Sonodynamic-chemotherapy synergy with chlorin e6-based carrier-free nanoparticles for non-small cell lung cancer

Abstract

Sonodynamic therapy (SDT), an emerging cancer treatment with significant potential, offers the advantages of non-invasiveness and deep tissue penetrability. The method involves activating sonosensitizers with ultrasound to generate reactive oxygen species (ROS) capable of eradicating cancer cells, addressing the challenge faced by photodynamic therapy (PDT) where conventional light sources struggle to penetrate deep tissues, impacting treatment efficacy. This study addresses prevalent challenges in numerous nanodiagnostic and therapeutic agents, such as intricate synthesis, poor repeatability, low stability, and high cost, by introducing a streamlined one-step assembly method for nanoparticle preparation. Specifically, the sonosensitizer Chlorin e6 (Ce6) and the chemotherapy drug erlotinib are effortlessly combined and self-assembled under sonication, yielding carrier-free nanoparticles (EC-NPs) for non-small cell lung cancer (NSCLC) treatment. The resulting EC-NPs exhibit optimal drug loading capacity, a simplified preparation process, and robust stability both in vitro and in vivo, owing to their carrier-free characteristics. Under the synergistic treatment of sonodynamic therapy and chemotherapy, EC-NPs induce an excess of reactive oxygen in tumor tissue, prompting apoptosis of cancer cells and reducing their proliferative capacity. Both in vitro and in vivo experiments demonstrate superior therapeutic effects of EC-NPs under ultrasound conditions compared to free Ce6. In summary, our research findings highlight that the innovatively designed carrier-free sonosensitizer EC-NPs present a therapeutic option with commendable efficacy and minimal side effects.

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1. Introduction

It is estimated that there are ∼2.2 million new cases of lung cancer worldwide each year, with an associated mortality of up to 1.79 million cases.¹ Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers and is currently the main cause of cancer-related deaths.² Common clinical treatments modalities include surgery in the early stages, radiation therapy, and chemotherapy, etc. However, these approaches often face challenges such as limited efficacy or serious side effects.³ This underscores the imperative need to develop alternative effective therapeutic strategies.

In recent years, research on non-invasive cancer therapies has been receiving increasing attention.^4,5 Among these, photodynamic therapy (PDT) stands out as a non-invasive treatment technique applicable to superficial skin cancers. Under the excitation of a specific wavelength of light, the photosensitizer can generate cytotoxic reactive oxygen species (ROS), effectively causing the demise of cancer cells.^6,7 However, due to the limited penetration capability of light in tissues, the therapeutic efficacy for tumor tissues located at deeper sites is often compromised.^8,9 Sonodynamic therapy (SDT), also a new type of non-invasive therapy, can activate photosensitizers to produce ROS through ultrasound stimulation.¹⁰ Due to the physical characteristics of ultrasound, it can achieve superior penetration in soft tissues. Compared with PDT, SDT exhibits enhanced tissue penetration, making it a promising alternative to PDT.^11–13 However, in certain cases, relying solely on SDT for anticancer treatment may yield limited efficacy and struggle to inhibit tumor growth and recurrence. Therefore, SDT may need to be combined with other anti-cancer treatment modalities to enhance the therapeutic outcomes.

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that often plays a role in the proliferation, invasion, and metastasis activities of cancer cells, showing high expression characteristics (40–80%) in NSCLC.¹⁴ Erlotinib is a tyrosine kinase inhibitor approved by the United States Food and Drug Administration (FDA). It is used as a first-line drug in clinical treatment for advanced metastatic pancreatic cancers and NSCLC.^15,16 By binding reversibly to the EGFR tyrosine kinase at the adenosine triphosphate (ATP) binding site, erlotinib effectively blocks the signalling pathway, curtailing cell proliferation and metastasis. Furthermore, this inhibition may trigger apoptotic cell death pathways, contributing to the suppression of tumor progression. The FDA has previously approved the combination of erlotinib with gemcitabine for the treatment of patients with late-stage pancreatic cancer.¹⁷ This prompts us to consider whether it can be combined with other treatment modalities to further enhance its therapeutic efficacy.

The nanodrug delivery system has become a hot topic in current anti-cancer drug research¹⁸ due to their numerous advantages, such as improving drug solubility, prolonging the blood circulation half-life of drugs, possessing targeted delivery capabilities, and simultaneous delivery of multiple formulations at the same time. Common drug delivery carriers include liposomes,¹⁹ polymer nanoparticles,²⁰ and inorganic carriers,²¹etc. Among them, Chlorin e6 (Ce6), as a photosensitizer and sonosensitizer, is widely utilized in numerous studies on anti-cancer nanomedicines related to PDT or SDT.^22–24 For instance, Huang and colleagues designed a lipid-based nanoparticle, P-aPD-L1/C, loaded with aPD-L1 and Ce6. This nanoparticle exhibited dual sensitivity to pH and MMP-2, and it had been used for the synergistic treatment of tumors through SDT and immunotherapy.²⁵ Han et al. developed nanoparticles using PEG-coated IR780 and Ce6 for sonodynamic therapy.²⁶ These strategies demonstrate the potential of Ce6 as a sonosensitizer in SDT, prompting us to consider the possibility of developing simpler and more efficient drug delivery systems for Ce6.

In recent years, carrier-free nanoparticles formed by the self-assembly or co-assembly of pure drugs or prodrugs have attracted widespread attention.^27–29 By utilizing hydrophobic interactions and/or π–π stacking, unmodified drug molecules can spontaneously assemble into uniformly distributed nanoparticles without the need for any stabilizers.^30–32 Consequently, the preparation process is relatively straightforward, and the drug loading capacity is high.³³ In addition, the incorporation of functional drug components typically endows carrier-free nanomedicines with a variety of therapeutic modalities, such as photodynamic therapy, sonodynamic therapy, and synergistic chemotherapy.³⁴ As a result, the co-assembly behavior of carrier-free nanoparticles provides an effective platform for dual-drug or multi-drug combination therapy, addressing the challenges associated with the synergistic delivery of multiple drugs in cancer treatment.

In this study, we employed hydrophobic interactions and π–π stacking to self-assemble Ce6 and erlotinib into carrier-free nanoparticles, denoted as EC-NPs, thereby integrating sonodynamic therapy and chemotherapy into a unified approach. The resulting EC-NPs exhibit excellent dispersibility and stability in solution, and their surface, being devoid of carrier modification, ensures an optimal drug loading efficiency for the nanoparticles. Additionally, the prepared nanoparticles possess effective passive targeting capabilities based on the enhanced permeability and retention (EPR) effect.³⁵ Upon uptake by cancer cells and activation by acoustic waves in the specific tumor region, EC-NP will induce excess generation of reactive oxygen species by Ce6, promoting apoptosis of cancer cells, thereby achieving sonodynamic therapy. Meanwhile, erlotinib, as an EGFR-TKI, competes with ATP for binding to the intracellular catalytic domain of the epidermal growth factor receptor tyrosine kinase, inhibiting phosphorylation and blocking downstream signalling, thus suppressing tumor growth. Therefore, these two approaches enable synergistic treatment through chemotherapy and sonodynamic therapy (Fig. 1).

Fig. 1 Schematic diagram illustrating the self-assembly of EC-NPs and their combined sonodynamic-chemotherapy treatment of established NSCLC tumors.

2. Materials and methods

2.1 Materials

Chlorin e6 (Ce6) was acquired from Macklin Reagent Company (China). Erlotinib was sourced from Aladdin Reagent Company (China). Dimethyl sulfoxide (DMSO) was obtained from National Medicines Chemical Reagents Co., Ltd (China). 2,5-Diphenyltetrazolium bromide (MTT) was procured from Sigma-Aldrich (USA). 1,3-Diphenylisobenzofuran (DPBF) was purchased from Macklin Reagent Company (China).

2.2 Preparation of EC-NPs

The preparation process of EC-NPs is straightforward. In a typical procedure, Ce6 (100 μL, 50 mM in DMSO) and erlotinib (100 μL, 25 mM in DMSO) are dissolved in DMSO and mixed together. The resulting mixture is then slowly added dropwise into a glass vial containing deionized water (4 mL) under ultrasonic conditions, and sonicated for 10 minutes. Next, the solution undergoes dialysis using a dialysis tube (MWCO, 3.5 kDa) for 24 hours to remove DMSO and unencapsulated free drugs. After centrifugation at 3000 rpm for 10 minutes, the nanoparticles are obtained by collecting the supernatant.

2.3 Characterization of nanoparticles

The absorbance of Ce6 and erlotinib was measured using a UV-1900i spectrophotometer (Shimadzu, Japan). The size and polydispersity index (PDI) of the nanoparticles in the solution (0.5 mg mL⁻¹) were measured by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, UK). The morphology of the nanoparticles was observed using a JEM-1400 Plus transmission electron microscope (TEM) (JEDL, Japan). For sample preparation, 10 μL of micellar solution (0.5 mg mL⁻¹) was placed on a copper grid and dried for observation.

2.4 In vitro stability of EC-NPs

In brief, 1 mL of micelles (0.1 mg mL⁻¹) was placed in a constant temperature shaking incubator. The dynamic light scattering (DLS) analysis of the solution was conducted on the 1st, 3rd, 5th, and 7th days, resulting in a 7-day in vitro particle size change curve. Additionally, 1 mL of micelles (0.1 mg mL⁻¹) was combined with 10% fetal bovine serum and subjected to a constant temperature shaking incubator. The DLS analysis of the solution was performed on the 1st, 2nd, and 3rd days, resulting in a 3-day serum stability curve.

2.5 ROS generation by EC-NPs

The singlet oxygen indicator fluorescent probe 1,3-diphenylisobenzofuran (DPBF) is used to detect the ROS generated by EC-NPs under ultrasonic action. Specifically, DPBF (25 μM, 1 mL) and EC-NPs (10 μM, 1 mL) are co-dissolved and then sonicated. The absorbance change of the solution is measured every 30 seconds using a UV spectrophotometer.

2.6 Hemocompatibility of EC-NPs

The hemocompatibility of EC-NPs was determined through a hemolysis experiment. 1 mL of rabbit blood was taken and sodium heparin was added to prevent coagulation. After dilution with PBS and centrifugation to remove the supernatant, the resulting red blood cells were washed multiple times and then diluted with PBS for later use. For the positive control group, 0.2 mL of diluted blood was mixed with 0.8 mL of pure water, and its absorbance at a wavelength of 540 nm was measured using an enzyme-linked immunosorbent assay reader, ensuring that the absorbance was between 0.5 and 0.55. For the negative control group, 0.2 mL of diluted blood was mixed with 0.8 mL of PBS. The experimental group was set with EC-NPs concentrations ranging from 0.625 μM to 10 μM, each taking 0.8 mL and adding 0.2 mL of diluted blood. After the samples were left standing for 3 hours, they were centrifuged to precipitate the red blood cells, and the supernatant was taken to measure the absorbance at 540 nm using an enzyme-linked immunosorbent assay reader. The percentage of hemolysis was calculated using the following formula:

In the formula, OD_S represents the absorbance of the experimental group, OD_P represents the absorbance of the positive control group, and OD_N represents the absorbance of the negative control group.

2.7 Cell culture

Non-small cell lung cancer A549 cells were cultured in RPMI-1640 medium containing 10% (v/v) fetal bovine serum and 1% (v/v) penicillin–streptomycin antibiotic at 37 °C in a 5% CO₂ atmosphere, for subsequent cell experiments and animal experiments.

2.8 In vitro cellular uptake and intracellular ROS detection

A549 cells are seeded in a 12-well plate, with 1 × 10⁵ cells per well. For the cell uptake experiment, after 24 hours, the culture medium is removed and replaced with a medium containing EC-NPs and free Ce6 (Ce6 concentration of 10 μM). The cells are then incubated for 0.5, 2, and 4 hours respectively, and images are taken using a fluorescence microscope.

For intracellular ROS detection, after incubating with a medium containing EC-NPs and free Ce6 (Ce6 concentration of 10 μM) for 24 hours, DCFH-DA is added and incubated for another 2 hours. The well plate is then irradiated for 5 minutes under an ultrasonic environment of 2 W cm⁻², 1 MHz, and 20% duty cycle. After washing 3 times with PBS buffer, the cells are observed under an inverted fluorescence microscope.

For quantitative analysis, flow cytometry is used. The cells, after drug and ultrasound treatment, are treated with trypsin, then collected by centrifugation, and recorded by flow cytometry (BD FACS Aria III).

2.9 MTT assay

The MTT method was employed to assess the survival rate of A549 cells. In both the presence and absence of ultrasound, the cytotoxicity of EC-NPs, free Ce6, and free erlotinib on A549 cells was investigated in a dose-dependent manner. A549 cells were seeded in a 96-well plate at a density of 8 × 10³ cells per well and incubated at 37 °C in a 5% CO₂ atmosphere for 24 hours. After the initial 24 hour period, the original culture medium was aspirated, and medium containing the drug was added. The concentrations of EC-NPs (determined by the Ce6 concentration) and free Ce6 ranged from 10 μM to 0.625 μM, while the concentration of free erlotinib ranged from 5 μM to 0.3125 μM.

Following drug addition, incubation continued for another 24 hours. Subsequently, the well plate was subjected to irradiation for 5 minutes under an ultrasound environment (2 W cm⁻², 1 MHz, 20% duty cycle) in the ultrasound treatment group, while the non-ultrasound treatment group did not undergo this step. After an additional 2 hours, the original culture medium was removed, and 110 μL of medium containing 5 mg mL⁻¹ MTT was added, followed by a 4 hour incubation. Post-incubation, 100 μL of DMSO was added to dissolve the formazan in the well plate. Finally, the absorbance of the cell solution at 490 nm was measured using an enzyme-linked immunosorbent assay reader to calculate cell viability. Cell viability (%) was determined using the following formula:

In the formula, N_s, N_C, and N represent the absorbance of the sample group, the control group, and the cell-free group.

2.10 Cell apoptosis

A549 cells were plated in a 6-well plate at a density of 2 × 10⁵ cells per well. Following a 24 hour incubation, the culture medium was replaced with medium containing EC-NPs (Ce6 concentration of 10 μM) and incubated for an additional 24 hours. Subsequently, the well plate underwent a 5 minute irradiation under ultrasound conditions (2 W cm⁻², 1 MHz, 20% Duty cycle). The apoptosis induced by ROS was assessed using an Annexin V-FITC/PI Cell Apoptosis Detection Kit and analysed through flow cytometry.

2.11 In vivo studies

All experiments were approved by the Experimental Animal Ethics Committee of Wuhan University of Technology. For tumor modelling, 6-week-old female BALB/C-nu mice (Wuhan Center for Disease Control & Prevention, China.) were subcutaneously injected with A549 cells (2 × 10⁶ cells, dissolved in 0.1 mL PBS) on the right dorsal side. Treatment experiments were conducted when the tumor volume in the mice reached 100 mm³.

Fluorescence imaging was performed using an IVIS Spectrum (PerkinElmer, USA). In brief, EC-NPs (5 mg kg⁻¹) were intravenously injected into the mice. The fluorescence imaging system was used to image at specific time points (0, 3, 6, 9, 24, and 48 hours). After intravenous administration for 48 hours, various organs and tumors are removed, washed three times in PBS, and then imaged ex vivo using the fluorescence imaging system.

Tumor-bearing mice were randomly divided into 4 groups, with 4 mice in each group: (I) control group (II) free Ce6 + ultrasound group (III) EC-NPs group (IV) EC-NPs + ultrasound group. The drug groups were intravenously injected with 5 mg kg⁻¹ of EC-NPs, and the control group was injected with the same volume of PBS solution. After 24 hours of injection, the ultrasound group and the EC-NPs + ultrasound group were irradiated for 5 minutes at the tumor site under an ultrasound condition (2 W cm⁻², 1 MHz, 20% Duty cycle). To evaluate the efficacy of SDT, the length and width of the tumor were measured with a calliper to monitor the tumor volume over time, and the weight change of the mice was also monitored. The tumor volume was calculated using the following formula:

The tumor growth curve was plotted as a function of the average tumor volume of each group over time.

Thereafter, all mice were sacrificed, and the heart, liver, lungs, kidneys, spleen, and tumor tissues were collected for hematoxylin and eosin (H&E) analysis after the treatment.

3. Results and discussion

3.1 Preparation and characterization of EC-NPs

Due to the amphiphilic nature of Ce6 molecules, we can utilize hydrophobic interactions to encapsulate the hydrophobic drug erlotinib, thereby achieving the formation of nanoparticles EC-NPs through the modulation of the self-assembly process. Specifically, by adjusting the feed ratio, the characteristics of the generated nanoparticles can be tailored. We found that within the molar feed ratio range of Ce6 : erlotinib from 2 : 1 to 1 : 2, relatively stable nanoparticles could be formed (Fig. 2a). Beyond this ratio, nanoparticles tended to become less stable or prone to precipitation, leading to synthetic failure. The hydrodynamic diameters of the nanoparticles measured by dynamic light scattering (DLS) were 112.5 nm (C2E1), 165.7 nm (C1E1), and 205.2 nm (C1E2), respectively. As the proportion of Ce6 increased, the particle size decreased (Table 1). With a higher Ce6 ratio, increased hydrophobic interactions and π–π stacking between Ce6 and erlotinib resulted in more compact nanoparticles. Among the three formulations, C2E1 exhibited a smaller particle size and a more uniform polydispersity index (PDI), thus C2E1 was selected for subsequent experiments. As shown in Fig. 2b, TEM images displayed that EC-NPs presented a relatively uniform spherical morphology. Furthermore, UV spectrum demonstrated that the formation of EC-NPs did not change the characteristics of Ce6 and erlotinib (Fig. 2c). By plotting standard curves based on UV absorption spectra (Fig. S1, ESI†), the drug loading efficiency of EC-NPs could be calculated. Additionally, we employed DPBF as a reactive oxygen species (ROS) probe to test the ability of EC-NPs to generate ROS under ultrasound stimulation (Fig. 2d). The results showed that as the ultrasound time increased, the absorbance value around 420 nm gradually decreased, indicating that DPBF was consumed by the ROS generated with EC-NPs. This confirmed the effective generation of reactive oxygen species by EC-NPs under ultrasound, demonstrating their potent sonodynamic therapeutic capability. To validate whether the prepared nanoparticles exhibit favourable storage stability and in vivo stability, we conducted continuous monitoring of the particle size. The results revealed that the particle size and polydispersity index (PDI) of EC-NPs remained relatively unchanged over 7 days in aqueous solution (Fig. 2e). Additionally, in simulated blood conditions (PBS solution containing 10% serum), the particle size and PDI of EC-NPs remained stable over 3 days (Fig. S3, ESI†), affirming the excellent stability of the nanoparticles. The zeta potential of EC-NPs is −11.2 ± 0.9 mV. This slightly negative surface charge property contributes to the prolonged blood circulation time due to its low phagocytic uptake capacity.³⁶ Furthermore, to assess the feasibility of subsequent in vivo experiments, we conducted a blood compatibility test for EC-NPs. Hemolysis assays were employed to evaluate the in vivo blood compatibility of the micelles. The results demonstrated that the hemolysis rates caused by EC-NPs at various concentrations were all below 5% (Fig. 2f), indicating their good blood compatibility and suitability for future in vivo applications.

Fig. 2 Characterization of EC-NPs. (a) Particle size of EC-NPs prepared at different feed ratios. (b) Typical TEM image of EC-NPs. (c) UV absorption spectra of erlotinib, chlorin e6, and EC-NPs. (d) ROS release over time under ultrasound stimulation of EC-NPs tested using DPBF. (e) Changes in particle size and PDI of EC-NPs in solution within 7 days. (f) Evaluation of blood compatibility of EC-NPs and free Ce6.

Table 1 Drug encapsulation efficiency and physical chemical properties of EC-NPs prepared at different feed ratios

Erlotinib : Ce6 ratio	Erlotinib encapsulation efficiency (%)	Ce6 encapsulation efficiency (%)	Size (nm)	PDI
1 : 2	24.68	45.88	112.5	0.14
1 : 1	30.23	39.18	165.7	0.21
2 : 1	35.91	34.03	205.2	0.19

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3.2 In vitro cellular uptake and ROS generation of EC-NPs

To validate the cellular uptake of EC-NPs by A549 tumor cells, we employed fluorescence microscopy to observe the intracellular behaviour of EC-NPs and Free Ce6 at different time points. As shown in the Fig. 3a, both free Ce6 and EC-NPs exhibited time-dependent cellular uptake behaviour, and visually, EC-NPs showed a stronger fluorescence intensity within cells compared to free Ce6, indicating easier cellular uptake. This may be attributed to the poor solubility of Ce6 itself, leading to self-aggregation and an increased particle size, making it difficult for tumor cells to uptake. In contrast, EC-NPs, with good solubility and stability, are more easily internalized by cells. Quantitative analysis using flow cytometry also confirmed this observation (Fig. 3c). At the same time points, the EC-NPs group exhibited a stronger fluorescence signal than the free Ce6 group, demonstrating the easier internalization of EC-NPs by A549 cells.

Fig. 3 In vitro cellular uptake and ROS generation of EC-NPs. (a) Fluorescence images illustrating the uptake of Free Ce6 and EC-NPs by A549 cells at various time points. (b) Fluorescence microscope observation depicting A549 cells’ uptake of Free Ce6 and EC-NPs (left); DCF fluorescence inside the cells following ultrasound activation (2 W cm⁻², 1 MHz, 20% Duty cycle) (middle); combination of the two images (right). Scale bars: 50 μm. (c) Flow cytometry quantitative analysis of A549 cells' uptake of Free Ce6 and EC-NPs; (d) flow cytometry quantitative analysis of ROS production in cells after Free Ce6 uptake and ultrasound irradiation; (e) flow cytometry quantitative analysis of ROS production in cells after EC-NPs uptake and ultrasound irradiation.

In addition, we used the DCFH-DA probe for quantitative detection of intracellular ROS generation. Since DCFH-DA itself is non-fluorescent and can freely pass through the cell membrane, it is hydrolyzed by intracellular esterases to generate DCFH, which cannot pass through the cell membrane, effectively trapping the fluorescent probe inside the cells. ROS can oxidize non-fluorescent DCFH, producing fluorescent DCF. Thus, we could assess the changes in fluorescence within cells through fluorescence microscopy and flow cytometry to determine the amount of ROS generated. As shown in Fig. 3b, fluorescence microscopy images revealed that both free Ce6 and EC-NPs produced DCF green fluorescence signals inside the cells under ultrasound stimulation. When overlaid with EC-NPs fluorescence images and bright-field images, all three completely matched, indicating that ROS was generated by intracellular Ce6 under ultrasound. Furthermore, EC-NPs produced a stronger fluorescence signal within cells compared to free Ce6, likely due to the higher cellular uptake of EC-NPs, as evidenced by quantitative analysis using flow cytometry (Fig. 3d and e). These results demonstrate that EC-NPs, compared to free Ce6, address the issue of poor delivery efficiency caused by low water solubility, making them more easily taken up by cells and resulting in a stronger SDT effect, highlighting their excellent drug delivery capability.

3.3 In vitro cytotoxicity and cell apoptosis of EC-NPs

Next, we assessed the toxicity of EC-NPs and free drugs on A549 cells under different conditions using the MTT assay. The concentration of EC-NPs was represented by the Ce6 concentration, with concentration gradients set at 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM. The concentration of free erlotinib was adjusted according to the erlotinib concentration in EC-NPs, corresponding to 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM. In the groups without ultrasound exposure (Fig. 4a), all three drugs exhibited low cell toxicity (<20%), but EC-NPs showed lower toxicity compared to the two free drugs. In the groups with ultrasound stimulation (Fig. 4b), EC-NPs demonstrated concentration-dependent cell toxicity, reaching 87% cell toxicity at 10 μM, showing a significant difference compared to free Ce6 and free Ce6 & erlotinib (p < 0.001). This indicates that EC-NPs have stronger cancer cell-killing ability compared to free monomeric drugs, possibly due to the water-soluble nature of the nanoparticles, making them more easily taken up by cells. Additionally, we validate the synergistic effect of Ce6 and erlotinib in EC-NPs in killing cancer cells by calculating the Combination Index (CI).³⁷ When CI > 1, it is generally considered that there is an antagonistic effect between the drugs; when CI = 1, it is considered that there is an additive effect between the drugs; and when CI < 1, it is considered that there is a synergistic effect between the drugs. Using Compusyn software to calculate the synergy between the two drugs in EC-NPs, we find that CI < 1 (Fig. S4, ESI†), indicating that the two drugs have a synergistic effect in the treatment process, achieving a therapeutic effect where 1 + 1 > 2.

Fig. 4 In vitro cytotoxicity and cell apoptosis of EC-NPs. (a) Cellular cytotoxicity of drugs at different concentrations to A549 cells. (b) Cytotoxicity of drugs at different concentrations to A549 cells after ultrasound irradiation (2 W cm⁻², 1 MHz, 20% Duty cycle) (***p < 0.001). (c) Flow cytometry analysis of cell apoptosis in different groups.

The cell apoptosis analysis shows (Fig. 4c) that the US group did not have a significant effect on cell apoptosis compared to the Control group; the EC-NPs and Free Ce6 groups caused about 10% of cells to enter the apoptosis process compared to the blank control group; Free Ce6 + US triggered 41.8% of cells to enter the apoptosis process; while the EC-NPs + US group triggered 71.9% of cells to enter the apoptosis process, all of which are consistent with the cytotoxicity data. These results prove that EC-NPs have excellent cancer cell killing ability under ultrasonic action, demonstrating their potential application in sonodynamic therapy.

3.4 In vivo study of EC-NPs

To investigate the in vivo biodistribution behavior of EC-NPs in tumor-bearing mice, we established subcutaneous xenograft models with A549 cells on nude mice. Using free Ce6 as a control, we intravenously injected EC-NPs into the mice through the tail vein and performed in vivo fluorescence imaging at different time points within 0–48 hours and ex vivo fluorescence imaging of organs after 48 hours. As shown in Fig. 5a, mice injected with free Ce6 exhibited only a small amount of fluorescence signal in the abdominal liver, with almost no fluorescence signal at the tumor site. This indicates that free Ce6 is quickly cleared from the bloodstream after injection and has difficulty accumulating at the tumor site, making it unsuitable for direct application in in vivo SDT. In comparison to free Ce6, EC-NPs demonstrated significant accumulation at the tumor site. The fluorescence intensity of EC-NPs at the tumor site showed a time-dependent pattern, increasing initially and then decreasing, suggesting that nanoparticles can be transported from the bloodstream to the tumor site and gradually cleared during prolonged circulation.

Fig. 5 In vivo Study of EC-NPs. (a) In vivo fluorescence imaging of mice injected with free Ce6 and EC-NPs at different times. (b) The average fluorescence intensity at the tumor site of mice injected with free Ce6 and EC-NPs at different times (***p < 0.001). (c) Ex vivo fluorescence imaging of organs and tumors. (d) The average ex vivo fluorescence signal intensity of various organs and tumors in mice injected with free Ce6 and EC-NPs (**p < 0.01, ***p < 0.001). (e) Time-related curves of tumor volume in mice from different treatment groups (***p < 0.001). (f) Time-related curves of weight changes in mice from different treatment groups.

The curve depicting the average fluorescence signal intensity at the tumor site over time (Fig. 5b) also illustrates that free Ce6 has weaker accumulation at the tumor site, while the accumulation of EC-NPs reaches its maximum 24 hours after injection. Therefore, we selected 24 hours post-injection as the optimal time for subsequent ultrasound treatment. Additionally, after 48 hours, the mice were euthanized, and ex vivo fluorescence imaging of the tissues was performed (Fig. 5c). In vitro fluorescence imaging of organs showed that after 48 hours, there was almost no accumulation of Free Ce6 in various organs and tumors, while EC-NPs significantly accumulated in the tumor site, with the degree of accumulation second only to the liver as a metabolic organ. With quantitative analysis of the fluorescence intensity in each organ. As shown in Fig. 5d, the fluorescence of EC-NPs at the mouse tumor site was prominent. This indicates that nanoparticles can effectively accumulate at the tumor site.

To assess the sonodynamic therapy (SDT) efficacy of EC-NPs, tumor-bearing mice were randomly divided into four groups, each consisting of 4 mice: (I) control group; (II) free Ce6 injection followed by ultrasound (free Ce6 + US); (III) EC-NPs injection only (EC-NPs); (IV) EC-NPs injection followed by ultrasound (EC-NPs + US). On day 0, mice in groups II, III, and IV received intravenous injections of free Ce6 and EC-NPs at a Ce6 dose of 5 mg kg⁻¹. On day 1, ultrasound irradiation was applied to groups II and IV. Subsequently, the mice were monitored daily for changes in body weight and tumor volume. The tumor volume change curve (Fig. 5e) showed that, except for group IV, the tumor volume in the other groups exhibited rapid growth. In contrast, the nanoparticle ultrasound treatment group demonstrated a significantly lower tumor growth rate than the other groups. This suggests that EC-NPs under ultrasound exposure can effectively inhibit or even eradicate tumor growth. In fact, the tumors in this group nearly disappeared after treatment, leaving only minimal protrusions on the skin. Furthermore, the body weight monitoring data (Fig. 5f) indicated that there were no significant changes in mouse body weight in all groups, suggesting that these treatments had no obvious side effects on the mice. In addition, H&E staining of the tumor site (Fig. 6) confirmed the substantial elimination of tumor cells in group IV. Histological sections of other organs demonstrated that the combined action of EC-NPs and ultrasound did not cause significant damage to the mouse heart, liver, spleen, lungs, and kidneys, indicating the effectiveness and safety of SDT.

Fig. 6 H&E stained sections of various organs and tumors from different groups of mice. Scale bars: 100 μm.

4. Conclusion

In conclusion, this study engineered a carrier-free nanoparticle, EC-NPs, using Chlorin e6 and erlotinib for sonodynamic therapy. This carrier-free nanoparticle preparation method is simple and exhibits excellent drug loading capacity and stability. Under ultrasound exposure, EC-NPs can generate a significant amount of reactive oxygen species (ROS), effectively inhibiting the growth of tumor cells. Moreover, this nanoparticle demonstrates good biocompatibility and tumor accumulation capabilities. Its cytotoxicity is low without ultrasound triggering, making its impact on normal cells negligible. The combination of nanoparticles and ultrasound effectively suppressed tumor growth in the A549 tumor model, demonstrating its outstanding therapeutic efficacy and biological safety, with potential clinical applications.

Author contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The research was supported by the National Natural Science Foundation of China (No. 52003211), Natural Science Foundation of Hubei Province (No. 2020CFB418), the Guangdong Basic and Applied Basic Research Foundation (2024A1515011550), and the Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Soochow University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4tb00009a

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