Dual-responsive peptide nanoplatform for improved lung cancer therapy via the synergistic suppression of autophagy and drug efflux

Abstract

The efficacy of traditional chemotherapy in the treatment of non-small cell lung cancer (NSCLC) is often compromised by poor targeting, barriers posed by the tumor microenvironment, and active drug efflux. Furthermore, tumor cells significantly reduce their sensitivity to chemotherapeutic drugs by activating protective autophagy pathways, which is a key mechanism contributing to treatment failure. We designed and synthesized a smart responsive amphiphilic peptide, Pep1, featuring targeted modification with the Arg-Gly-Asp (RGD) peptide and triphenylphosphonium (TPP). Pep1 efficiently coloads paclitaxel (PTX) and hydroxychloroquine (HCQ) (PH/Pep1) and self-assembles into spherical nanoparticles. In vitro and in vivo experiments confirmed that PH/Pep1 not only efficiently entered lung cancer cells through active targeting to impair mitochondrial function but also inhibited autophagy, thereby preventing the clearance of damaged mitochondria. Through a “damaging mitochondria and inhibiting autophagy” synergistic mechanism, PH/Pep1 ultimately efficiently induced tumor cell apoptosis. Moreover, PH/Pep1 underwent a morphological transformation from nanospheres to nanofibers upon sequential stimulation by alkaline phosphatase (ALP) and reduced glutathione (GSH), which significantly increased drug retention and accumulation within tumor tissues. In a Lewis lung carcinoma (LLC) cell tumor-bearing mouse model, PH/Pep1 demonstrated the strongest tumor growth inhibition effect and good biosafety. In summary, the “dual-targeted/dual-responsive” synergistic therapeutic strategy proposed in this study provides a novel approach for designing highly efficient and precise nanodelivery platforms.

Graphical abstract

1. Introduction

Lung cancer remains a leading malignancy worldwide and is characterized by high incidence and mortality rates [1,2]. Approximately 85% of lung cancer cases are non-small cell lung cancer (NSCLC) [3,4]. Currently, chemotherapy continues to play a vital role in clinical lung cancer treatment [5]. However, tumor cells can weaken the cytotoxic effects of chemotherapy drugs by activating a series of complex cellular stress response pathways [[6], [7], [8]]. Among these, the induced activation of autophagy—a highly conserved intracellular degradation process—is considered one of the key mechanisms through which cancer cells maintain survival under chemotherapy stress [9,10]. Under normal conditions, autophagy maintains intracellular homeostasis by clearing damaged organelles such as mitochondria [11]. However, within the tumor microenvironment, this process is hijacked by cancer cells to counteract the cellular damage induced by chemotherapy drugs [12,13]. Specifically, mitochondrial autophagy can selectively eliminate mitochondria rendered dysfunctional by chemotherapeutic drugs, preventing the release of proapoptotic factors and thereby helping cells evade apoptosis [14,15]. Therefore, inhibiting autophagy and inducing mitochondrial damage have emerged as potential strategies for increasing the efficacy of chemotherapy.

Hydroxychloroquine (HCQ), an autophagy inhibitor, can suppress autophagy induced by the tumor microenvironment (TME) [16]. HCQ blocks the degradation of damaged organelles and proteins by lysosomes through the inhibition of acid-dependent lysosomal enzymes, thereby inhibiting mitochondrial autophagy [17,18]. Paclitaxel (PTX) kills tumors by interfering with microtubule dynamics, leading to G2/M cell cycle arrest and apoptosis [19]. Within cells, mitochondria are transported along microtubules to areas with high energy demands [20]. PTX disrupts the dynamic equilibrium of microtubules by suppressing depolymerization, preventing the proper distribution and transport of mitochondria, thereby impairing the cellular energy supply [21,22]. Additionally, when chemotherapy drugs (such as paclitaxel) attack tumor cells, many damaged mitochondria are generated [23]. HCQ inhibits the clearance of these damaged mitochondria, leading to their accumulation within cells and thereby exacerbating apoptosis [24]. However, both HCQ and PTX face challenges such as poor water solubility, short circulation times in vivo, lack of tumor targeting, and systemic toxicity caused by nonspecific distribution, which severely limit their clinical application potential [25].

The emergence of smart nanomedicine delivery systems has presented new opportunities to overcome the aforementioned challenges [26,27]. Functionalized peptide-based nanocarriers can significantly increase drug bioavailability [28]. Among these, Arg-Gly-Asp (RGD) peptides can specifically recognize integrins (such as αvβ3) that are highly expressed on the surface of various tumor cells, including NSCLC cells [29,30]. Moreover, the triphenylphosphonium (TPP) is often used as a mitochondrion-targeting group because of its high affinity for the negatively charged mitochondrial membrane [31,32]. Integrating RGD peptides with TPP molecules into a single delivery system enables multilevel targeted drug delivery, thereby synergistically increasing therapeutic efficacy.

Moreover, the efficacy of chemotherapy is often compromised by insufficient drug accumulation within tumor tissues, limited penetration depth [33], and rapid clearance by efflux pumps such as P-glycoprotein, all of which negatively affect therapeutic outcomes [34,35]. Alkaline phosphatase (ALP) is an important independent prognostic biomarker of cancer [36]. Elevated ALP levels have been observed in lung cancer patients [37]. Reduced glutathione (GSH) is among the most abundant antioxidants within cells [38], and its production has been demonstrated to be increased in cancer cells [39,40]. The development of small-molecule peptides that undergo structural transformation upon induction by ALP- and GSH-mediated catalysis may offer novel approaches for increasing drug accumulation [41].

In recent years, various nanoparticle delivery systems based on autophagy inhibitors have been reported, such as those utilizing liposomes or hyaluronic acid-modified carriers to achieve tumor accumulation [42,43], and these studies have provided valuable strategies for the delivery of autophagy inhibitors to tumors. We previously developed a pH-responsive peptide nanoplatform that ruptures and releases the encapsulated drugs in the slightly acidic tumor microenvironment, relying primarily on the EPR effect to increase tumor accumulation [44]. In addition, plasma amine oxidase (PAO) has been used to induce morphological changes to increase enrichment; however, PAO is abundant in blood and lacks specificity for the tumor microenvironment [45].

On the basis of the above analysis, in this study, a novel amphiphilic peptide (Pep1) comodified with RGD and TPP was designed and synthesized for the codelivery of PTX and HCQ (PH/Pep1). We hypothesize that this nanosystem can achieve efficient tumor targeting and, in response to stimulation by both ALP and GSH in the tumor microenvironment, undergo a morphological transformation that releases the encapsulated drugs, resulting in the killing of tumor cells and mitochondrial damage. The simultaneous inhibition of protective autophagy and prevention of the clearance of damaged mitochondria by cells could synergistically increase therapeutic efficacy in the context of NSCLC (Fig. 1). Compared with existing systems that rely on a single target or lack responsive regulation, this dual-target/dual-response strategy offers significant advantages in terms of delivery specificity and drug release precision.

Fig. 1.

PH/Pep1 responds to ALP and GSH in the tumor microenvironment and undergo a morphological transformation. PH/Pep1 actively targets tumor cells. Within cells, the spherical nanoparticles transform into fibrous aggregates with a high aspect ratio, prolonging the drug retention time. PH/Pep1 inhibits cellular autophagy, thereby increasing the efficacy of PTX. Moreover, Pep1 damages mitochondria, exhibiting increased tumor cell killing efficacy.

2. Materials and methods

2.1. Materials

PTX was purchased from Macklin (Shanghai, China). HCQ and coumarin 6 (Cou6) were purchased from Aladdin (Shanghai, China). DAPI, Ad-GFP-LC3B, and cell cycle and apoptosis assay kits were purchased from Biyuntian Biotechnology Co., Ltd. (Shanghai, China). Human non-small cell lung cancer cells (A549), human umbilical vein endothelial cells (HUVECs), and Lewis lung carcinoma (LLC) cells were obtained from the BeNa Culture Collection Center (BNCC). Pengyue Laboratory Animal Breeding Co., Ltd. (Jinan, China) provided C57BL/6 mice. The peptides were purchased from ChinaPeptides Biotechnology Co., Ltd. (QYAOBIO, Shanghai, China).

2.2. Preparation of the peptide nanocarriers

0.25 mg of each peptide (Pep1 or Pep2) was dissolved in 1 mL of HEPES buffer (25 mM, pH 7.4) to prepare 0.25 mg/mL peptide solutions. To obtain PTX-loaded peptide solutions (PTX/Pep1 and PTX/Pep2), 0.05 mg of PTX was dissolved in 10 μL of dimethyl sulfoxide (DMSO) and 990 μL of peptide solution was added for incubation at room temperature. The volume ratio of DMSO was 1% (v/v). To obtain peptide solutions loaded with PH (PH/Pep1 or PH/Pep2), 0.05 mg of each PTX and HCQ was dissolved in 10 μL of DMSO, these solutions were combined, 980 μL of peptide solution was added, and the mixture was incubated at room temperature.

2.3. Mass spectrometry and high-performance liquid chromatography

Chromatographic analysis of the Pep1 and Pep2 peptide solutions with or without ALP and GSH was performed using a Thermo Fisher Ultra 3000 system (Thermo Fisher Scientific, USA) [46]. The molecular weight (Mw) of the purified peptides was determined using electrospray ionization mass spectrometry (ESI-MS).

2.4. Transmission electron microscopy (TEM)

Drug-loaded peptide solution samples with or without ALP and GSH (PH/Pep1 and PH/Pep2) were prepared. An electron microscopy copper grid was placed on top of an appropriate amount of sample, and the sample was incubated for 5 min to ensure complete adsorption. After adsorption, 100 μL of staining solution was added, and the copper mesh was placed on top for 5 min of incubation. After staining was complete, the copper mesh was removed, and the sample was prepared for transmission electron microscopy observation.

2.5. Zeta potential and particle size analysis

The zeta potentials of the pure peptides and drug-loaded peptides before and after ALP and GSH addition were measured using a Zetasizer Nano ZS90 (Malvern, UK). ImageJ software was used to determine the particle size distribution. The data were analyzed using Origin 8.5 software.

2.6. CCK-8 assay

A549 cells were seeded into a 96-well plate and incubated overnight. After washing three times with PBS, 100 μL of drug solution was added per well and incubated for 12 h. Then 10 μL of CCK-8 reagent was added and incubated for 3 h at 37°C. The absorbance was recorded at 450 nm.

2.7. Cellular uptake assay

An A549 cell suspension (200 μL/well) was added to an 8-well plate and incubated overnight. Next, 1/20 of the volume of drug-loaded peptide (Cou6, Cou6/Pep2, or Cou6/Pep1) was added to the wells. After 2 h and 15 min of incubation, the wells were drained and washed with PBS. Two hundred microliters of 4% paraformaldehyde solution was added to each well, and the plate was incubated for 15 min for cell fixation. The 4% paraformaldehyde solution was removed, and the cells were washed twice with PBS. DAPI staining solution was added, and the cells were incubated at 37 °C for 15 min, washed twice with PBS, and observed under an upright fluorescence microscope.

2.8. Cell retention assay

An A549 cell suspension (200 μL/well) was added to an 8-well plate and incubated overnight. Next, 1/20 of the volume of drug-loaded peptide (Cou6, Cou6/Pep2, or Cou6/Pep1) was added to the wells. Two hours later, the liquid was removed from the wells, and the cells were rinsed with PBS. The cells were cultured in complete medium for 60 h or 72 h. Then, the culture medium was aspirated, the cells were washed twice with PBS, 200 μL of 4% paraformaldehyde was added to each well, and the cells were fixed in an incubator for 15 min. After the 4% paraformaldehyde solution was removed, the cells were washed twice with PBS. DAPI staining solution was added, and the cells were incubated for 15 min, washed twice with PBS, and observed under an upright fluorescence microscope.

2.9. Ad-GFP-LC3B infection

The MOI for A549 cells was determined through preliminary experiments to establish optimal infection conditions. After the cells were thawed on ice, the virus was added to the cells at the appropriate proportion. Twenty-four hours after infection, the adenovirus solution was removed, and 2 mL of complete medium was added to each well. The cells were cultured for another 24 h, after which cell growth and fluorescent protein expression were assessed. Subsequent infection experiments were conducted under these conditions. Next, the cells were fixed with 4% paraformaldehyde and washed with PBS. DAPI staining solution was added, and the cells were incubated. The fluorescence intensity of LC3B was observed under a fluorescence microscope.

2.10. Western blotting

A549 cells were seeded into a 6-well plate and cultured overnight. Different drugs mixed with serum-free medium at a ratio of 1:12000 were added to the appropriate group. The cells were incubated at 37 °C for 24 h. Then, protein samples were collected, and their concentrations were determined. After the protein samples were denatured, electrophoresis gels were prepared, and electrophoresis was performed after sample loading. The membrane was transferred, blocked, and then incubated sequentially with primary and secondary antibodies. Finally, the color development reaction was conducted.

2.11. JC-1 assay

A549 cells were placed in an eight-well plate and incubated overnight. The medium was removed, and the cells were rinsed once with PBS. Different drugs mixed with serum-free medium at a ratio of 1:12000 were added to each group. The cells were incubated at 37 °C for 20 min. The medium was removed, and the cells were rinsed twice with prepared JC-1 staining buffer (10 μg/mL). Next, 500 μL of PBS was added to each well, and the cells were visualized using an inverted fluorescence microscope.

2.12. Cell cycle assessment

After cells had fully adhered to the bottom of a T25 flask, they were digested with trypsin and centrifuged. After centrifugation, the supernatant was discarded. PBS (1 mL) was added, and the cells were gently resuspended by pipetting and then centrifuged again. After the PBS was discarded, 1 mL of prechilled 70% ethanol was added, and the cells were gently mixed by pipetting and then incubated at 4 °C overnight for fixation. After centrifugation, the fixative was discarded. Next, 0.5 mL of propidium iodide staining solution was added to each sample tube and gently mixed with the cell pellet, followed by incubation at 37 °C in the dark for 30 min. The cells were assessed using a flow cytometer at an excitation wavelength of 488 nm.

2.13. Cell scratch assay

A549 cells were seeded into a six-well plate and cultured overnight. The cell layer was scratched with a pipette tip at the 0-h time point. The drug was mixed with serum-free medium at a ratio of 1:12000 and then added to each group. After 24 or 48 h, the medium was aspirated, and the cell layer was examined under an inverted microscope. The scratch area was quantified using ImageJ software.

2.14. Apoptosis assay

A549 cells were seeded at a density of 2 × 10⁵ cells per well in a 6-well plate and incubated overnight at 37 °C. Different drugs were subsequently mixed with serum-free medium at a ratio of 1:12000 and added to each group of cells. After incubation for 24 or 48 h, the cells were collected and stained with annexin V-FITC and PI for 10 min. Finally, apoptosis was assessed using flow cytometry (FCM).

2.15. In vivo imaging

LLC cells (5 × 10⁶) were subcutaneously injected into the right axillary region of C57BL/6 mice to establish an ectopic tumor model. After being labeled with the fluorescent dye DiR, the samples were divided into three groups: DiR, DiR/Pep1, and DiR/Pep2. After tumor-bearing mice received tail vein injection of the appropriate material, the fluorescence signals were detected at 4, 8, 12, 24, 48, and 72 h using small animal imaging equipment (PerkinElmer IVIS Lumina III). Then, ex vivo fluorescence imaging of the major organs and tumors of the mice was performed, and frozen tumor sections were prepared. Approval for this study was obtained from the Ethics Committee of Shandong Second Medical University.

2.16. Assessment of the antitumor effects in mice

An ectopic tumor model was established in mice using a previously described method. After the tumors were allowed to grow for approximately 7 days and the volume reached 100 mm³, treatment was initiated according to the assigned groups. Injections were given every other day for a total of 7 injections. The mice were weighed, and the tumor volume was measured before injection. Following treatment, H&E staining and immunohistochemical staining of the primary organs and tumors were performed.

$$\text{Tumor}\text{inhibition}\text{rate}=\left(1-\text{tumor}\text{weight}\text{in}\text{the}\text{experimental}\text{group}/\text{tumor}\text{weight}\text{in}\text{the}\text{control}\text{group}\right)\times 100\%\text{.}$$

2.17. Immunohistochemical staining

Paraffin-embedded tumor sections were dewaxed and rehydrated. The sections were repaired with citrate buffer at elevated temperatures, washed with PBS after they cooled, and blocked with BSA. Next, the sections were incubated with a primary antibody overnight at 4 °C. After the sections were washed with PBS, they were incubated with the secondary antibody at room temperature. The sections were subjected to DAB staining, followed by hematoxylin counterstaining, differentiation, and counterstaining with eosin. The sections were dehydrated in gradient ethanol solutions and cleared in xylene. Finally, the sections were mounted with neutral resin and examined under an upright microscope.

2.18. Immunofluorescence staining

After antigen retrieval, the tumor sections were incubated with PBS containing 0.1% Triton X-100 at room temperature for 15 min and then washed three times with PBS. The sections were blocked at room temperature for 30 min using goat or bovine serum. The sections were incubated with primary antibody overnight at 4 °C and then rinsed three times with PBS. The sections were incubated with the fluorescently labeled secondary antibody at room temperature in the dark for 60 min and then washed three times with PBS. The nuclei were stained with DAPI for 15 min, followed by three washes with PBS. After the slide was covered with an anti-fluorescence quenching cover slip, it was allowed to dry in air in the dark before observation under an upright fluorescence microscope.

2.19. Data analysis

Statistical analysis of the data was performed with GraphPad Prism and Origin 8.5 software. Statistical comparisons were performed using Student's unpaired t-test, one-way ANOVA, and two-way ANOVA with repeated measures. Tukey's post hoc test was also used. The sample size for each statistical analysis was ≥3.

3. Results and discussion

3.1. Synthesis and characterization of amphiphilic peptides

To develop an intelligent drug delivery system that is responsive to the tumor microenvironment, we designed and synthesized the experimental peptide Pep1 (sequence: (TPP)-IIISIG(p-Y)C–CKKKKKRGD-NH₂), which consists of an RGD targeting peptide, a mitochondrion-targeting TPP modification, a phosphorylated tyrosine residue, and two cysteine residues linked by a disulfide bond. RGD and TPP were attached to the C-terminus and N-terminus of Pep1, respectively, for targeting purposes [47,48]. IIISIG provides the driving force for hydrophobic self-assembly, while charged residues (K/R) increase water solubility [44]. The inclusion of phosphorylated tyrosine (p-Y) residues cleavable by ALP and disulfide bonds (C-C) cleavable by GSH into the peptide chain enables sequential morphological transformations and drug release [49,50]. Pep2 (sequence: Ac-KKKDPVLP-NH₂), lacking both the RGD peptide and TPP, served as the control peptide (Fig. S1–S2).

The purity and molecular weight of the amphiphilic peptides were assessed through high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI‒MS) [51]. The results confirmed the successful synthesis of these amphiphilic peptides with purities exceeding 95% (Fig. S3–S6). The morphologies of the drug-loaded amphiphilic peptides during self-assembly and after enzymatic modification were observed using transmission electron microscopy (TEM) [52]. PH/Pep1 and PH/Pep2 self-assembled into spherical particles in HEPES buffer (Fig. 2A). Under a single stimulus (ALP or GSH), PH/Pep1 underwent a partial morphological transformation, with the spherical nanoparticles partially converting into nanofibers. Under dual stimulation (ALP + GSH), the spherical nanoparticles disappeared and numerous fibrillar aggregates appeared (Fig. 2A and S7-S11). In contrast, PH/Pep2 did not exhibit a change in morphology upon ALP and GSH stimulation, and its spherical structure was maintained (Fig. 2A and S12-S14). These results indicate that Pep1 is responsive to ALP and GSH, and the spherical nanoparticles undergo a morphological transformation. We further designed Pep3 ((AVL)₂KKKY), which contains only unmodified Tyr without the targeting moiety, and Pep4 (Ac-IIIIKKKK(p-Y)RGD-NH₂), which contains the RGD peptide and p-Tyr. The morphology of Pep3 remained unchanged before and after the addition of ALP, whereas the spherical Pep4 particles transformed into fibrous structures (Fig. S15–S16). Furthermore, PH/Pep1 maintained its spherical shape under physiological conditions (PBS or 10% FBS), indicating its stability (Fig. S17 and Table S1).

Fig. 2.

Characterization and analysis of amphiphilic peptides and cytotoxicity assessments. (A) TEM images of PH/Pep1, PH/Pep2, and samples treated with ALP and GSH. (B) Zeta potentials of Pep1 and PH/Pep1 in the presence or absence of ALP and GSH after incubation for different durations. (C) Zeta potentials of Pep2 and PH/Pep2 in the presence or absence of ALP and GSH after incubation for different durations. Cumulative release rates of (D) HCQ and (E) PTX under different conditions. Survival rates of (F) HUVECs and (G) A549 cells after 12 h of incubation. Scale bar: 100 nm.

The zeta potentials of the peptides and drug-loaded peptides were measured. Both Pep1 and Pep2 exhibited negative potentials. Following loading with PTX and HCQ, the potentials of both Pep1 and Pep2 increased. After the amphiphilic peptides were loaded with the drugs, the potential of PH/Pep1 was positive. After ALP and GSH were added and allowed to react, the potential of PH/Pep1 became negative, which may be related to drug release. However, the changes in the potential of PH/Pep2 were not significant (Fig. 2B and C). We also evaluated the release behavior of PTX and HCQ from the amphiphilic peptides, and after 72 h of dialysis, the cumulative release rates of HCQ and PTX in the absence of ALP and GSH were approximately 27.4% and 29.0%, respectively. However, the cumulative release rates of HCQ and PTX in the presence of ALP and GSH were approximately 68.9% and 66.3%, respectively. Furthermore, the cumulative release rate under dual stimulation (ALP + GSH) was greater than that under each stimulus alone (Fig. 2D and E). Under the combined effects of ALP and GSH, the small-molecule peptides underwent morphological changes, thereby releasing the encapsulated drugs PTX and HCQ. The encapsulation efficiencies of HCQ and PTX in PH/Pep1 were determined to be 80.8% and 66.5%, respectively, with drug loadings of 13.5% and 11.1%, respectively.

The viability of HUVECs, BEAS-2B cells, and A549 cells was assessed using a CCK-8 assay. HUVECs and BEAS-2B cells in both the Pep1 and Pep2 groups exhibited high activity (Fig. 2F and S18). Pep1 is significantly more toxic to A549 cells than Pep2, Pep3 and Pep4 (Fig. 2G and S19). In the hemolysis assays, the solutions in all the groups were clear and the hemolysis rates were all less than 5% (Fig. S20).

Changes in the peptides in the presence of ALP and GSH were detected via HPLC. ALP induces the dephosphorylation of p-Y [49], whereas GSH induces the cleavage of disulfide bonds [50]. After ALP and GSH were added to Pep1, HPLC analysis revealed new peaks (Figs. S21–S22), indicating the formation of peptides with increased hydrophobicity, which altered its morphology upon self-assembly [53]. However, no new peaks appeared after incubation of Pep2 with ALP and GSH (Fig. S23). Circular dichroism (CD) analysis revealed an increase in β-sheet content within the secondary structure of Pep1 upon the addition of ALP and GSH (Fig. S24). The morphological changes in Pep1 induced by ALP and GSH may be related to alterations in its primary structure.

3.2. Internalization and retention of Pep1 and Pep2 in A549 cells

Cou6 was used to label Pep1 and Pep2 to evaluate the uptake of the drug-loaded peptides by A549 cells [54]. After 0.25 h of co-incubation, the Cou6/Pep1 group exhibited the strongest fluorescence signal in A549 cells (Fig. 3A and S25). At 2 h, fluorescence increased in both groups (Fig. 3B and S26). The FCM results validated this trend: the fluorescence intensity of the Cou6/Pep1 group was 1.25 times greater than that of the Cou6/Pep2 group after 0.25 h of incubation (Fig. 3C and D); after 2 h, that of the former was 1.04 times greater than that of the latter (Fig. 3E and F). Compared with Pep2, Pep1 more effectively promoted drug uptake by A549 cells, particularly during the initial phase, and exhibited stronger tumor-targeting capacity.

Fig. 3.

Uptake and retention of small-molecule peptides in A549 cells. Upright fluorescence microscopy images of A549 cells treated with free Cou6, Cou6/Pep2, and Cou6/Pep1 for (A) 0.25 h and (B) 2 h. The nuclei are stained blue, and Cou6 appears green. (C) FCM analysis of A549 cells after 0.25 h of incubation with different formulations and (D) quantitative analysis. (E) FCM analysis of A549 cells after 2 h of incubation with different formulations and (F) quantitative analysis. (G) Upright fluorescence microscopy images of A549 cells after 60 h of incubation. The nuclei are stained blue, and Cou6 appears green. Scale bar: 100 μm ns, P > 0.05, ∗P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

The retention of the drug-loaded small-molecule peptides in A549 cells was also evaluated, and the fluorescence decreased in all groups after 60 h. However, fluorescence persisted in the Cou6/Pep1 and Cou6/Pep2 groups, with the Cou6/Pep1 group exhibiting stronger fluorescence intensity than that of the Cou6/Pep2 group (Fig. 3G and S27). After 72 h, the fluorescence intensity in the Cou6/Pep1 group was 2.09 times greater than that in the Cou6/Pep2 group (Fig. S28–S29). The fluorescence of the free Cou6 group was consistently the weakest at both 60 h and 72 h. These experimental results confirm that Pep1 exhibits excellent uptake efficiency and long-lasting retention in A549 cells. After A549 cells were coincubated with PH/Pep1, the cells were lysed and examined by TEM, revealing many nanofibers in the field of view (Fig. S30).

3.3. PH/Pep1 inhibits autophagy in cancer cells in vitro and damages the mitochondria

The adenovirus Ad-GFP-LC3B allows expression of the GFP-LC3B fusion protein, enabling the detection of cellular autophagy post-infection by labeling autophagosomes [55,56]. As clearly shown in the figure, compared with the control group, the treatment groups exhibited significantly greater fluorescence intensity. Among these groups, the PH/Pep1 group presented the greatest fluorescence intensity (Fig. 4A and B). These results indicate that compared with the other treatments, PH/Pep1 treatment resulted in the greatest number of autophagosomes. As a key protein in autophagy, LC3B exists in cells in two forms: LC3B-I and LC3B-II [57]. Once autophagy is activated, LC3B-I is converted into LC3B-II, which localizes to autophagosome membranes [58,59]. Autophagosomes subsequently fuse with lysosomes, and LC3B-II is degraded [60]. Therefore, the content of LC3B-II directly reflects the number of autophagosomes [61]. The expression of autophagy protein LC3B-II was measured via Western blot analysis [62]. The lowest expression of the LC3B-II protein was detected in the control group, whereas the highest expression was detected in the PH/Pep1 group (Fig. 4C and D). These results indicate that PH/Pep1 promotes the accumulation of autophagosomes.

Fig. 4.

PH/Pep1 inhibits autophagy in cancer cells and damages mitochondria. (A) Fluorescence images of A549 cells after transfection with Ad-GFP-LC3 adenovirus in each group, with selected regions in the merged images shown at higher magnification; and (B) quantitative analysis. (C) Analysis of LC3B and p62 protein expression by Western blotting. (D) Quantitative analysis of LC3B-II protein expression. (E) Quantitative analysis of p62 protein expression. (F) Effects of different treatments on the mitochondrial membrane potential. Scale bar: 100 μm ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Both the PTX/Pep2 group and the PTX/Pep1 group exhibited elevated LC3B-II levels. On this basis, p62 protein expression was analyzed to determine the degree of autophagy [62]. Measurement of p62 expression confirmed the inhibitory effect of PH/Pep1 on autophagy in A549 cells. The p62 protein is a key substrate in autophagy, and inhibiting autophagy leads to its accumulation [63,64]. The results revealed that p62 levels were significantly reduced in both the PTX/Pep2 and PTX/Pep1 groups, with the lowest expression observed in the PTX/Pep1 group and the highest in the PH/Pep1 group (Fig. 4C and E). The increased levels of LC3B-II and decreased levels of p62 in the PTX/Pep1 group indicate that protective autophagy is activated: the accumulation of LC3B-II reflects increased autophagosome formation, whereas the decrease in p62 expression indicates that autophagosomes have fused with lysosomes and completed substrate degradation [65]. In contrast, in the PH/Pep1 group, HCQ prevented the clearance of autophagosomes, leading to the sustained accumulation of LC3B-II; moreover, the degradation of p62 was inhibited, resulting in its massive accumulation.

To evaluate the blockade of autophagic flux, LysoTracker Red staining and Ad-mCherry-GFP-LC3B transfection assays were performed. LysoTracker Red staining revealed varying degrees of increased red fluorescence in the autophagy-activated groups (PTX/Pep2 and PTX/Pep1), indicating an increase in the number of autophagolysosomes; in contrast, the red fluorescence intensity decreased significantly in the autophagy-inhibited groups (PH, PH/Pep2, and PH/Pep1), with the weakest signal observed in the PH/Pep1 group (Fig. S31–S32). Transfection experiments using Ad-mCherry-GFP-LC3B confirmed the significant increases in green fluorescence and yellow puncta in the PH/Pep1 group, indicating the successful inhibition of autophagosome and lysosome fusion. In contrast, the PTX/Pep1 group exhibited weaker green fluorescence and red puncta, indicating that the autophagosomes had fused with lysosomes and the smooth progression of autophagic flux (Fig. S33). HCQ blocks the fusion of autophagosomes with lysosomes and that PH/Pep1 has the strongest inhibitory effect [17,18].

To evaluate the ability of Pep1 to target mitochondria, Cou6 was encapsulated within Pep1, and its colocalization with mitochondria was detected. The green fluorescence in both the free Cou6 group and the Cou6/Pep2 group was relatively weak, whereas the intense green fluorescence in the Cou6/Pep1 group showed significant overlap with the red fluorescence from mitochondria, indicating that Pep1, which contains a TPP moiety, has good mitochondrial targeting ability (Fig. S34). Because TPP and the RGD peptide are located at opposite ends of the Pep1 chain, mitochondrial targeting efficiency may be limited.

Exogenous stimuli such as drugs can damage mitochondria [23]. To assess mitochondrial function, the fluorescent probe JC-1 was used to detect changes in the mitochondrial membrane potential [66,67]. The control group had the brightest red fluorescence and the faintest green fluorescence, indicating that no mitochondrial damage had occurred in these A549 cells. The fluorescence in the Pep2 group was not significantly different from that of the control group. Compared with the control and Pep2 groups, the Pep1 group exhibited less intense red fluorescence and more intense green fluorescence. These results indicate that TPP-modified Pep1 can cause a certain degree of mitochondrial damage. Further assessments revealed that the Pep1 nanocarrier systems (P/Pep1 and PH/Pep1) caused greater mitochondrial damage than the Pep2 nanocarrier systems (P/Pep2 and PH/Pep2) did. Among these groups, the PH/Pep1 group exhibited the weakest red fluorescence intensity but the strongest green fluorescence intensity (Fig. 4F and S35). These findings indicate a significant reduction in mitochondrial membrane potential and severe mitochondrial damage.

Mitochondrial damage typically manifests as elevated levels of reactive oxygen species (ROS) and decreased levels of ATP within the cell [68,69]. The ROS levels in the Pep1 group were significantly greater than those in the Pep2 group. Among all the experimental interventions, PTX/Pep1 and PH/Pep1 both increased ROS levels, with the highest ROS levels detected in the PH/Pep1 group (Fig. S36–S37). Moreover, ATP assays revealed that ATP levels were significantly lower in the Pep1 group than in the Pep2 group, whereas ATP synthesis was lowest in the PH/Pep1 group (Fig. S38). Mitochondrial damage leads to inhibition of the cellular energy supply, which may impair the function of P-glycoprotein (P-gp), thereby impeding tumorigenesis and tumor progression [70,71]. The WB results revealed that the P-gp levels in the Pep1 group were significantly lower than those in the Pep2 group, and the lowest levels were detected in the PH/Pep1 group (Fig. S39–S40). Pep1 causes mitochondrial damage and that PH/Pep1 induces the most severe mitochondrial damage. Mitochondrial colocalization and JC-1 assays revealed that, compared with Pep1, single-targeted, single-responsive Pep4 has weaker mitochondrial targeting ability and causes less mitochondrial damage (Fig. S41–S43). Mitochondrial damage was more severe in the PH/Pep1 group than in the PTX/Pep1 group, suggesting that the synergistic effects of HCQ-mediated autophagy inhibition together with the deleterious effects of Pep1 and PTX contributed to the increased damage.

Time-course analysis confirmed that mitochondrial damage precedes autophagy inhibition (Fig. S44–S45). From 0 to 6 h, the mitochondrial membrane potential decreased in both the PTX/Pep1 group and the PH/Pep1 group, and autophagy blockade was not yet detected. By 12 h, intact autophagic flux was observed in the PTX/Pep1 group (reduced levels of p62), accompanied by less severe damage, whereas PH/Pep1 intervention led to the inhibition of autophagy (increased levels of p62) and exacerbated damage.By 24 h, the mitochondrial membrane potential in the PTX/Pep1 group remained relatively stable, and the p62 level decreased further, indicating autophagic clearance. However, the mitochondrial membrane potential in the PH/Pep1 group continued to decrease while p62 accumulation increased, indicating progressive injury. Mitochondrial damage occurred first and was aggravated by subsequent autophagy inhibition, supporting a synergistic “damage-inhibition-clearance failure” process.

3.4. PH/Pep1 inhibits tumor cell migration and proliferation

The cell cycle plays a crucial role in tumor development [72]. When the cell cycle is assessed via FCM, if the drug is effective, the proportion of cells in the G2/M phase significantly increases, whereas the proportion of cells in the G0/G1 phase correspondingly decreases [73]. Our results revealed that after treatment with different drugs, significant cell cycle inhibition was not detected in the control group or the free drug combination group (PH). The proportions of cells in the G1 phase in the P/Pep2, P/Pep1, and PH/Pep2 groups were 10%, 10%, and 9%, respectively, and those in the G2 phase were 47%, 52%, and 52%, respectively. All three treatments inhibited the cell cycle. Notably, the proportion of cells in the G1 phase was 6% in the PH/Pep1 group, and this group exhibited the highest proportion of cells in the G2 phase (69%) (Fig. S46), indicating that PH/Pep1 has the strongest ability to inhibit the cell cycle.

The migratory capacity of tumor cells is a key factor in cancer progression and metastasis [74]. Therefore, inhibiting tumor cell migration is crucial for antitumor therapy. A scratch assay was performed on A549 cells after different treatments. Cell migration was the most significant in the blank control group, whereas cell migration in the other groups was inhibited to varying degrees. Notably, in the PH/Pep1 group, the area of the scratch zone showed almost no reduction, indicating the most pronounced inhibition of cell migration and the lowest wound healing rate (Fig. 5A and B). Next, we investigated the in vitro toxic effects of each group on tumor cells. After A549 cells were cocultured with HEPES, PH (PTX and HCQ), P/Pep2, P/Pep1, PH/Pep2, or PH/Pep1 for 24 h, the control group (HEPES) exhibited the lowest apoptosis rate (1.79%). The apoptosis rate in the PH group was 4.85%, which was 2.7 times greater than that in the control group. The apoptosis rates after treatment with the Pep2 nanocarrier systems (P/Pep2 and PH/Pep2) were 7.24% and 9.18%, respectively. The apoptosis rates after treatment with the Pep1 nanocarrier systems (P/Pep1 and PH/Pep1) were 12.58% and 15.67%, respectively. Compared with the Pep2 system, the Pep1 nanocarrier system better induced apoptosis. Furthermore, the antitumor efficacy of the nanocarrier system loaded with both drugs was superior to that of the system loaded with PTX only. Among these groups, the PH/Pep1 group exhibited the highest apoptosis rate and demonstrated the strongest tumor-killing capacity (Fig. 5C and S47). After 48 h, the apoptosis rates in all groups increased, with the highest rate of 18.02% observed in the PH/Pep1 group (Fig. 5D and S48).

Fig. 5.

In vitro antitumor activity of PH/Pep1. (A) Wound healing capacity of A549 cells in different treatment groups and (B) quantitative analysis at 48 h. (C) Assessment of apoptosis in A549 cells after 24 h in different treatment groups. (D) Assessment of apoptosis in A549 cells in different treatment groups at 48 h. Scale bar: 400 μm ∗P < 0.05, ∗∗P < 0.01.

3.5. Retention of the drug-loaded peptides in vivo

To investigate the retention of Pep1 and Pep2 in vivo, an LLC tumor-bearing mouse model was established. Pep1 and Pep2 were labeled with the fluorescent dye DiR [75]. In vivo fluorescence imaging revealed that the fluorescence intensity of the tumors in the mice injected intravenously with DiR-labeled Pep1 gradually increased, peaking at 72 h. The fluorescence intensity of the latter two groups remained consistently lower than that of the DiR/Pep1 group (Fig. 6A). These results indicate that compared with the free drug and Pep2, Pep1 exhibits superior tumor targeting and drug retention effects. Afterward, the mice were sacrificed, and the tumors and main organs were collected for ex vivo fluorescence imaging. The tumors of the mice administered free DiR exhibited the weakest fluorescence. DiR accumulation in the DiR/Pep1 group was 1.61 times greater than that in the DiR/Pep2 group. Moreover, the weakest fluorescence signals in other organs were detected in the DiR/Pep1 group, indicating minimal accumulation in nontumor organs (Fig. 6B and C). These results indicate that the nanocarrier Pep1 can efficiently deliver drugs to tumor tissues. The tumor tissue was subsequently processed into frozen sections for analysis. Observation using an upright fluorescence microscope revealed that DiR/Pep1 exhibited prolonged retention within tumor tissue (Fig. 6D and E).

Fig. 6.

Investigation of the tumor accumulation of drug-loaded particles. (A) In vivo fluorescence imaging was conducted on LLC tumor-bearing mice following the injection of free DiR, DiR-labeled Pep2, and DiR-labeled Pep1. (B) Ex vivo fluorescence images of major organs and tumor tissues from LLC tumor-bearing mice obtained at 72 h postinjection. (C) Semiquantitative analysis of the fluorescence distribution in major organs and tumor tissues. (D) Fluorescence images of frozen sections from each tumor group and (E) quantitative analysis. Scale bar: 200 μm ∗P < 0.05.

3.6. In vivo antitumor effects

The in vivo safety and pharmacokinetics of PH/Pep1 were evaluated. Blood tests revealed no significant differences among the three groups of mice, indicating that PH/Pep1 did not induce apparent acute toxicity (Fig. S49). The pharmacokinetic results revealed that compared with PH/Pep2, PH/Pep1 had higher plasma concentrations but lower concentrations in urine and feces, suggesting slower clearance (Fig. S50).

The in vivo therapeutic efficacy of PH/Pep1 in LLC tumor-bearing mice was subsequently investigated. At the end of the treatment cycle, the tumor volume was greatest in the saline group. In the drug treatment groups, tumor growth was inhibited, and the tumor suppression effect was markedly better in the drug-loaded peptide groups than in the free PH group. Among these treatments, the Pep1 nanocarrier systems demonstrated significantly better therapeutic efficacy than the Pep2 nanocarrier systems did.

The tumor suppression rate in the PH/Pep1 group was 88.2%, and the PH/Pep1 group exhibited the slowest tumor growth rate and the highest tumor inhibition rate (Fig. 7A–D). The tumor suppression rate in the PH/Pep1 group was 1.36 times that in the PH/Pep2 group. The tumor inhibition rate in the PTX/Pep1 group was 76.6%, which was higher than that in the PH/Pep2 group. Furthermore, throughout the entire treatment period, the body weights of the mice remained stable (Fig. 7E).

Fig. 7.

Tumor suppression effect of PH/Pep1. (A) Photographs of mouse tumors collected after treatment. (B) Tumor volumes and (C) weights. (D) Tumor inhibition rates in different treatment groups. (E) Curves of the changes in mouse body weight. (F) TUNEL and Ki-67 immunohistochemical staining images of tumor tissue. Scale bar: 100 μm ∗P < 0.05, ∗∗∗P < 0.001. n ≥ 6.

To further evaluate the in vivo antitumor activity of PH/Pep1, tumor tissues were subjected to immunohistochemical staining (Fig. 7F). The Ki-67 expression level in the PH/Pep1 group was significantly reduced, with the lowest positive rate among all the groups. Moreover, the TUNEL assay results revealed the highest positivity rate in the PH/Pep1 group, suggesting the greatest number of apoptotic cancer cells, demonstrating the strongest tumor-killing capacity. H&E staining was subsequently performed on the tumors and major organs of the mice (Fig. 8), which revealed that compared with the tumor sections from the other groups, those from mice treated with PH/Pep1 contained more apoptotic tumor cells and more extensive areas of tumor necrosis, indicating the greatest degree of histological damage. These findings were consistent with the immunohistochemical results. Additionally, examination of H&E-stained sections of major organs revealed no significant histological damage in the PH/Pep1 group, indicating its excellent biocompatibility. Furthermore, lung H&E samples revealed intact alveolar structures and no significant pulmonary nodules in the PH/Pep1 group. These results strongly indicate that PH/Pep1 effectively inhibits tumor growth without significant side effects.

Fig. 8.

H&E-stained sections of tumors and major organs. Scale bar: 100 μm.

3.7. PH/Pep1 suppresses tumors in vivo by inhibiting autophagy and inducing mitochondrial damage

To investigate the in vivo effects of blocking autophagy and damaging mitochondria, staining analyses were performed on tumor tissues. Immunohistochemical staining revealed that the protein LC3B, an autophagy marker, was most highly expressed in the PH/Pep1 group (Fig. 9A). Additionally, tumor immunofluorescence staining for the autophagy substrate p62 showed that the PH/Pep1 group exhibited stronger fluorescence than the other groups did (Fig. 9B and S51). These two indicators collectively demonstrated that PH/Pep1 effectively suppressed autophagy by impeding the conversion of autophagosomes into autophagolysosomes. Tumor fluorescence staining for P-gp suggested that PH/Pep1 caused the greatest damage to mitochondria (Fig. 9C and S52) [70,71].

Fig. 9.

Antitumor effects of PH/Pep1 in vivo. (A) Immunohistochemical staining images of LC3B in tumor tissue. Immunofluorescence staining images of (B) p62 and (C) P-gp in tumor tissue. Scale bar: 100 μm.

In this study, a multifunctional peptide delivery system, PH/Pep1, that can target tumors, is responsive to the tumor microenvironment, and self-assembles into spherical nanoparticles under physiological conditions, was successfully developed. Within the tumor microenvironment, PH/Pep1 sequentially responded to the elevated levels of ALP and GSH, undergoing a morphological transition from spherical nanoparticles to fibrous aggregates accompanied by charge reversal, thereby enabling efficient drug release.

At the cellular and animal levels, PH/Pep1 significantly increased drug uptake by lung cancer cells through active targeting and demonstrated superior intracellular and tumor retention capabilities. In vivo distribution experiments confirmed that this system achieved specific, long-lasting accumulation at tumor sites. PH/Pep1 synergistically killed tumor cells by increasing chemical toxicity, inhibiting autophagy, and damaging mitochondria. On the one hand, PH/Pep1 effectively blocked autophagic flux, manifested by the accumulation of the LC3B-II and p62 proteins, thereby inhibiting protective autophagy in tumor cells. On the other hand, PH/Pep1 caused a significant decrease in the mitochondrial membrane potential, leading to the accumulation of dysfunctional mitochondria and exacerbating oxidative stress and apoptosis. Moreover, PH/Pep1 downregulated P-gp expression and reduced drug efflux. In vivo experiments demonstrated that in solid tumors, drug accumulation in the Pep1 group was 4.45 times greater than that in the free drug group and 1.61 times greater than that in the Pep2 group. In vivo experiments demonstrated that PH/Pep1 significantly inhibited tumor growth without causing significant systemic toxicity.

4. Conclusion

In this study, we successfully developed a peptide nanoconjugate delivery system, PH/Pep1, that combines active targeting with microenvironment-responsive morphological transformation. This delivery system achieved targeted enrichment in tumor tissues, in situ morphological transformation in response to tumor microenvironment signals (ALP/GSH), and long-term retention. ALP and GSH sequentially convert PH/Pep1 from spherical nanoparticles into nanofibers and increase the fiber diameter, enabling sustained drug release and long-term drug retention. In vitro and in vivo evaluations thoroughly confirmed the high efficacy of this strategy: PH/Pep1 directly induced cellular damage while simultaneously blocking autophagy, thereby inhibiting cellular self-repair mechanisms, which subsequently drives the accumulation of toxic substances and amplifies apoptotic signals. A synergistic “damage-suppression-clearance” amplification loop was established, resulting in potent tumor cell toxicity. In summary, the PH/Pep1 nanoplatform demonstrated tumor-targeted drug delivery, microenvironment-triggered release, and prolonged drug retention. By synergistically regulating autophagy and mitochondrial function, PH/Pep1 exhibited significantly increased antitumor efficacy, offering a novel therapeutic strategy for killing tumor cells by inducing autophagy inhibition and mitochondrial damage.

CRediT authorship contribution statement

Ziru Liu: Investigation, Methodology, Writing – original draft. Hongjie Li: Data curation, Investigation, Methodology. Wei Gao: Funding acquisition, Resources. Wenhao Wang: Funding acquisition, Resources. Chuntao Li: Investigation, Validation. Haiqin Huang: Investigation, Validation. Jin Liu: Investigation, Validation. Junjie Li: Investigation, Validation. Huiying Jia: Investigation, Validation. Cheng Ma: Investigation. Yuanxin Song: Validation. Changgang Sun: Funding acquisition, Supervision. Tongyi Sun: Supervision. Jingkun Bai: Conceptualization, Investigation, Methodology, Writing – review & editing.

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.