of the CTS-S-CPT@IR 780 NPs for SDT ROS-activatable CPT prodrug release and local tumor therapy

1. Introduction

Melanoma tumors are malignant [[1], [2], [3], [4]] and caused by the malignant proliferation of epidermal melanocytes [5]. Melanoma is currently the fifth most common tumor disease in the United States [6] and is highly aggressive with a high mortality rate [[5], [6], [7], [8], [9]]. Moreover, melanoma has a high incidence in developed countries including the United States, Australia, Spain and others, being particularly common in Caucasian and elderly individuals. According to the most recent SEER data, approximately 97,920 new melanoma cases were diagnosed in the United States in 2022 [10].

Early resection of melanoma has shown significant therapeutic effects [11]. However, owing to its pathological characteristics, complete resection of melanoma tumors is very difficult and often accompanied by a high rate of recurrence; moreover, the therapeutic effect varies greatly among individuals [[12], [13], [14], [15]]. Although melanoma treatment has seen breakthroughs [9,13,15,16], tremendous therapeutic effects remain difficult to achieve, and new tumor treatment methods and novel synergistic therapies are urgently needed.

Small-molecule prodrugs have certain drawbacks, such as a short half-life and premature drug activation. As a result, these small-molecule drugs can be formulated into nanoprodrug delivery systems to increase their therapeutic efficacy. However, it is crucial that prodrugs release the active drug promptly and accurately at the tumor site while remaining biologically inactive in normal tissues to achieve effective antitumor outcomes [[17], [18], [19], [20], [21], [22], [23], [24], [25]].

Sonodynamic therapy (SDT) involves both sonochemical reactions and photoacoustic chemical reactions [[26], [27], [28], [29], [30]]. When ultrasound (US) irradiation is applied in the presence of a sensitizer, the above reactions occur, resulting in cytotoxicity to kill tumor cells. SDT, a noninvasive treatment strategy proposed by Yumita in 1989 [31], can penetrate deep tissues [[32], [33], [34]]. Compared with photodynamic therapy, SDT has a greater penetration capability with lower phototoxicity and better patient compliance [35]. SDT can kill tumor cells through actions such as acoustic cavitation, the generation of singlet oxygen, mechanical damage, acoustic thermal effects, and the induction of apoptosis [36,37]. Some people opt for sonodynamic therapy to treat tumors; however, they confront challenges such as significant toxicity and poor bioavailability of small molecule medications [28,29,37].

To overcome these challenges, a ROS-responsive nanodrug delivery system (CTS-S-CPT@IR 780 NPs) was designed in this study. This nanosystem self-assembles into nanoparticles (NPs), covalently binds the poorly soluble antitumor drug camptothecin (CPT) and encapsulates the poorly soluble sonosensitizer IR 780 [38] to reduce drug leakage and systemic toxicity. The nanodrug drug delivery system has the following characteristics: CPT is covalently bound to form a prodrug; combined chemotherapy and SDT can be achieved; drugs are released in response to ROS; the NPs became passively enriched in the tumor site; and the NPs containing the sonosensitizer IR 780 self-assembled. The advantages of these NPs is that they can form prodrugs through covalent bond formation, and their synthesis route is simple, easy to operate, and suitable for the preparation of many drugs. The formation of covalent bonds to CPT can prevent drug leakage in normal tissues and reduce toxic side effects, and the generated amphiphilic copolymers can self-assemble to encapsulate the insoluble acoustic sensitizer IR 780 under mild conditions to achieve drug loading. Owing to the enhanced permeability and retention (EPR) effect in tumors, NPs can passively target the tumor site and reach an effective concentration. At this time, SDT at the tumor site can activate the sonosensitizer IR 780 to release large amounts of ROS and heat, inducing apoptosis. ROS can also cleave carbon‒sulfur bonds and release the chemotherapeutic drug CPT. In combination with topoisomerase 1, CPT can inhibit tumor growth and proliferation, and effectively treat melanoma [39]. Moreover, the fluorescence signal of the IR 780 allows the NPs to be precisely tracked at the tumor sites, thereby achieving combined chemotherapy and SDT for melanoma treatment.

2. Results

2.1. Fabrication and ROS catalytic mechanism of the CTS-S-CPT@IR 780 NPs

The design and construction of CTS-S-CPT in this project are shown in Fig. 1, and the reaction mechanism is shown in Fig. 1A–D. CPT-S-COOH was obtained after the addition of CPT [[39], [40], [41]] during the synthesis of compound 1 and the tert-butyl group was removed. After the carboxyl group was exposed, it reacted with the amino group in chitosan [[42], [43], [44]] to form an amide bond, yielding CTS-S-CPT. Chitosan derivatives play an important role in targeting drugs, prolonging release, and enhancing drug absorption. At present, these derivatives are mainly used to delay drug release time, prepare targeted drugs, and serve as thrusters for gene therapy. In the field of drug carriers, chitosan derivatives are widely used in microspheres, nanoparticles, micelles and gel. The size of these particles is usually between 1 and 500 nm. These small-sized nanoparticles can reach their targets and release drugs through various biological barriers [45]. The core-shell structure micelles have excellent stability, tissue permeability, and the ability to continuously release drugs [46]. Amphiphilic chitosan can form micelles through self-assembly, which enhances its solubility, biological activity, and ability to encapsulate lipophilic drugs [47].

Fig. 1.

(A–D) CTS-S-CPT synthetic route.

The morphology of the CTS-S-CPT@IR 780 NPs was observed via TEM (Fig. 2A–D and S10A-C). The CTS-S-CPT@IR 780 NPs were uniform, solid, circular particles on the nanometer scale, with a particle size between 100 and 200 nm. The edges were smooth and black, indicating that an outer film encapsulated IR 780 [38,[48], [49], [50]]. The CTS-S-CPT@IR 780 NPs (Fig. 2E) ranged from 100 to 200 nm in size, which is suitable to exploit the EPR effect [51] in tumors. The polydispersity index (PDI) of those nanoparticles is 0.2739 ± 0.005 by dynamic light scattering (DLS). We also compared the ζ potential (ZP) before and after NPs formation, as shown in Fig. 2G. Upon assembly, the ZP of the CTS-S-CPT@IR 780 NP increased significantly, indicating that NPs formation changed the electric charge. The ZP of approximately 14 mV (Fig. 2F) indicates that the NPs are stable. In addition, the good stability of the CTS-S-CPT@IR 780 NPs was demonstrated by measuring the change in particle size over seven days (Fig. 2H and S8). As shown in Fig. 2I, the size of the NPs before and after freeze-drying further revealed their good stability [21]. The encapsulation efficiency and loading efficiency curve was shown in Fig. 3A and B.

Fig. 2.

(A–D) TEM images of the CTS-S-CPT@IR 780 NPs (scale bar: 100 nm). (E) Hydrodynamic diameters and (F) ζ potentials of the CTS-S-CPT@IR 780 NPs. (G) Ζeta potentials of I, CTS-S-CPT, II, the CTS-S-CPT NPs, III, CTS-S-CPT@IR 780, and IV, the CTS-S-CPT@IR 780 NPs (n = 3). (H) Stability of the CTS-S-CPT@IR 780 NPs in vitro (n = 3). (I) Stability before and after freeze-drying (n = 3).

Fig. 3.

(A) Drug loading in the CTS-S-CPT@IR 780 NPs (n = 3). (B) Encapsulation efficiency in the CTS-S-CPT@IR 780 NPs (n = 3). (C) In vitro cumulative CPT release profiles (n = 3). (D) In vitro cumulative IR 780 release profiles (n = 3). (E) Time-dependent ¹O₂ generation via IR 780 upon irradiation with US at 1.5 W/cm². (F) Time-dependent ¹O₂ generation via IR 780 upon irradiation with US at 1.5 W/cm² (n = 3). (G) Power density-dependent ¹O₂ generation of IR 780 upon irradiation with US for 3 min. (H) Power density-dependent ¹O₂ generation of IR 780 upon irradiation with US for 3 min (n = 3). (I) Concentration-dependent ¹O₂ generation of IR 780 upon irradiation with US for 3 min at 1.5 W/cm² (n = 3).

To assess the ability of the CTS-S-CPT@IR 780 NPs to act as sonosensitizers to produce ROS and the underlying mechanism, the persistence of CPT and IR 780 and the US-mediated drug release behavior from the CTS-S-CPT@IR 780 NPs were evaluated in simulated body fluids with or without US irradiation for 3 min at different sampling time points (Fig. 3C and D). The 48-h drug release rate under US irradiation was greater than that without. These results indicate that the release of CPT is associated with the promotion of carbon‒sulfur bond cleavage by ROS [52]. After 48 h, a large amount of the drug was released. The groups in which the Fenton reaction, which is dependent on H₂O₂ and Fe³⁺, occurred indicated that a large amount of CPT was released, while the groups without the ability for SDT and ROS production displayed lower drug release rates. CTS-S-CPT possessed the sulfur atom, increasing the probability of being attacked by ROS. Under sonodynamic conditions, large amounts of ROS and heat are released to promote tumor cell apoptosis, while ROS cleave the carbon‒sulfur bond to release CPT for chemotherapy. To further confirm our hypothesis, we conducted mass spectrometry analysis of the CTS-S-CPT activited by ROS, providing evidence for the carbon-sulfur bond cleavage (Fig. S13). Those result indicates that less of the drug leaks out and there are fewer toxic side effects to normal tissue.

ROS generation is a key indicator of the efficiency of SDT [[53], [54], [55]]. The ¹O₂ probe singlet oxygen sensor green (SOSG) was used to investigate the generation of ¹O₂ via fluorescence spectrometry. Produced ¹O₂ can react with SOSG, resulting in increased fluorescence intensity. As shown in Fig. 3E and F, the fluorescence intensity increased with increasing SDT treatment time. Moreover, we increased the SDT power and found that the fluorescence intensity increased, as shown in Fig. 3G and H. In addition, the fluorescence intensity of SOSG increased with increasing IR 780 concentration (Fig. 3I). These results indicated that nanoparticles produce ROS under US.

2.2. In vitro antitumor effects of the CTS-S-CPT@IR 780 NPs

Given the effective sonosensitizer properties, we next used CTS-S-CPT@IR 780 NPs for cancer therapy in vitro. The extent of the internalization of IR 780 and IR 780-loaded NPs into B16 cells was investigated qualitatively via confocal microscopy. Weak fluorescence intensity was detected after incubation for 4 h (Fig. 4A). With increasing time, the intracellular fluorescence intensity gradually increased and finally peaked at 8 h, suggesting that the CTS-S-CPT@IR 780 NPs can be enriched in tumor cells. Endocytosis of the IR 780-loaded NPs was confirmed by flow cytometry (Fig. 4B), indicating that the CTS-S-CPT@IR 780 NPs can be absorbed by B16 cells with maximum uptake at 8 h. We used Label-free live cell microscopy system to monitor intracellular drug release, achieving more convenient and real-time fluorescence image compared with traditional fluorescence microscope (Fig. S14).

Fig. 4.

I, Control, II, the CTS-S-CPT NPs, III, CTS-S-CPT@IR 780, and IV, the CTS-S-CPT@IR 780 NPs + US. (A) Cellular uptake of CTS-S-CPT@IR 780 NPs observed via CLSM after various durations of coincubation (scale bar: 20 μm). (B) Cellular uptake of CTS-S-CPT@IR 780 NPs observed by flow cytometry after various durations of coincubation. (C) Representative images of calcein AM/PI double staining showing live/dead cells (scale bar: 200 μm). (D) Intracellular ROS generation capacity of the different treatments, as determined by CLSM (scale bar: 20 μm). (E) Statistical analysis of ROSsignal intensities in F. (F) Intracellular ROS generation in different groups observed by flow cytometry. (G) Mitochondrial membrane potential determined by JC-1 staining after various treatments. (H) Flow cytometry analysis of B16 cell apoptosis after various treatments.

The ability of the NPs to kill B16 cells was evaluated via fluorescence micrographs, as shown in Fig. 4C, which were obtained after staining with calcein-AM and PI. The group treated with CTS-S-CPT@IR 780 NPs plus US irradiation presented most dead cell markers, which proved that this treatment had the strongest B16 cell killing effect.

We next investigated the generation of intracellular ROS during SDT with CTS-S-CPT@IR 780 NPs via confocal laser scanning microscopy (CLSM), using DCFH-DA as the indicator. In the confocal images shown in Fig. 4D, we noted the bright green fluorescence of oxidized DCF (the products of DCFH-DA oxidation by ROS). However, there were negligible changes in the signal intensity in the control nondrug and non-US irradiated groups. This experiment clearly demonstrated that the NPs can interact with ultrasonic waves to produce large amounts of ROS in the cell. We also used flow cytometry combined with the probe DCFH-DA to analyze ROS production quantitatively, as shown in Fig. 4E and F.

Mitochondria are sensitive to ROS [56]. The intracellular red to green fluorescence ratio of JC-1 can be applied to detect changes in the MMP and monitor mitochondrial function. Fig. 7A and B shows that the ratio of red fluorescence to green fluorescence was significantly lower in B16 cells treated with the CTS-S-CPT@IR 780 NPs, indicating a reduction in the MMP. Flow cytometry confirmed these results (Fig. 4G). Mitochondrial dysfunction is an result of cellular oxidative damage, which is due to increased oxidative stress. A decrease in the electrochemical gradient (ΔΨm) is the major cause of endogenous mitochondrial damage.

Fig. 7.

I, Control, II, the CTS-S-CPT NPs, III, CTS-S-CPT@IR 780, and IV, the CTS-S-CPT@IR 780 NPs + US. (A) Mitochondrial membrane potential following JC-1 staining after various treatments (scale bar: 50 μm). (B) Statistical analysis of the JC-1 monomer and aggregate signal intensities in A. (C) Uptake of CTS-S-CPT@IR 780 NPs by 3D melanoma tumor spheroids observed via CLSM after various durations of coincubation (scale bar: 50 μm). (D) Statistical analysis of the CTS-S-CPT@IR 780 NP signal intensities in C. (E) Fluorescence image of 3D A375 cell spheroids stained with calcein-AM and propidium iodide after different treatments, in which green and red indicate live and dead cells, respectively (scale bar: 50 μm). (F) Fluorescence images of JC-1-stained 3D A375 cell spheroids, with the red and green channels indicating J-aggregates and JC-1 monomers, respectively (scale bar: 50 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Flow cytometry analysis with Annexin V/PI demonstrated that apoptosis markedly increased after treatment with CTS-S-CPT@IR 780 NPs + US compared with the control (Fig. 4H). When the MMP decreases, the cells enter an early apoptotic state; thus, this nanocarrier loaded with photosensitizer can exert significant toxic effects on tumor cells.

2.3. In vivo antitumor effects of the CTS-S-CPT@IR 780 NPs

To evaluate the antitumor effects of the drugs in vivo, fluorescence imaging was subsequently performed to monitor the in vivo distribution of the CTS-S-CPT@IR 780 NPs (50 μg/g, 100 μL) after intravenous injection into B16 melanoma tumor-bearing mice at excitation/emission wavelengths of 710 nm and 790 nm, respectively (Fig. 5B). The tumor site in the CTS-S-CPT@IR 780 NP group presented strong fluorescence signals after 6 h (Fig. 5F). These results demonstrated that the CTS-S-CPT@IR 780 NPs could allow high-resolution microscopic imaging of the tumor site. The experimental results reflect not only the advantages of fluorescence imaging upon deeper tissue penetration but also the remarkable ability of the CTS-S-CPT@IR 780 NPs to target B16 melanoma cells. After 24 h, only a weak fluorescence signal remained, and most of the NPs had been degraded and excreted, proving their good tumor enrichment and biosafety.

Fig. 5.

I, Control, II, CPT, III, CPT + IR 780 + US, IV, the CTS-S-CPT NPs, V, CTS-S-CPT@IR 780 NPs, VI, the CTS-S-CPT@IR 780 NPs + US. (A) Treatment scheme. (B) Fluorescence images of B16 tumor-bearing mice after intravenous injection of CTS-S-CPT@IR 780 NPs (n = 3). (C) Photographs of the tumors after various treatments (n = 5). (D) Changes in the body weights of the mice during treatment (n = 5). (E) Profiles of mouse tumor growth during treatment (n = 5). (F) Quantified tumor fluorescence intensities in B (n = 3). (G) Weights of the tumors after various treatments (n = 5).

To examine the therapeutic effect of the CTS-S-CPT@IR 780 NPs + US for melanoma treatment, the drug of six groups (PBS, CPT, CPT + IR 780 + US, CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs and CTS-S-CPT@IR 780 NPs + US) were intravenously injected into the tumor-bearing mice on the 7th day after the subcutaneous injection of B16 cells. Fig. 5A shows an experimental diagram of the treatment process. Compared with that in the other groups, the remarkable antitumor efficiency of CTS-S-CPT@IR 780 NPs + US was demonstrated by the decrease in tumor volume, with significant inhibition and elimination in vivo (Fig. 5C, E and G).

The toxicity of these NPs was subsequently investigated in vivo. There was no decrease in the weights of the mice in each group during treatment, which demonstrates the low toxicity of the CTS-S-CPT@IR 780 NP + US treatment (Fig. 5D). Histological analysis was performed via H&E staining of the main organs after treatment to study acute and chronic organ damage. No tissue necrosis was observed in the main organs (heart, liver, spleen, lung or kidney) of the six groups (Fig. S7), demonstrating that the CTS-S-CPT@IR 780 NPs have no significant side effects in vivo.

Tumor sections from the mice were stained with H&E and subjected to TdT-mediated dUTP nick-end labeling (TUNEL) and Ki-67 assays to evaluate the pathological, necrotic/apoptotic and proliferative activity changes in the tumor lesions (Fig. 6A). The substantial reduction in tumor nuclei, cell death, and histological lesion disintegration confirmed toxicity to the tumor lesions. The results obtained from the proliferation and apoptosis assays with ki-67 and TUNEL staining, respectively, were in good agreement with the mouse treatment results, which confirmed that CTS-S-CPT@IR 780 NPs with SDT contributed to efficient tumor necrosis, thus providing a promising noninvasive targeted melanoma treatment strategy.

Fig. 6.

(A) TUNEL and Ki67 staining of tumor tissues after various treatments (scale bar: 50 μm) (n = 5). (B) Immunofluorescence of P16 in 3D melanoma spheroids treated with different NPs (scale bar: 50 μm). (C) Immunofluorescence of Ki67 in 3D melanoma spheroids after various treatments (scale bar: 50 μm).

2.4. 3D melanoma spheroid penetration and antitumor studies

To investigate the cancer killing effects, CTS-S-CPT@IR 780 NPs were applied to 3D melanoma spheroids. NPs penetrability was evaluated by constructing 3D A375 tumor cell spheroids in vitro to simulate tumor tissue. As shown in Fig. 7C, after 24 h of coincubation with the 3D tumor cell spheroids, the strong fluorescence of IR 780 from the CTS-S-CPT@IR 780 NPs entered the center of the 3D tumor cell spheroid, indicating that the CTS-S-CPT@IR 780 NPs were taken up by the spheroids. Quantitative analysis revealed the efficient tumor accumulation of these NPs (Fig. 7D).

A change in the MMP alters the electrochemical gradient (ΔΨm), which is the initial stage in apoptosis. The changes in MMP in the 3D melanoma spheroids were assessed via the dye JC-1. Here, cells with red fluorescence were abundant among the control spheroids, indicating that there was little alteration in the MMP. Spheroids treated with CTS-S-CPT@IR 780 NPs + US fluoresced green and thus presented a change in the MMP, indicating the initiation of apoptosis (Fig. 7F). The calcein-AM/PI double-staining assay revealed that, compared with the control, the CTS-S-CPT@IR 780 NP + US treatment markedly reduced the relative spheroid viability (Fig. 7E). The expression of proliferation markers (p16 and Ki67) decreased in the samples treated with the CTS-S-CPT@IR 780 NPs + US compared with the control samples (Fig. 6B and C). These results indicated that the nanoparticles are able to enter 3D melanoma shperoid and have a killing effect.

3. Conclusion

We successfully prepared the CTS-S-CPT@IR 780 NP drug delivery system for the treatment of melanoma with combined chemotherapy and SDT, screened the preparation conditions, and performed morphological and structural characterization and in vitro and in vivo evaluations. It can effectively inhibit tumor growth and kill tumor cells. In summary, this nanodrug delivery system has four objectives. a) NPs target and accumulate at tumor sites via the EPR effect. b) CPT is conjugated to the NPs via an amide bond to reduce systemic toxicity. c) Amphiphilic self-assembly results in the formation of NPs loaded with the photosensitizer IR 780. d) Under sonodynamic conditions, large amounts of ROS and heat are released to promote tumor cell apoptosis, while ROS cleave the carbon‒sulfur bond to release CPT for chemotherapy. Therefore, the CTS-S-CPT@IR 780 NPs designed and prepared in this study provide a new approach for combined tumor therapy.

4. Materials and methods

4.1. Materials

CPT was purchased from Aladdin Industrial Co., Ltd. (C111281, Shanghai, China). IR 780 was obtained from GLPBIO (GC48396, America). Glycol chitosan (CTS) was purchased from Macklin Co., Ltd. (G810700, Shanghai, China). Trifluoroacetic acid was procured from Macklin Co., Ltd. (T822118, Shanghai, China). N-Hydroxysuccinimide (NHS) was obtained from TCI Co., Ltd. (H0623, Shanghai, China). 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was supplied by Bide Pharmatech Co., Ltd. (BD19757, Shanghai, China). Thiolactic acid was obtained from Aladdin Industrial Co., Ltd. (T162374, Shanghai, China). Tert-butyl acrylate was procured from Macklin Co., Ltd. (B802797, Shanghai, China). Triethylamine was obtained from Aladdin Industrial Co., Ltd. (T103284, Shanghai, China). 4-Dimethylaminopyridine supplied by Macklin Co., Ltd. (D807273, Shanghai, China).

4.2. Methods

4.2.1. Preparation of CTS-S-CPT

We adopted a simple and convenient method to synthesize CTS-S-CPT. One gram (9.42 mmol) of mercaptopropionic acid and 1.204 g (9.42 mmol) of tert-butyl acrylate were added to a 100 mL flask, followed by the addition of 1 mL of triethylamine [57,58]. Then, 10 mL of dichloromethane was added, and the mixture was heated in a 60 °C oil bath for 12 h. After cooling to room temperature, the solution was diluted with an additional 100 mL of dichloromethane. The mixture was extracted three times with 100 mL of distilled water. Four grams of dry sodium sulfate was added to the organic layer to remove any remaining moisture, which was subsequently filtered and vacuum dried for 24 h to obtain an oily liquid referred to as compound 1.

A total of 0.10 g (0.426 mmol) of compound 1 and 0.1 g (0.52 mmol) of EDC were accurately weighed into a 100 mL flask, 60 mL of dichloromethane (DCM) was added, and the mixture was stirred at room temperature for 30 min. Then, 0.12 g (0.345 mmol) of CPT and 0.063 g (0.52 mmol) of 4-dimethylaminopyridine (DMAP) were added, and the mixture continued to stir at room temperature for 48 h [59]. Excess methanol was added to completely dissolve the material until the solution became clear and transparent. One gram of silica gel powder was added and dry-loaded onto a silica gel column with DCM: CH₃OH at a ratio of 1: 5. The first product obtained from the column was collected and named compound 2.

After compound 2 was dissolved in 20 mL of DCM, this solution was added dropwise to 10 mL of trifluoroacetic acid, and the mixture was stirred overnight. After rotary evaporation was performed, the mixture was passed through a silica gel column. The second product was collected using DCM: CH₃OH (200: 1) as the eluent, followed by collection of the first product using DCM: CH₃OH (200: 1) as the eluent; this product was referred to as compound 3.

100 mg of CTS was dissolved in 7 mL of dimethyl sulfoxide (DMSO) with stirring for 12 h. Then, compound 3 (11.176 mg, 0.22 μM, 508 g/mol), EDC (6.326 mg, 191.7 g/mol) and NHS (3.798 mg, 115.09 g/mol) were each dissolved in 1 mL of DMSO [60]. These solutions were added dropwise into the 7 mL of CTS solution at room temperature with stirring for an additional 24 h. Afterward, the product mixture was dialyzed against a CH₃OH: H₂O (1: 1) solution in a bag with a molecular weight cutoff of 3500 kDa for two days, followed by dialysis against H₂O for another two days before being freeze-dried to obtain CTS-S-CPT [61].

4.2.2. Characterization of the CTS-S-CPT@IR 780 NPs

The particle size and ZP of the prepared CTS-S-CPT@IR 780 NPs were measured on a Zetasizer Nano ZS90 (Malvern Instruments Ltd., U.K.). Distilled water was added to the dried powder samples with sonication before measurement. The shape and surface morphology of the CTS-S-CPT@IR 780 NPs were observed via transmission electron microscopy (TEM; Tecnai G2 F30; FEI Company, Hillsboro, Oregon). To prepare the TEM samples, an appropriate amount of particles was suspended in distilled water. After sonication, a drop of the sample suspension was deposited onto a carbon-coated copper grid and dried overnight before observation.

The degree of CPT modification in the CTS-S-CPT@IR 780 NPs was determined via high-performance liquid chromatography (HPLC) (LC1200, Agilent Technologies) [52,62,63]. HPLC analysis of the CPT loading was performed on a reversed-phase C18 column (250 × 4.6 mm², 5 μm; Phenomenex, USA) with a mobile phase consisting of CH₃OH and H₂O (80: 20, v/v) operated at a flow rate of 1.0 mL/min. The column effluents were monitored at 254 nm with a UV detector. During drug release from the CTS-S-CPT@IR 780 NPs, all of the supernatants and washings were collected. The concentration of CPT in the solution was calculated via a calibration curve and the CPT release rate was calculated on the basis of the following equation: release rate (%) = $\frac{\text{weight}\text{of}\text{CPT}\text{in}\text{the}\text{solution}}{\text{total}\text{weight}\text{of}\text{CPT}}$ × 100 % [64].

4.2.3. Drug loading content (DLC) and encapsulation efficiency (EE)

The DLC and EE of CTS-S-CPT@IR 780 were determined via UV spectrophotometry with a detection wavelength of 780 nm, as follows [65].

$$\text{DLC}(\%)=\frac{\text{weight}\text{of}\text{the}\text{drug}\text{in}\text{the}\text{nanospheres}}{\text{total}\text{weight}\text{of}\text{the}\text{nanospheres}}\times 100\%$$ (1)

$$\text{EE}(\%)=\frac{\text{weight}\text{of}\text{the}\text{drug}\text{in}\text{the}\text{nanospheres}}{\text{weight}\text{of}\text{the}\text{fed}\text{drug}}\times 100\%$$ (2)

4.2.4. In vitro drug release profile

A simple method was used to investigate CPT release in vitro. Briefly, 10 mg of accurately weighed lyophilized sample was dispersed in 10 mL of simulated body fluid (PBS + 0.5 % Tween 80; pH 7.4) [66]. After thorough mixing, 1.5 W/cm² US irradiation was applied for 5 min, after which the sample was equally portioned into eight Eppendorf (EP) tubes. The samples were placed in a constant-temperature oscillator and incubated at 37 °C with constant-temperature oscillation (100 rpm) for 2, 4, 6, 8, 12, 24, 36, or 48 h. After each time point, one EP tube was removed and centrifuged at 15000 rpm for 10 min. Then, the supernatant (0.6 mL) was collected and mixed with an equal volume of methanol. The mixture was filtered through a 0.22 μm filter membrane before being transferred to a 1.5 mL vial. The amount of CPT in the supernatant was evaluated via HPLC [67].

4.2.5. Binding affinity analysis

The binding affinities of CPT and Bio-DNA towards Top1, respectively, were measured by Bio-Layer Interferometry (BLI) using an Octet RED96 instrument (FortéBio, Pall Life Sciences). All assays were performed in D-PBS with 0.05 % Tween-20 (assay buffer). The final volume for all the solutions was 200 μL per well. Assays were performed at 25 °C in solid black 96-well plates (Geiger Bio-One). Bio-DNA was loaded onto the surface of SA Cpature Biosensors for 300 s. The Top1 was used in a Ni-NTA Cpature Biosensors for 300 s. The interaction was followed for 300 s. Data were analyzed using FortéBio data acquisition software v.8.1 and were fitted using a global fit 1: 1 model.

4.2.6. Cell culture

B16 (CL-0029), HUVEC (CP-H082) and A-375 (CL-0014) cells were kindly provided by WuhanPricella Biotechnology Co.,Ltd. All of the cell lines were cultured at 37 °C incubator with 5 % CO₂. B16 melanoma cells were maintained in vitro with 1640 medium supplemented with 10 % (v/v) fetal bovine serum (FBS) and the antibiotics penicillin (100 U/mL) and streptomycin (100 μg/mL). Then A-375 and HUVEC cells were incubated in vitro with DMEM medium supplemented with 10 % (v/v) fetal bovine serum (FBS) and the antibiotics penicillin (100 U/mL) and streptomycin (100 μg/mL).

4.2.7. In vitro cellular uptake of the CTS-S-CPT@IR 780 NPs

CTS-S-CPT@IR 780 NPs were prepared and incubated with B16 melanoma cells cultured for different durations (2 h, 4 h, 6 h, and 8 h). The cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI), and the locations of DAPI and IR 780 fluorescence were observed with a confocal laser scanning microscope (LSN880, Zeiss, Germany).

4.2.8. Intracellular drug release behavior

The intracellular drug release behavior of drug-loaded nanoparticles can be observed by fluorescence microscopy. A375 cells were seeded in glass bottom culture plate at a density of 2 × 10⁵ cells per well and incubated with a culture medium for 24 h. After the medium was removed, 1 mL of CTS-S-CPT@IR 780 NPs medium containing a IR 780 concentration of 4 μM was added. The cells were continued to be incubated for 6 h. Finally, the cells were washed three times with PBS, and then the fluorescence images of the cells were obtained by Label-free live cell microscopy system (IDT channels, SC3000-pro, Zircon Optoelectronics (Suzhou) Co., Ltd.).

4.2.9. Cytotoxicity test

HUVEC cells were selected for cytotoxicity test. The basal medium of the HUVEC cells line is DMEM. Logarithmic cells were collected and inoculated on 96-well cell culture plate at a density of 5000 cells/well. After incubating for 24 h, culture medium containing different concentrations of CPT, IR 780 and NPs (0, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 μM) was replaced. Then, the cells were exposed to US irradiation (1.0 MHz, 1.5 W/cm², 50 % duty cycle) for 2 min, followed by an additional 8 h of incubation. The cell activity was measured by CCK8 method with 4 parallel holes for each concentration.

4.2.10. Live/dead cell assays

A Calcein/propidium iodide (PI) Cell Viability/Cytotoxicity Assay Kit (Beyotime, C2015L) was used according to the manufacturer's instructions. Briefly, B16 cells were seeded at a density of 1 × 10⁵ cells per well in a 12-well plate and subjected to a 6 h of treatment with CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs or CTS-S-CPT@IR 780 NPs + US. The cells were then stained with live/dead stain. The green and red fluorescence signals from calcein-AM and PI, respectively, were detected via CLSM. Then, 3D tumor spheroids were cultured with different samples, including CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs and CTS-S-CPT@IR 780 NPs + US, for 24 h. Then, the spheroids were exposed to US irradiation (1.0 MHz, 1.5 W/cm², 50 % duty cycle) for 5 min, followed by an additional 8 h of incubation. After washing with PBS three times, calcein AM and PI were used to stain the spheroids. The spheroids were examined via a high-content screening system (Opera Phenix Plus).

4.2.11. Measurement of the mitochondrial membrane potential (MMP) with JC-1

The MMP of the cells was determined via JC-1 (5′,6′,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) staining. After B16 cells were incubated with CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs, or CTS-S-CPT@IR 780 NPs + US for 8 h, the drug-containing media was discarded. The cells were subsequently incubated with JC-1 solution for 30 min, after which the JC-1 dye was removed, and the cells were washed twice with JC-1 buffer. Finally, CLSM was used to visualize the cells in the culture media. In addition, following 24 h of treatment with the CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs or CTS-S-CPT@IR 780 NPs + US, the treated spheroids were harvested, washed and stained with JC-1 staining solution in the dark for 4 h. Green and red fluorescence signals were detected via a high-content screening system (Opera Phenix Plus).

4.2.12. Detection of intracellular ROS

After B16 cells were treated with CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs, or CTS-S-CPT@IR 780 NPs + US for 8 h, media containing 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used instead of media containing drugs to culture the cells for 30 min. Then, the intracellular ROS level was determined via CLSM and quantitatively analyzed via flow cytometry.

4.2.13. Assessment of B16 cell apoptosis

B16 cell apoptosis was assessed via flow cytometry following experimental treatment. The cells were treated with trypsin without ethylenediamine tetraacetic acid (GIBCO), collected, and washed twice with ice-cold PBS. The cells were then resuspended in 1 × binding buffer to a concentration of 1 × 10⁶ cells/mL. Annexin V and PI (5 μL of each) were added to 500 μL of cell suspension in a flow tube, mixed well, and incubated for 10 min at room temperature in the dark. Five hundred microliters of binding buffer was then added, the tubes were mixed well, and the samples were analyzed for apoptosis via flow cytometry. Each experiment was repeated three times for statistical analysis.

4.2.14. Establishment of the in vivo tumor model

Female C57BL/6 mice aged 4–6 weeks were purchased from Guangdong Medical Experimental Animal Center and housed in specific pathogen-free (SPF) facilities with controlled temperature (25 ± 2 °C), humidity (55 ± 5 %) and light (12 h-light-dark cycle). During acclimatization for 7 days, animals were fed a standard laboratry diet (Corn 39.12 %, Bran 20 %, Flour 15 %, Soybean meal 16 %, Soybean oil 1.33 %, Fish meal 4 %, Stone powder 1.5 %, Calcium bicarbonate 2 %, Additive 1 %, Choline 0.05 %). Water and food were supplied ad libitum. All the protocols for the in vivo experiments were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University with protocol number SYSU-IACUC-2023-000198. After acclimatization for 7 days, approximately 5 × 10⁵ B16 cells were suspended in 100 μL of PBS and subcutaneously injected into the backs of the mice to establish a tumor model. Animal welfare was evaluated daily, which considered body weight, the animals' general condition, and tumor growth.

4.2.15. Biodistribution analysis

Randomly chosen mice were intravenously injected with CTS-S-CPT@IR 780 NPs at an IR 780 concentration of 3 μg/g. The biodistribution of CTS-S-CPT@IR 780 was observed via an in vivo imaging system (LB-983-NC100, Berthold, Germany).

4.2.16. In vivo tumor therapy

Tumor-bearing mice with a tumor volume of approximately 50 mm³ were randomly divided into six groups, and their body weights and tumor volumes before treatment were recorded. The tumor-bearing mice were intravenously injected with PBS, free CPT, free CPT + free IR 780 + US, CTS-S-CPT NPs, CTS-S-CPT@IR 780 NPs, or CTS-S-CPT@IR 780 NPs + US. The mice were treated once every other day for six consecutive days at an IR 780 dosage of 3 μg/g (or equivalent IR 780 dosage for the IR 780-loaded NPs). We prepare the IR 780 or CPT solution using a mixture of 2 % DMSO, 20 % PEG300, 5 % Tween-80, and 73 % PBS. The tumor volumes and body weights of the mice were recorded every day to observe tumor inhibition in each group. The tumor volume was calculated according to formula V = L × W²/2, where L and W denoted the maximum tumor length and width. On the 10th day, all the mice were sacrificed, and their tumor tissues were removed for to be weighed and photographed.

4.2.17. Histological examination

For the tumor therapy experiments described above, after the mice were sacrificed, the hearts, livers, spleens, lungs, kidneys, and tumor tissues were excised, fixed with 4 % formalin, embedded in paraffin, sliced into thin sections, and stained with hematoxylin and eosin (H&E).

4.2.18. 3D spheroid model

A375 melanoma spheroids (approximately 50 μm) were cultured in Basement Membrane Matrix HC (HY-K6005, MedChemExpress, America) for seven days in 96-well cell culture plates. The cells that grew as a monolayer were detached via trypsin and then centrifuged. Spheroids were seeded by diluting the single-cell suspensions in the liquid media (50 % Dulbecco's modified eagle medium (DMEM) with 10 % FBS and 50 % Basement Membrane Matrix HC), and 8 μL of the suspension (1 × 10⁷ cells per mL) was added to each well of the 96-well plates. The plates were subsequently incubated at 37 °C for 30 min to allow the matrix gel to solidify, after which 150 μL of complete culture media was added and the plates were incubated at 37 °C. The medium (150 μL) was removed every other day and replaced with fresh medium.

4.2.19. 3D melanoma tumor spheroid penetration

A 3D tumor spheroid model of A375 cells (approximately 50 μm) was established in vitro. The tumor spheroids were incubated with CTS-S-CPT@IR 780 NPs for 0, 4, 8, 12, 24, or 36 h, and the penetration depth of IR 780 fluorescence was evaluated with a high-content screening system (Opera Phenix Plus).

4.2.20. Immunohistochemistry (IHC)

The spheroids were fixed in Immunol Staining Fix Solution (Beyotime, P0098) overnight at 4 °C, followed by rinsing with Immunol Staining Wash Buffer (Beyotime, P0106). After incubation in goat serum (ZSGB-BIO, ZLI-9021) for 8 h, the spheroids were incubated overnight at 4 °C with primary antibodies against Ki67 (1: 1000, Beyotime, AB2008) and p16 (1: 1000, Beyotime, AF1069) for 24 h. Thereafter, the spheroids were incubated with a goat anti-rabbit secondary antibody (1: 1000, FITC-labeled goat anti-rabbit IgG (H + L), Beyotime, A0562) for 8 h at room temperature. Images were acquired with a high-content screening system (Opera Phenix Plus).

CRediT authorship contribution statement

Ying Wang: Writing – original draft, Methodology, Conceptualization. Yujie Wang: Writing – review & editing, Methodology. Yao Yang: Methodology, Conceptualization. Ling Leng: Writing – review & editing, Conceptualization.

Statistical analysis

All error bars shown in the Figures represented the standard deviation. Graph Pad Prism 10.1.0 and Origin Pro 2021 were used for data statistics and statistical significance calculation. Statistical analysis was completed by one-way analysis of variance (ANOVA) for calculating the P value. P value < 0.05 was considered as significant. ns means no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Funding

This work was supported by Beijing Municipal Science & Technology Commission (Z231100007223009, L.L.), the National Natural Science Foundation of China (82341079, L.L.), the National Key R&D Program of China (2024YFA1108400, L.L.), the CAMS Innovation Fund for Medical Sciences (CIFMS) (2023-I2M-QJ-001 and 2023-I2M-3-002, L.L.), and National High Level Hospital Clinical Research Funding (2025-PUMCH-C-016, L.L.).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.