Targeted dual-responsive nanocapsules for synergistic cisplatin and paclitaxel prodrug delivery in the treatment of lung cancer

Introduction

Lung cancer is the leading cause of cancer-related mortality worldwide (1). Its disease burden is shaped by evolving risk factors, demographic shifts, and disparities in healthcare access. Globally, approximately 2.2 million new cases and 1.8 million attributable deaths were recorded in 2020 (1). Although age-standardized incidence and mortality rates have declined in some high-income countries owing to effective tobacco control measure, the absolute number of cases continues to rise, driven by population growth and aging (2,3). Notably, the epidemiological landscape of lung cancer has undergone significant changes. In Finland, for instance, lung adenocarcinoma has surpassed squamous cell carcinoma as the most common histological subtype, while incidence has risen among females (4). In Singapore, there has been a marked increase in incidence among women aged 30–49 years and nonsmokers, with 81.6% of patients being diagnosed at advanced stages (5). These trends underscore the urgent need for the development of precise intervention strategies tailored to specific population characteristics.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases (6). Lung adenocarcinoma, the predominant subtype in nonsmokers, is closely associated with driver gene mutations including EGFR, ALK, and ROS1 (7). For patients with advanced NSCLC, platinum-based combination chemotherapy remains the first-line standard of care. Although adding pemetrexed to platinum chemotherapy has been shown to significantly improve survival outcomes in patients with advanced NSCLC, it also increases the risk of high-grade adverse events (8). To address this efficacy-toxicity dilemma, more precise and less toxic therapeutic approaches need to be devised.

Paclitaxel (PTX) and cisplatin (Pt) are core first-line chemotherapeutic agents in the treatment of NSCLC, acting synergistically via complementary mechanisms: PTX stabilizes microtubules to inhibit cell division, while cisplatin induces DNA cross-linking to block replication (9,10). However, the clinical application of both drugs is hampered by poor water solubility, lack of targeting specificity, and severe dose-limiting toxicities, including neurotoxicity, nephrotoxicity, and myelosuppression (11,12). Nanotechnology offers a promising solution to overcoming the limitations of conventional chemotherapy (13). As indicated by Guo et al. (14), the unique pathophysiological features of the tumor stroma—such as hypoxia, an acidic pH gradient, abnormal redox homeostasis, and leaky vasculature—provide ideal targets in the design of intelligent nanocarriers. Specifically, stimulus-responsive nanocarriers can achieve spatiotemporally controlled drug release through the cleavage of chemical bonds in response to these distinct tumor microenvironmental cues (15).

For active targeting, arginine-glycine-aspartic acid (RGD) peptides specifically recognize αvβ3 integrin receptors, which are highly expressed on tumor cells and neovascular endothelial cells (16). Lin et al. (17) demonstrated that RGD-modified gold nanoparticles loaded with SAHA (RGD-AuNPs-SAHA) can significantly enhance cellular uptake in A549 cells, leading to the downregulation of HIF-1α and VEGF expression and inducing marked DNA damage and apoptosis. Similarly, Muradova et al. (18) found that cyclic RGD (cRGD)-modified AGuIX-Bi nanoparticles could promote tumor-specific accumulation via integrin-mediated endocytosis, converting immunologically “cold” tumors into “hot” ones and increasing CD8⁺ T-cell infiltration. In a related study, Liu et al. (19)developed manganese oxide nanoparticles functionalized with a platinum (IV) prodrug (DHP/MnO₂NP). This system allowed for the precise release of cisplatin and Mn²⁺ driven by pH and redox dual responsiveness, synergistically activating the STING pathway and antitumor immune responses. Collectively, these studies indicate that integrating active targeting with microenvironment-responsive drug release within a single nanoplatform constitutes an effective strategy for improving the efficacy of chemotherapy in lung cancer.

However, the nanocarriers developed thus far still require optimization in terms of drug-loading capacity, dual-stimulus responsiveness, and the synergistic integration of targeting moieties (20). To address these limitations, we constructed novel acid-sensitive lipid-polymer nanocapsules co-loading with paclitaxel and cisplatin and modified with RGD (designated as PTX/Pt@RGD). This system was designed as follows: a redox-sensitive paclitaxel prodrug (PTX-SS-PEG-RGD), where PTX is conjugated to PEG-RGD via a disulfide (SS) bond, with PEG representing polyethylene glycol, was synthesized, while cisplatin was encapsulated within acid-sensitive lipid-polymer hybrid nanovesicles containing PCL-hyd-PEG (poly(ε-caprolactone)-hydrazone-polyethylene glycol) with pH-responsive hydrazone bonds. By integrating RGD-mediated active targeting, pH/redox dual-responsive drug release, and the synergistic chemotherapeutic action of PTX and cisplatin, we sought to develop a nanosystem capable of synergistic and low-toxicity nano-co-delivery for the treatment of lung cancer (Figure 1A,1B). We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0967/rc).

Figure 1 Schematic illustration of the design and tumor-targeting mechanism of PTX/Pt@RGD nanocapsules. (A) Proposed mechanism of action of PTX/Pt@RGD for targeted lung cancer therapy. (B) Schematic illustration of the dual-responsive targeting and therapeutic effects of PTX/Pt@RGD in A549 lung cancer cells. GSH, glutathione; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

Methods

Experimental materials

Polymers and lipids were purchased from Xi’an Ruixi Biotechnology Co., Ltd. (Xi’an, China) and included PCL-hyd-PEG-COOH (cat no. R-PLZ1137-4K), PTX-SS-PEG-COOH (cat no. R-EL-090), and DSPE-cRGD (cat no. R9970).

Among the drugs and chemicals used in this study, cisplatin (purity ≥99%) was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China); Aladdin Reagent Co., Ltd. (Shanghai, China) provided PTX (cat. no. P106869), lecithin (cat. no. L105732), cholesterol (cat. no. C657147), chloroform (cat. no. C1506275), and methanol (cat. no. M116115); and indocyanine green (ICG) was sourced from Merck KGaA (cat. no. I2633; Darmstadt, Germany).

For the cell culture reagents, Gibco (Thermo Fisher Scientific, Waltham, MUA, USA) provided the fetal bovine serum (FBS; cat. no. 10099141) and RPMI 1640 medium (cat. no. 11875093), while Kaiji Biotechnology (Nanjing, China) provided phosphate-buffered saline (PBS; cat. no. KGL2207), penicillin-streptomycin (cat. no. KGL2303), and trypsin (cat. no. KGL2103).

Antibodies included primary antibodies against CD31 [cat. no. 11265-1-P; rabbit polyclonal antibody; Western blotting (WB) 1:500; immunohistochemistry (IHC) 1:100; Proteintech, Rosemont, IL, USA], Ki-67 (cat. no. 27309-1-AP; rabbit polyclonal antibody; IHC 1:200; Proteintech), PCNA (cat. no. 10205-2-AP; rabbit polyclonal antibody; IHC 1:500; Proteintech), Bcl-2 (cat. no. 12789-1-AP; WB 1:1,000; Proteintech), Caspase-9 (cat. no. 10380-1-AP; WB 1:1,000; Proteintech), Bax (cat. no. 50599-2-Ig; WB 1:2,000; Proteintech), COX-2 (cat. no. 12375-1-AP, WB 1:1000; Proteintech), and β-actin (cat. no. 66009-1-Ig; WB 1:5,000; internal control), as well as horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (cat. no. SA00001-2; WB 1:5,000; Proteintech).

Kits and probes included Cell Counting Kit-8 (CCK-8) cell proliferation assay kit (cat. no. KGA9310; Kaiji Biotechnology), reactive oxygen species (ROS) probe (DCFH-DA; cat. no. KGA7308; Kaiji Biotechnology), JC-1 mitochondrial membrane potential assay kit (cat. no. KGA1904; Kaiji Biotechnology), crystal violet (cat. no. KGE1202; Kaiji Biotechnology), paraformaldehyde (cat. no. KGC3121; Kaiji Biotechnology), DNA double-strand break staining kit (γ-H2AX; cat. no. KGA9505; Kaiji Biotechnology), hematoxylin and eosin (H&E) staining kit (cat. no. KGA501; Kaiji Biotechnology), and WB gel preparation kit (cat. no. KGC4407; Kaiji Biotechnology).

Other materials included Transwell inserts [8-μm pore size; cat. no. 3422; (Corning Inc., Corning, NY, USA)] and dialysis bags [molecular weight cutoff (MWCO) 3500 Da; cat. no. D9277; Merck KGaA].

Preparation of PTX/Pt@RGD

PTX/Pt@RGD was fabricated with a thin-film hydration method and ultrasonic emulsification. Briefly, 25 mg of lecithin, 25 mg of PCL-hyd-PEG-COOH, 20 mg of PTX-SS-PEG-RGD, and 10 mg of cholesterol were accurately weighed and dissolved in 5 mL of chloroform. The solution was sonicated for 5 min and concentrated in a rotary evaporator at 40 ℃ for 30 min until a uniform lipid film was formed. A total of 10 mg of cisplatin was dissolved in 4 mL of PBS (pH 7.4), added to the lipid film, and hydrated for 30 min at 37 ℃ with shaking at 100 rpm. The mixture was then ultrasonicated with a probe sonicator (20 kHz; VCX 500 Ultrasonic Processor; Sonics & Materials, Newtown, CT, USA) at 30% amplitude for 5 min (5 s on and 2 s off) to regulate particle size. Free (unencapsulated) drugs were removed via ultrafiltration with 3-kDa centrifugal filter units, at 4,000 ×g for 20 min at 4 ℃. The final suspension was filtered through a 0.22-μm polyethersulfone membrane and stored at 4 ℃ away from light.

Quantification of PTX and Pt in nanocapsules

The contents of PTX and Pt in the nanocapsules were determined by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity system (Agilent Technologies, Santa Clara, CA, USA); the nanocapsules were first dissolved in a solution of acetonitrile and water (1:1, v/v) and filtered through a 0.22-μm membrane. For PTX, separation was performed on a Spherisorb NH2 column (0.46 cm × 30 cm) using methanol-water (85:15, v/v) containing 1.26 g/L sodium chloride as the mobile phase at a flow rate of 0.8 mL/min, with UV detection at 227 nm (column temperature, 31 ℃; injection volume, 20 μL). For Pt, samples were derivatized with sodium diethyldithiocarbamate (DDTC) at 70 ℃ for 30 min to form the Pt(DDTC)2 complex prior to injection; the derivative was separated on a Hypersil ODS2 C18 column (250 mm × 4.6 mm, 5 μm) using methanol-water (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min, with UV detection at 254 nm (column temperature, 35 ℃; injection volume, 20 μL). Both drugs were quantified by the external standard method. All measurements were performed in triplicate. Drug loading efficiency (DLE) and encapsulation efficiency (EE) were calculated as follows:

DLE(%)=weightofdruginnanocapsulesweightofnanocapsules×100%[1]

EE(%)=weightofdruginnanocapsulesweightofdrugininitialfeed×100%[2]

Characterization of nanocapsules

Morphology was observed with a transmission electron microscope (TEM; HT7800; Hitachi, Tokyo, Japan) operated at 80 kV. Samples were stained with 2% uranyl acetate. Hydrodynamic particle size and zeta potential were measured via dynamic light scattering (DLS) with the Zetasizer Nano ZS90 (Malvern Panalytical, Malvern, UK) at 25 ℃ with a 173° backscatter angle. Each measurement was performed in triplicate.

For storage stability, the particle size of PTX/Pt@RGD in PBS was monitored at 4 ℃ for over 15 days by DLS. For serum stability, PTX/Pt@RGD was incubated in complete culture medium (RPMI 1640 containing 10% FBS) at 37 ℃, and the particle size was measured at 0, 6, 24, 48, and 72 h. For in vitro drug release, 10 mg of PTX/Pt@RGD was placed in a dialysis bag (MWCO 3500 Da) and immersed in 50 mL of PBS (pH 7.4 or 6.5) containing 0.5% Tween 80 to maintain sink conditions. The systems were incubated at 37 ℃ with gentle shaking (100 rpm). At predetermined time points (0, 0.5, 1, 2, 4, 8, 12, 24, and 48 h), 1 mL of release medium was withdrawn and replaced with an equal volume of fresh prewarmed buffer. The concentrations of released PTX and Pt were determined by HPLC as described above. All experiments were performed in triplicate.

To evaluate the redox-responsiveness of the disulfide linkage, the in vitro release of PTX and Pt was further examined in PBS (pH 7.4) containing 0.5% Tween 80 with or without 10 mM glutathione (GSH) under the same conditions (37 ℃, 100 rpm). GSH solution was freshly prepared before use. At the predetermined time points, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh prewarmed buffer containing the corresponding GSH concentration. The released PTX and Pt were quantified by HPLC as described above. All experiments were performed in triplicate.

Cell culture

A549 human lung adenocarcinoma cells (cat. no. CCL-185; Manassas, VI, USA) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin (100 U/mL). Cells were maintained at 37 ℃ in a humidified atmosphere containing 5% CO₂. All experiments were performed with cells in the logarithmic growth phase.

CCK-8 cytotoxicity assay and calculation of half maximal inhibitory concentration

A549 cells were seeded into 96-well plates at 3×10³ cells per well. After 12 h of attachment, cells were treated with serial dilutions (0, 0.0004, 0.002, 0.01, 0.025, 0.05, 0.25, 1, 5, and 10 mg/mL) of Blank@RGD, PTX/Pt@NP, PTX/Pt@RGD, free PTX, free cisplatin, and free PTX + cisplatin (2:1 mass ratio) for 24 h. Following treatment, 10 μL of CCK-8 reagent was added to each well and incubated for 2 h at 37 ℃. Absorbance at 450 nm was measured with a microplate reader (BioTek Synergy H1, Agilent Technologies). Cell viability was normalized to untreated control cells. The half-maximal inhibitory concentration (IC₅₀) was calculated by nonlinear regression fitting of the dose–response curves using a four-parameter logistic model.

Transwell invasion assay

For the invasion assay, 100 μL of Matrigel (cat. no. 356234; Corning) diluted 1:8 in serum-free medium was added to the upper chamber of Transwell inserts (8-μm pore size) and allowed to solidify for 2 h at 37 ℃. A total of 5×10⁴ A549 cells in 200 μL of serum-free RPMI 1640 medium were seeded into the upper chamber. The lower chamber was filled with 600 μL of medium containing 10% FBS as a chemoattractant. Cells were then treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. After treatment, non-invading cells on the upper membrane surface were removed with a cotton swab. Invading cells on the lower membrane surface were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 20 min. Stained cells were imaged under an inverted microscope (Olympus IX73), and five random fields per well were counted.

Intracellular ROS detection

A549 cells were seeded in 12-well plates at 1×10⁵ cells/well and allowed to attach for 12 h. Cells were then treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. After treatment, cells were incubated with 10 μM of DCFH-DA in serum-free medium for 30 min at 37 ℃ in the dark. Cells were then washed three times with PBS. Fluorescence images [excitation/emission wavelengths (Ex/Em) =488/525 nm] were captured with a fluorescence microscope (Eclipse Ti2, Nikon, Tokyo, Japan). Mean fluorescence intensity was quantified with ImageJ software (US National Institutes of Health, Bethesda, MD, USA).

Mitochondrial membrane potential (ΔΨm) detection

A549 cells were seeded in 12-well plates at 1×10⁵ cells/well and allowed to attach for 12 h. Cells were then treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. Following treatment, cells were stained with JC-1 (5 μg/mL) for 20 min at 37 ℃ in the dark according to the manufacturer’s protocol (Kaiji Biotechnology, Nanjing, China; cat. no. KGA1904). After washing twice with JC-1 buffer, cells were observed under a fluorescence microscope. The red (Ex/Em =540/590 nm; J-aggregates) and green (Ex/Em =485/530 nm; monomer) fluorescence intensities were measured, and the red:green ratio was calculated to reflect ΔΨm.

DNA damage detection (γ-H2AX foci)

Cells cultured on coverslips were treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin for 1 h at room temperature. Anti-γ-H2AX antibody (1:500; cat. no. KGA9505; Kaiji Biotechnology) was incubated overnight at 4 ℃, followed by incubation with Alexa Fluor 488-conjugated secondary antibody (1:200) for 1 h at 37 ℃. Nuclei were counterstained with DAPI (1 μg/mL) for 5 min. Fluorescence images were captured with a confocal microscope (LSM 800, Zeiss, Oberkochen, Germany). The number of γ-H2AX foci per nucleus was counted in at least 100 cells per group.

Western blot analysis

A549 cells were seeded in 6-well plates and treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. Total protein was extracted with RIPA lysis buffer (cat. no. P0013B; Beyotime Biotechnology, Shanghai, China) containing protease inhibitor cocktail (cat. no. 04693159001; Roche, Basel, Switzerland). Protein concentration was quantified with a bicinchoninic acid (BCA) kit (cat. no. P0012S; Beyotime Biotechnology, Shanghai, China). Equal amounts of protein (30 μg per lane) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (0.45 μm; Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature and incubated with primary antibodies overnight at 4 ℃ with gentle shaking. After three washes with TBST (10 min each), membranes were incubated with HRP-conjugated secondary antibody (1:5,000) for 1 h at room temperature. Protein bands were visualized via enhanced chemiluminescence (ECL; cat. no. WBKLS0500; Millipore) on a ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Band intensities were analyzed with ImageJ software and normalized to β-actin.

Flow cytometry for detecting apoptosis

Apoptosis was quantified via Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) double staining. A549 cells were treated with free PTX, free cisplatin, free PTX + cisplatin (PTX/cisplatin mass ratio of 2:1), or PTX/Pt@RGD at a total drug concentration of 0.05 mg/mL for 24 h; untreated cells served as the control group. After treatment, cells were harvested by trypsinization without EDTA, washed twice with cold PBS, and resuspended in 1× binding buffer at a concentration of 1×10⁶ cells/mL. Cells were stained with 5 μL of Annexin V-FITC and 5 μL of PI for 15 min at room temperature in the dark. Fluorescence was measured within 1 h with a flow cytometer (FACSCanto II, BD Biosciences, Franklin Lakes, NJ, USA). For each sample, 20,000 events were acquired. Forward scatter and side scatter were used for gating to exclude debris and cell doublets. Early apoptotic cells were defined as those that were annexin V⁺ and PI⁻, while late apoptotic cells were defined as those that were Annexin V⁺ and PI⁺. The total apoptosis rate was calculated as follows: total apoptosis rate (%) = [(early apoptotic cells + late apoptotic cells) / total events] ×100%. Data were analyzed with FlowJo v. 10 software (Waters Corporation, Milford, MA, USA).

Evaluation of in vivo targeting

For targeting evaluation, when tumor volume reached 150 mm³, mice were randomly divided into three groups (three mice per group): (I) PBS; (II) nontargeted PTX/Pt (without RGD modification); and (III) PTX/Pt@RGD. ICG was loaded into nanocapsules at 0.1% w/w. ICG-labeled formulations (equivalent to 200 μg ICG per mouse) were injected via the tail vein. In vivo fluorescence imaging was performed with an IVIS Spectrum imaging system (Revvity, Waltham, MA, USA) at 1, 4, 12, 24, and 48 h postinjection (Ex/Em =710/780 nm). Regions of interest (ROIs) were drawn around the tumor, and mean fluorescence intensity was quantified with Living Image software (Revvity).

Tumor-bearing mouse model

All animal experiments were approved by the Experimental Animal Management and Ethics Committee of Bengbu Medical University (approval No. 087, issued in 2024), and were conducted in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition). Female BALB/c-nu mice (6 weeks old; body weight 18–22 g) were purchased from the Laboratory Animal Center of Yangzhou University. Mice were housed under specific pathogen-free conditions with a 12-h light-dark cycle at 22±2 ℃ and 55%±5% humidity and provided libitum access to sterilized food and water.

For tumor inoculation, 1×10⁶ A549 cells suspended in 100 μL of PBS were injected subcutaneously into the right flank. When the tumor volume reached approximately 100 mm³ (calculated as length × width²/2), mice were randomly assigned to 5 groups (n = 6 per group): (I) vehicle (PBS); (II) free cisplatin (5 mg/kg); (III) free PTX (10 mg/kg); (IV) free PTX + cisplatin (10 and 5 mg/kg, respectively); and (V) PTX/Pt@RGD (at an equivalent PTX dose of 10 mg/kg). Drug administration started on the same day of randomization. All drugs were administered via tail vein injection at a volume of 200 μL per mouse once every two days (on Days 0, 2, 4, 6, 8, 10, and 12) for a total of 7 injections over 14 days. Tumor volumes and body weights were recorded every 2 days. At the end of the experiment, mice were euthanized via intraperitoneal injection of pentobarbital sodium (150 mg/kg), and tumor tissues were excised for further analysis.

Histopathology and IHC

At the end of the treatment period, tumors and major organs (heart, liver, spleen, lungs, and kidneys) were excised, fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sectioned at 4-μm thickness. HE staining was performed according to the manufacturer’s protocol (cat. no. KGA501; Kaiji Biotechnology).

For IHC, tumor sections were deparaffinized, rehydrated, and subjected to antigen retrieval in citric acid buffer (10 mM; pH 6.0) with a pressure cooker for 3 min. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min. Sections were incubated with primary antibodies against CD31 (1:100), Ki-67 (1:200), and PCNA (1:500) overnight at 4 ℃ and then with HRP-conjugated secondary antibody (1:500) for 30 min at 37 ℃. Signal was developed with DAB substrate (Kaiji Biotechnology) for 1–3 min, and nuclei were counterstained with hematoxylin. Images were acquired with a slide scanner (Aperio AT2, Leica, Wetzlar, Germany), and the percentage of positively stained area was quantified with ImageJ software (with the IHC profiler plugin).

Statistical analysis

All statistical analyses were performed with GraphPad Prism 9.0. Data were presented as the mean ± standard deviation (SD) from at least three independent experiments (n≥3), unless otherwise specified. The normality of data distribution was assessed with the Shapiro-Wilk test. Homogeneity of variance was evaluated via the Levene test. Pairwise comparisons between two independent groups were performed with the two-tailed Student t-test when data were normally distributed with equal variances; otherwise, the Mann-Whitney U test was used. For multi-group comparisons, one-way analysis of variance (ANOVA) followed by the Tukey post hoc test was applied (21). A P value <0.05 was considered statistically significant.

Results

Characterization of PTX/Pt@RGD

TEM imaging showed regular spherical morphology (Figure 2A). DLS analysis revealed a mean particle size of 99.4±8.4 nm with a PDI of 0.41±0.04 and a zeta potential of –32.9±3.5 mV (Figure 2B,2C). The drug-loading efficiency and EE of PTX were 2.60%±0.53% and 44.68%±1.67%, respectively. The corresponding values for Pt were 6.37%±1.08% and 48.71%±2.08%, respectively. The in vitro drug release assay confirmed the pH-responsive release of the loaded drugs. At pH 6.5 (tumor-mimicking), 47.31% of PTX and 40.82% of Pt were released within 0.5 h, whereas at physiological pH 7.4 the cumulative release at 0.5 h was only 11.89% and 10.78%, respectively (Figure 2D,2E). Both drugs exhibited a rapid initial release at pH 6.5 followed by sustained release, reaching 72.09% (PTX) and 70.14% (Pt) at 48 h, whereas release at pH 7.4 remained slower throughout (71.91% and 69.41% at 48 h). These results demonstrate that the release of PTX and Pt is selectively accelerated under acidic tumor conditions, supporting the pH-responsive design of PTX/Pt@RGD. The redox-responsiveness of the disulfide linkage was further evaluated in the presence of 10 mM GSH (Figure 2F,2G). The release of PTX was markedly accelerated in GSH-containing medium: 28.04% of PTX was released within 0.5 h in the presence of 10 mM GSH, compared with 11.89% in GSH-free medium, and the 48-h cumulative release increased from 71.63% to 80.26% (statistically significant). These results confirm the redox-triggered release of PTX mediated by the disulfide bond in PTX-SS-PEG-RGD. Similarly, the release of Pt was accelerated in GSH-containing medium (27.83% within 0.5 h vs. 11.35% in GSH-free medium; 88.31% vs. 69.47% at 48 h), indicating that GSH triggered the release of both drugs from the nanocapsule. The particle size of PTX/Pt@RGD in PBS increased only slightly from 100.6 to 111.3 nm over 15 days of storage at 4 ℃, indicating good storage stability. Upon incubation in complete culture medium at 37 ℃, the particle size gradually increased from 98.4 to 124.3 nm over 72 h with no visible precipitation, which is likely attributable to the adsorption of serum proteins onto the nanoparticle surface (protein corona formation) rather than particle aggregation (Figure S1).

Figure 2 Characterization of PTX/Pt@RGD. (A) TEM image of PTX/Pt@RGD. Scale bar =100 nm. (B) Hydrodynamic diameter distribution of PTX/Pt@RGD as measured by DLS. (C) Zeta potential distribution of PTX/Pt@RGD as measured by DLS. (D,E) In vitro pH‑responsive cumulative release profiles of PTX and Pt at pH 7.4 and pH 6.5. (F,G) Redox‑triggered cumulative release of PTX and Pt in the absence or presence of GSH. DLS, dynamic light scattering; GSH, glutathione; PTX, paclitaxel; Pt, cisplatin; RGD, arginine-glycine-aspartic acid; TEM, transmission electron microscopy.

In vivo tumor targeting ability of PTX/Pt@RGD

In vivo fluorescence imaging was performed to evaluate whether RGD modification enhances the tumor-specific accumulation of the nanocapsules. ICG-labeled PTX/Pt@RGD or non-targeted PTX/Pt (without RGD) were injected into the tail vein of A549 tumor-bearing mice. As shown in Figure 3, tumor fluorescence intensity in the PTX/Pt@RGD group peaked at 24 h post-injection [(2.05±0.05) ×10⁶ p/s/cm²/sr], which was significantly higher than that in the non-targeted group [(1.42±0.03) ×10⁶; P<0.05]. The tumor-to-background ratio (TBR) of the PTX/Pt@RGD group was 14.0±0.4, higher than the 9.7±0.2 in the non-targeted group (P<0.05). These results demonstrate that RGD modification enables active tumor targeting of PTX/Pt@RGD.

Figure 3 In vivo tumor targeting of PTX/Pt@RGD in A549 xenograft-bearing mice. (A) Representative in vivo fluorescence images of mice at indicated time points after intravenous injection of PBS, non-targeted PTX/Pt nanoparticles, or PTX/Pt@RGD (n=3 per group). Fluorescence signals were monitored with an IVIS Spectrum imaging system (Ex/Em =710/780 nm). (B) Quantitative analysis of fluorescence intensity at the tumor site over time. Data are presented as the mean ± SD (n=3). Statistical significance was determined via two-way ANOVA followed by the Tukey post hoc test. ***, P<0.001 compared to the non-targeted group at the same time point. ANOVA, analysis of variance; Ex/Em, excitation/emission; PBS, phosphate-buffered saline; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid; SD, standard deviation.

In vitro antitumor efficacy

Cytotoxicity and inhibition of invasion

The biocompatibility of the nanocarrier was first evaluated by CCK-8 assay using the drug-free, RGD-modified carrier (Blank@RGD) against A549 cells. Blank@RGD exhibited negligible cytotoxicity across all tested concentrations, with cell viability remaining above 76.93% even at the highest concentration tested (10 mg/mL), confirming the favorable biosafety of the lipid–polymer nanocapsule platform (Figure 4A). The cytotoxic activity of PTX/Pt@RGD and the control formulations was then determined by CCK-8 assay. All drug-loaded groups inhibited A549 cell viability in a concentration-dependent manner. The IC₅₀ values were 18.11, 30.47, 72.33, 95.71, and 106.72 μg/mL for PTX/Pt@RGD, PTX/Pt@NP, the free PTX/Pt combination, free cisplatin, and free PTX, respectively (Figure 4A,4B). PTX/Pt@RGD exhibited the strongest antiproliferative activity, being approximately 4.0-fold more potent than the free PTX/Pt combination (IC₅₀: 18.11 vs. 72.33 μg/mL), approximately 5.3-fold more potent than free cisplatin, and approximately 5.9-fold more potent than free PTX. These results demonstrate the combined advantages of nanoencapsulation, synergistic drug combination, and RGD-mediated active targeting.

Figure 4 Cytotoxicity and anti‑invasive effects of PTX/Pt@RGD in A549 cells. (A,B) CCK‑8‑based cell viability assays of A549 cells after 24 h treatment with different formulations. (A) Narrow linear concentration range (0–0.25 mg/mL). (B) Broad concentration range on log‑scale axis, fitted with 4‑parameter logistic (4PL) model. (C) Representative crystal‑violet‑stained images of Transwell invasion assays (magnification: 20×). (D) Quantitative statistics of invaded cell number per field. Data represent mean ± SD (n = 3; five random fields per well). One‑way ANOVA; ns, not significant; ****, P<0.0001. ANOVA, analysis of variance; CCK‑8, Cell Counting Kit‑8; PTX, paclitaxel; Pt, cisplatin; RGD, arginine-glycine-aspartic acid; SD, standard deviation.

Transwell invasion assay showed that PTX/Pt@RGD treatment reduced the number of migrated cells to 25.5±3.3 per field, significantly lower than that of the control group (77.7±4.0) and representing an invasion inhibition rate of 67.2% (P<0.001). No significant difference was found between the PTX/Pt@RGD and free PTX + cisplatin (27.5±3.2; P>0.05). The inhibition rates of PTX and cisplatin alone were 39.4% and 46.8%, respectively (Figure 4C,4D).

PTX/Pt@RGD induced apoptosis via the mitochondrial pathway

To elucidate the mechanisms underlying the enhanced antitumor activity of PTX/Pt@RGD, we examined the mitochondrial apoptotic pathway, which is known to be activated by both PTX and cisplatin.

Regarding ROS generation and mitochondrial dysfunction, DCFH-DA staining revealed that PTX/Pt@RGD treatment increased intracellular ROS levels to 109.33±5.99 (relative fluorescence intensity, a.u.), which was 2.45-fold higher than that of the control group (44.66±3.51; P<0.001) and significantly higher than that of the free combination group (92.44±11.28; 2.07-fold; P<0.05). Single-drug groups induced moderate ROS increase (1.59–1.61-fold vs. control; P<0.01) (Figure 5A). Consistent with elevated ROS levels, JC-1 staining showed that the red:green fluorescence ratio decreased dramatically to 0.50±0.06 in the PTX/Pt@RGD group (only 16.5% of the control level; P<0.001), indicating severe mitochondrial membrane potential (ΔΨm) loss. The free combination group had a ratio of 0.94±0.00 (31.0% of control; P<0.001), while the PTX/Pt@RGD group exhibited significantly greater ΔΨm reduction in comparison (P<0.01) (Figure 5B). These results suggest that PTX/Pt@RGD triggers potent oxidative stress and mitochondrial dysfunction.

Figure 5 PTX/Pt@RGD induces oxidative stress, mitochondrial dysfunction, DNA damage, and apoptosis in A549 cells. (A) PTX/Pt@RGD induced ROS generation in A549 cells. Representative fluorescence images of A549 cells stained with DCFH-DA after 24-h treatment with the indicated formulations (green fluorescence indicates ROS levels) (magnification: 20×). Quantitative analysis of relative DCFH-DA fluorescence intensity. Data are presented as the mean ± SD from three independent experiments (n=3). Statistical significance was determined via one-way ANOVA followed by the Tukey post hoc test. (B) PTX/Pt@RGD induced loss of mitochondrial membrane potential in A549 cells. Representative fluorescence images of A549 cells stained with JC-1 after 24-h treatment with the indicated formulations. JC-1 monomer emitted green fluorescence (Ex/Em = 490/530 nm), and JC-1 aggregates emitted red fluorescence (Ex/Em =490/590 nm) (magnification: ×20). Quantitative analysis of the red:green fluorescence intensity ratio. Data are presented as the mean ± SD from three independent experiments (n=3). Statistical significance was determined via one-way ANOVA followed by the Tukey post hoc test. (C) PTX/Pt@RGD induced DNA damage in A549 cells (γ-H2AX immunofluorescence). Representative immunofluorescence images of A549 cells stained with anti-γ-H2AX antibody (green) and DAPI (blue) after 24-h treatment with the indicated formulations (magnification: ×20). Quantitative analysis of relative DNA damage index (γ-H2AX/DAPI mean fluorescence intensity). Data are presented as the mean ± SD from three independent experiments (n=3). Statistical significance was determined via one-way ANOVA followed by the Tukey post hoc test. (D) Western blot analysis of apoptosis- and inflammation-related proteins in A549 cells after different treatments. Protein expression of Bax, Bcl-2, Caspase-9, and COX-2 was detected by Western blotting with β-actin as the loading control. Relative protein levels were quantified and normalized to β-actin. Data are shown as the mean ± SD (n=3). (E) PTX/Pt@RGD induced apoptosis in A549 cells. Flow cytometry analysis of apoptosis in A549 cells after treatment with control, PTX, Pt, PTX/Pt, or PTX/Pt@RGD. The bar graph shows the quantitative percentage of apoptotic cells. Data are presented as mean ± SD (n=3). ns, not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 compared to the control group. ANOVA, analysis of variance; Ex/Em, excitation/emission; Pt, cisplatin; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid; ROS, reactive oxygen species; SD, standard deviation.

Regarding DNA damage and apoptotic protein expression, γ-H2AX immunofluorescence demonstrated that PTX/Pt@RGD treatment produced a relative DNA damage index of 0.5091±0.0138 (12.21-fold vs. control), comparable to that of the free combination group (0.5106±0.0099; 12.24-fold vs. control), and both were significantly higher than that of the control group (0.0417±0.0011; P<0.001). No significant difference was observed between the PTX/Pt@RGD and free combination groups (P>0.05) (Figure 5C). Western blot analysis further showed that PTX/Pt@RGD treatment upregulated Bax (8.6-fold) and Caspase-9 (3.4-fold) and downregulated Bcl-2 (to approximately 12% of the control level), with more pronounced effects than those of the free combination group (Figure 5D). The single-drug groups only produced slight differences.

Cell apoptosis was examined via flow cytometry with Annexin V-FITC/PI staining. The total apoptosis rate in the control group was 29.61%±1.80%, that of the PTX/Pt@RGD group was 2.84-fold higher at 84.20%±6.19% (P<0.0001 higher), and that of the free combination group was 2.52-fold higher at 74.70%±4.11% (P<0.001). No significant difference was observed between the PTX/Pt@RGD and free combination groups (P>0.05). The apoptosis rates of PTX and cisplatin alone were 56.83% and 62.83%, respectively (Figure 5E).

Collectively, these results establish a causal cascade: PTX/Pt@RGD induces robust ROS generation, leading to mitochondrial membrane potential collapse, DNA damage, activation of the mitochondrial apoptotic pathway (upregulation of Bax and Caspase-9, downregulation of Bcl-2), and ultimately, potent cell apoptosis.

In vivo antitumor efficacy

Inhibition of tumor growth

The in vivo antitumor efficacy of PTX/Pt@RGD was evaluated in A549 tumor-bearing nude mice. Mice were treated with PBS (control), free PTX, free cisplatin, free PTX + cisplatin, or PTX/Pt@RGD via tail vein injection once every two days (days 0, 2, 4, 6, 8, 10, and 12), for a total of 7 injections over 14 days. Tumor volumes were measured every 2 days.

Tumors in the control group grew rapidly, reaching 720.8±18.5 mm³ by day 14. Free PTX and free cisplatin monotherapies exhibited moderate tumor growth inhibition, with final tumor volumes of 405.2±9.2 and 338.5±7.8 mm³, respectively, corresponding to tumor growth inhibition rates (TGIs) of 43.8% and 53.0%, (both P values <0.001 vs. control).

The free PTX + cisplatin combination group showed significantly enhanced efficacy, with a final tumor volume of 194.3±2.4 mm³ (TGI =73.0%; P<0.001 vs. control; P<0.001 vs. either monotherapy). PTX/Pt@RGD achieved comparable antitumor activity, with a final tumor volume of 189.5±1.9 mm³ (TGI =73.7%; P<0.001 vs. control). No statistically significant difference was observed between the PTX/Pt@RGD group and the free combination group at any measured time point (P>0.05 for all comparisons), indicating that the nanocapsule formulation preserved the synergistic efficacy of the dual-drug regimen (Figure 6A).

Figure 6 PTX/Pt@RGD inhibited tumor growth in A549 xenograft-bearing mice. (A) Left: tumor‑volume growth curves over treatment period; right: quantitative analysis of tumor volume on day 14. (B) Left: body‑weight change curves of mice during treatment; right: quantitative analysis of mouse body weight on day 14. Data are shown as mean ± SD (n = 6). ns, not significant; ****, P<0.0001. Pt, cisplatin; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid; SD, standard deviation.

The body weight of mice in the PTX/Pt@RGD group remained stable over the 14-day treatment (from 19.9±0.5 to 21.5±0.6 g), whereas the free drug groups exhibited marked body weight loss (free PTX/cisplatin combination: from 20.2±0.5 to 14.9±0.7 g; free cisplatin: from 19.8±0.5 to 15.8±0.6 g), indicating that the nanocapsule formulation markedly reduced the systemic toxicity of the free drugs (Figure 6B).

Histopathological and immunohistochemical analysis

H&E staining of tumor sections revealed that PTX/Pt@RGD treatment induced the largest area of tumor necrosis (characterized by nuclear pyknosis, karyorrhexis, and loss of cellular architecture), followed by the free combination group, while monotherapy groups displayed only scattered necrotic regions. HE staining of major organs (heart, liver, spleen, lungs, and kidneys) revealed no obvious pathological abnormalities in any treatment group, confirming the favorable biosafety profile of PTX/Pt@RGD (Figure 7A,7B).

Figure 7 Histopathological analysis of tumor tissues and major organs after treatment. (A) H&E staining of tumor tissues from different treatment groups (control, PTX, Pt, PTX/Pt, and PTX/Pt@RGD) (magnification: ×20). (B) H&E staining of major organs (heart, liver, spleen, lung, kidney) in control and PTX/Pt@RGD-treated mice, showing no obvious pathological damage (magnification: ×20). H&E, hematoxylin and eosin; Pt, cisplatin; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid.

IHC was performed to assess tumor proliferation and angiogenesis. The Ki-67-positive rate in the control group was 12.639%±0.426%. PTX single-agent treatment showed minimal inhibition, reducing Ki-67 expression to 12.152%±0.415% (inhibition rate =3.9%; P>0.05 vs. control). In contrast, free cisplatin alone reduced Ki-67 to 2.573%±0.090% (inhibition rate =79.6%; P<0.0001 vs. control), while the free PTX + cisplatin combination further reduced it to 1.850%±0.070% (inhibition rate =85.4%; P<0.001). PTX/Pt@RGD achieved a comparable Ki-67-positive rate of 1.820%±0.070% (inhibition rate =85.6%; P<0.001 vs. control), with no significant difference from the free combination group (P>0.05). The PCNA-positive rate decreased from 14.307%±0.450% in the control group to 1.802%±0.060% in the PTX/Pt@RGD group (inhibition rate =87.4%; P<0.001), with no significant difference in comparison with the free combination group (2.227%±0.075%; P>0.05). Regarding angiogenesis, the CD31-positive rate in the control group was 9.842%±0.316%. Free PTX produced modest inhibition (7.974%±0.322%; inhibition rate =19.0%; P<0.05 vs. control), while the effect produced by cisplatin was stronger (4.931%±0.170%; inhibition rate =49.9%; P<0.001). The free PTX + cisplatin combination reduced CD31 expression to 2.200%±0.080% (inhibition rate = 77.6%; P<0.001). Notably, PTX/Pt@RGD exhibited the most potent antiangiogenic effect, reducing CD31 expression to 2.150%±0.080% (inhibition rate =78.1%; P<0.001 vs. control), which was comparable to that of the free combination group (P>0.05) (Figure 8A-8D). These results indicate that PTX/Pt@RGD effectively suppresses tumor proliferation and angiogenesis, achieving efficacy comparable to that of the free drug combination.

Figure 8 PTX/Pt@RGD inhibited tumor angiogenesis and proliferation in A549 xenografts. (A) Representative IHC images of CD31, Ki-67, and PCNA (magnification: ×20). (B-D) Quantitative analysis of positive area percentages. Data are the mean ± SD (n=3). ***P<0.001, ****P<0.0001 vs. control; ns, not significant (one-way ANOVA with the Tukey post hoc test). ANOVA, analysis of variance; IHC, immunohistochemistry; Pt, cisplatin; PTX, paclitaxel; RGD, arginine-glycine-aspartic acid; SD, standard deviation.

Discussion

PTX/Pt@RGD exhibited a pH/redox dual-responsive drug release profile. Under acidic tumor-mimicking conditions (pH 6.5), both PTX and Pt were released markedly faster than at physiological pH 7.4 (Figure 2D,2E): the cumulative release reached ~47% (PTX) and ~41% (Pt) within 0.5 h at pH 6.5, compared with only ~12% and ~11% at pH 7.4, respectively, followed by sustained release reaching a plateau of ~70% by 48 h. The acidic tumor microenvironment (pH 6.5-6.8) has been widely exploited to trigger drug release from pH-responsive nanocarriers, enabling selective drug delivery while minimizing premature drug leakage under physiological conditions (22-25) The pH-triggered release of PTX/Pt@RGD is likely attributable to the acid-labile hydrazone linkage in the PCL-hyd-PEG polymer, which destabilizes the nanocapsule shell under acidic conditions (22). In addition, the disulfide bond in the PTX-SS-PEG-RGD prodrug conferred reduction-responsiveness: the release of PTX was markedly accelerated in the presence of 10 mM GSH (28.04% vs. 11.89% within 0.5 h), and the 48-h cumulative release was significantly increased (80.26% vs. 71.63%), demonstrating that the disulfide linkage acts as a redox-responsive release switch. The GSH-triggered release of both PTX and Pt (48-h cumulative release of Pt: 88.31% vs. 69.47% in GSH-free medium) suggests that the reducing environment facilitates the destabilization of the disulfide-based nanocapsule architecture, promoting the release of both the prodrug-conjugated PTX and the encapsulated Pt. Similar tumor-microenvironment-responsive nanocapsules, such as the cisplatin/doxorubicin prodrug-loaded nanocapsule (CIS-DOXp NC) reported by Jin et al. (26) and the RGD-modified pH/redox dual-responsive nanoparticles co-delivering paclitaxel and cisplatin described by Tian et al. (27), have demonstrated the broad applicability of exploiting intrinsic tumor stimuli for triggered drug release (28). In addition, the integration of the RGD targeting moiety in PTX/Pt@RGD adds an additional layer of tumor specificity, potentially enhancing tumor-specific accumulation beyond what is achievable by passive targeting and microenvironmental responsiveness alone (29). Collectively, these results reinforce the notion that designing nanocarriers to respond to intrinsic tumor microenvironmental cues—including acidic pH and redox potential—is a potent strategy for improving drug delivery precision and efficacy.

The CCK-8 assay revealed that PTX/Pt@RGD exhibited potent concentration-dependent cytotoxicity against A549 cells, with an IC₅₀ of 18.11 μg/mL, significantly lower than those of free PTX (106.72 μg/mL) and free cisplatin (95.71 μg/mL). Notably, the nanoformulation was approximately 4.0-fold more potent than the free PTX/Pt combination (IC₅₀: 18.11 vs. 72.33 μg/mL), indicating that the nanocapsule system not only preserves but significantly amplifies the synergistic interaction between PTX and cisplatin. This synergy arises from their complementary mechanisms: PTX arrests mitosis by stabilizing microtubules, while cisplatin induces genotoxic stress via DNA cross-linking (9,10). Notably, the efficacy of this combination has been historically reported to vary according to administration sequence. For instance, Liebmann et al. (30) demonstrated that administering cisplatin prior to PTX in A549 cells could lead to antagonism, whereas the reverse sequence yielded an additive effect. In our study, the simultaneous co-delivery achieved by PTX/Pt@RGD resulted in significant synergistic cytotoxicity. Although this does not represent a direct comparison to optimized sequential schedules, it suggests that the RGD-mediated targeted delivery and the consequent improved intracellular co-localization of both drugs within the same tumor cells may create a favorable condition for synergy to occur, potentially mitigating the strict dependence on external administration timing observed with free drugs. However, such speculation should be further confirmed through direct comparison with sequential dosing regimens.

The combination of paclitaxel and cisplatin was selected because it constitutes a first-line chemotherapeutic regimen for advanced NSCLC (9,10). However, when the two drugs are administered as free agents, their markedly different pharmacokinetic profiles, including differential plasma half-lives, tissue distribution, and protein binding, can lead to asynchronous and spatially discordant drug concentrations at the tumor site, compromising the optimal synergistic ratio; moreover, the schedule-dependent efficacy of this combination, as discussed above, further complicates free-drug regimens (30). The co-delivery of both drugs within a single nanocapsule overcomes these pharmacokinetic mismatches by: (I) ensuring simultaneous delivery of both agents to the tumor at a defined ratio through the enhanced permeability and retention effect; (II) enabling synchronized, stimuli-responsive release of both drugs within the tumor microenvironment, thereby promoting their intracellular co-localization; and (III) eliminating the dependence on external administration sequence, as the two drugs are co-administered and co-released within the same tumor cells (31,32). Consistent with this, PTX/Pt@RGD achieved synergistic cytotoxicity in vitro without sequential dosing, and in vivo it exhibited efficacy comparable to that of the free combination while markedly reducing systemic toxicity, as reflected by the stable body weight.

Recent evidence from molecular studies supports the effectiveness of RGD-modified active targeting. Sarıçam et al. (33) demonstrated that different configurations of RGD peptide on gold nanoparticles significantly affected cellular uptake and cell cycle arrest in A549 cells and that cRGD-NH₂ provided higher internalization and G0/G1 blocking capacity. Similarly, Lin et al. (17) reported that RGD-AuNPs-SAHA promoted drug uptake in both A549 cells and xenografts, leading to downregulation of HIF-1α and VEGF expression. Although these studies employed gold nanocarriers, the consistent targeting enhancement across different platforms underscores the robustness of the RGD strategy. The application of RGD modification is not limited to gold nanoparticles; similar targeting strategies have also been integrated with other therapeutic modalities, such as mitochondrial-targeted chemo-photothermal therapy (34). In our study, the TBR of PTX/Pt@RGD at 24 h was significantly higher than that of the non-targeted group, constituting in vivo evidence that aligns with the cellular-level findings reported by Sarıçam et al. (33) and Lin et al. (17). Collectively, these results indicate that the RGD-mediated αvβ3 integrin targeting strategy is an effective approach for tumor-specific drug delivery.

Mechanistically, PTX/Pt@RGD induced tumor cell death through multiple synergistic pathways. The ROS level in the nanocapsule group was 2.45-fold higher than that of the control, significantly exceeding that of the free combination group. This excessive oxidative stress impaired mitochondrial membrane integrity, as evidenced by a pronounced decrease in the JC-1 red:green ratio to 16.5% of the control level. Concurrently, γ-H2AX staining indicated a 12.21-fold increase in DNA double-strand breaks. This cascade of oxidative stress, mitochondrial injury, and DNA damage is consistent with the mechanism reported for RGD-AuNPs-SAHA (17), corroborating the broad applicability of this apoptosis-inducing pathway. In line with this, Western blot analysis revealed the upregulated expression of Bax and Caspase-9, along with the downregulated expression Bcl-2, confirming the activation of the mitochondrial endogenous apoptosis pathway (35). Collectively, these results demonstrate that PTX/Pt@RGD synergistically targets both mitochondrial and nuclear pathways, driving a robust apoptotic response. Taken together, these observations delineate a cascade in which PTX/Pt@RGD-induced ROS overproduction leads to mitochondrial membrane potential collapse and oxidative DNA damage, culminating in the activation of the mitochondrial intrinsic apoptotic pathway (as reflected by the marked upregulation of Bax and Caspase-9 and downregulation of Bcl-2; Figure 5D). In parallel, the upregulation of COX-2 is consistent with a ROS-driven inflammatory stress response accompanying apoptosis. We acknowledge that the causal relationship between these events was not directly verified by antioxidant- or inhibitor-based experiments, and that the release of cytochrome c was not examined; these aspects warrant further mechanistic investigation.

In vivo IHC was performed to evaluate tumor proliferation and angiogenesis. Free PTX alone exhibited minimal inhibition of Ki-67 (3.9%), which is consistent with its mechanism of arresting cells in the mitotic phase without promoting G0/G1 phase exit, in which Ki-67 expression is typically lost (36). In contrast, free cisplatin reduced Ki-67 expression by 79.6%, reflecting its ability to induce DNA damage and inhibit cell activity throughout the cell cycle. The combination of free PTX and cisplatin achieved 85.4% inhibition, while PTX/Pt@RGD showed comparable efficacy (85.6% inhibition). For PCNA, a marker of DNA replication, PTX/Pt@RGD achieved 87.4% inhibition, comparable to the free combination. Regarding angiogenesis, staining for CD31, a well-established marker for evaluating tumor angiogenesis, revealed that free PTX alone showed only modest inhibition (19.0%), while the effect produced by free cisplatin was stronger (49.9%). The combination of free PTX and cisplatin achieved 77.6% inhibition, and PTX/Pt@RGD showed comparable antiangiogenic efficacy (78.1% inhibition). These results suggest that PTX/Pt@RGD effectively suppresses both tumor cell proliferation and angiogenesis, achieving efficacy comparable to that of the free drug combination. The similar efficacy between PTX/Pt@RGD group and the free combination group, with the nanocapsule formulation requiring no external adjustment of drug sequence or ratio, indicates the practical advantage of this nanoplatform for in vivo application.

Histopathological analysis revealed no obvious pathological lesions in the major organs of mice treated with PTX/Pt@RGD after repeated administration. In addition, the body weight of the PTX/Pt@RGD-treated mice remained stable over the 14-day treatment period (19.9±0.5 to 21.5±0.6 g), whereas marked body weight loss (approximately 13–26%) was observed in the free drug groups, indicating that the nanocapsule formulation reduced the systemic toxicity of the free drugs. This finding aligns with the low systemic toxicity reported for other dual-responsive nano-combinations, such as the DDP-P/PTX NPs developed by Wang et al. (37). The favorable safety profile observed in our study and others is likely attributable to the core design strategy of combining stimulus-responsive release with active targeting. RGD-mediated targeting is expected to enhance tumor-specific accumulation, thereby reducing off-target distribution in healthy tissues and mitigating systemic side effects. This notion is further supported by the work on the CIS-DOXp nanocapsule (26), which was reported to achieve high tumor suppression without significant body weight loss. Collectively, these results attest to the advantage of integrating microenvironmental responsiveness with active targeting in the development of nano-drug delivery systems that are both potent and safe (38).

Several limitations of this study should be acknowledged (39). First, final tumor weights—the most direct indicators of tumor growth inhibition—were not recorded, while the findings on in vivo efficacy were primarily based on serial tumor volume measurements and HE staining. Second, although the sequential changes in ROS production, mitochondrial membrane potential, DNA damage, and apoptotic protein expression were characterized, the causal relationship between these events was not directly verified by antioxidant- or inhibitor-based experiments, and the release of cytochrome c from mitochondria was not examined. Third, the subcutaneous A549 xenograft model does not fully recapitulate the native lung tumor microenvironment, limiting the generalizability of our findings. Fourth, the PTX loading efficiency (2.60%) was relatively low, which is primarily attributable to the prodrug-based design in which PTX is covalently linked to the PEG-RGD segment, limiting the PTX mass fraction within the nanocapsule; the encapsulation efficiencies (44.68% for PTX and 48.71% for Pt) partly reflect the removal of unencapsulated drugs by ultrafiltration. Nevertheless, the loading parameters are within the range of clinically translated prodrug-type nanocarriers, and the formulation achieved in vivo efficacy comparable to that of the free drug combination. Future optimization—including increasing the PTX fraction of the prodrug, adjusting the material feed ratio, and applying active-loading strategies—should further improve drug loading and EE. Future studies should incorporate end-point tumor weighing, verify cytochrome c release, employ orthotopic or PDX models, and optimize drug loading through active loading strategies (40).

Conclusions

The PTX/Pt@RGD successfully integrates active targeting and pH/redox dual-responsive release, as demonstrated by comparative release studies at pH 6.5 and 7.4 and in the presence of GSH. In this study, it achieved a high tumor-to-background ratio and synergistic efficacy comparable to that of the free PTX/cisplatin combination, together with a favorable in vivo safety profile and the practical advantage of co-delivering both drugs without external adjustment of dosing sequence or ratio. Although the in vivo therapeutic benefit of RGD-mediated targeting beyond enhanced tumor-specific accumulation warrants further investigation, our findings support the proactive evaluation of this combinatorial nanoplatform for lung cancer combination chemotherapy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0967/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0967/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0967/prf

Funding: This study was supported by the Bengbu Medical University Key Natural Science Research Project (No. 2022byzd074).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2026-0967/coif). All authors report that this study was supported by the Bengbu Medical University Key Natural Science Research Project (No. 2022byzd074). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were approved by the Experimental Animal Management and Ethics Committee of Bengbu Medical University (approval No. 087, issued in 2024), and were conducted in compliance with the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2024;74:229-63. [Crossref] [PubMed]
2. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin 2024;74:12-49. [Crossref] [PubMed]
3. Smolarz B, Łukasiewicz H, Samulak D, et al. Lung Cancer-Epidemiology, Pathogenesis, Treatment and Molecular Aspect (Review of Literature). Int J Mol Sci 2025;26:2049. [Crossref] [PubMed]
4. Puuniemi L, Mäkinen J, Koivunen J, et al. Changing Epidemiology of Lung Cancer in Finland: A Registry-Based Study by Histology and Region. Cancer Control 2025;32:10732748251401264. [Crossref] [PubMed]
5. Juang YR, Hu M, Hui M, et al. Trends in lung cancer incidence and stage at diagnosis in Singapore: a population-based joinpoint regression analysis by age, sex, and smoking status. Lancet Reg Health West Pac 2026;66:101779. [Crossref] [PubMed]
6. Travis WD. Update on small cell carcinoma and its differentiation from squamous cell carcinoma and other non-small cell carcinomas. Mod Pathol 2012;25 Suppl 1:S18-30. [Crossref] [PubMed]
7. Rina A, Maffeo D, Minnai F, et al. The Genetic Analysis and Clinical Therapy in Lung Cancer: Current Advances and Future Directions. Cancers (Basel) 2024;16:2882. [Crossref] [PubMed]
8. Zhao Z, Yang C, Li J. Pemetrexed and platinum with or without pembrolizumab for advanced non-small-cell lung cancer (NSCLC): a systematic review and meta-analysis. Clin Transl Oncol 2025;27:2024-36. [Crossref] [PubMed]
9. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004;4:253-65. [Crossref] [PubMed]
10. Kelland L. The resurgence of platinum-based cancer chemotherapy. Nat Rev Cancer 2007;7:573-84. [Crossref] [PubMed]
11. Abu Rous F, Singhi EK, Sridhar A, et al. Lung Cancer Treatment Advances in 2022. Cancer Invest 2023;41:12-24. [Crossref] [PubMed]
12. DeRidder L, Rubinson DA, Langer R, et al. The past, present, and future of chemotherapy with a focus on individualization of drug dosing. J Control Release 2022;352:840-60. [Crossref] [PubMed]
13. Ashique S, Garg A, Mishra N, et al. Nano-mediated strategy for targeting and treatment of non-small cell lung cancer (NSCLC). Naunyn Schmiedebergs Arch Pharmacol 2023;396:2769-92. [Crossref] [PubMed]
14. Guo FY, Pan YZ. Targeting lung cancer utilizing smart nanoparticles: Nanomedicine meets tumor stroma. Bioorg Chem 2025;167:109202. [Crossref] [PubMed]
15. Cabral H, Kinoh H, Kataoka K. Tumor-Targeted Nanomedicine for Immunotherapy. Acc Chem Res 2020;53:2765-76. [Crossref] [PubMed]
16. Sun X, Gao W, Liu Y, et al. Mater Des. 2024;238:112619.
17. Lin J, Huang X, Wang X, et al. RGD-Modified Gold Nanoparticles Loaded with SAHA Remodel the Hypoxic Inflammatory Microenvironment via Inhibiting HIF-1α-VEGF Signaling to Enhance Radiosensitivity in NSCLC. Int J Nanomedicine 2025;20:14753-85. [Crossref] [PubMed]
18. Muradova Z, Carmès L, Brown N, et al. Targeted-theranostic nanoparticles induce anti-tumor immune response in lung cancer. J Nanobiotechnology 2025;23:466. [Crossref] [PubMed]
19. Liu XM, Zhu ZZ, He XR, et al. NIR Light and GSH Dual-Responsive Upconversion Nanoparticles Loaded with Multifunctional Platinum(IV) Prodrug and RGD Peptide for Precise Cancer Therapy. ACS Appl Mater Interfaces 2024;16:40753-66. [Crossref] [PubMed]
20. Tang L, Luo Y, Luo W, et al. Pt(IV) Prodrug Photoactivation: A Promising Strategy for Cancer Therapy. Curr Protein Pept Sci 2024;25:797-813. [Crossref] [PubMed]
21. TUKEY JW. Comparing individual means in the analysis of variance. Biometrics 1949;5:99-114. [PubMed]
22. Wang S, Liu L, Tian L, et al. Elucidation of Spatial Cooperativity in Chemo-Immunotherapy by a Sequential Dual-pH-Responsive Drug Delivery System. Adv Mater 2024;36:e2403296. [Crossref] [PubMed]
23. Guo Z, Huang T, Lv X, et al. Tumor microenvironment-activated polypeptide nanoparticles for oncolytic immunotherapy. Biomaterials 2025;314:122870. [Crossref] [PubMed]
24. Li Q, Shi J, Ruan X, et al. Hypoxia- and pH-Responsive Metal-Phenolic Network-Engineered Ferroptosis-Immuno Microspheres for Transarterial Chemoembolization in Hepatocellular Carcinoma. Adv Funct Mater 2025;35:e10798.
25. Srinivasarao DA, Shah S, Famta P, et al. Unravelling the role of tumor microenvironment responsive nanobiomaterials in spatiotemporal controlled drug delivery for lung cancer therapy. Drug Deliv Transl Res 2025;15:407-35. [Crossref] [PubMed]
26. Jin Y, Wang Y, Liu X, et al. Synergistic Combination Chemotherapy of Lung Cancer: Cisplatin and Doxorubicin Conjugated Prodrug Loaded, Glutathione and pH Sensitive Nanocarriers. Drug Des Devel Ther 2020;14:5205-15. [Crossref] [PubMed]
27. Tian J, Min Y, Rodgers Z, et al. Co-delivery of paclitaxel and cisplatin with biocompatible PLGA-PEG nanoparticles enhances chemoradiotherapy in non-small cell lung cancer models. J Mater Chem B 2017;5:6049-57. [Crossref] [PubMed]
28. Li K, Gui S, Wang N, et al. Sequential pH/GSH-responsive stealth nanoparticles for co-delivery of anti-PD-1 antibody and paclitaxel to enhance chemoimmunotherapy of lung cancer. Eur J Med Chem 2025;285:117273. [Crossref] [PubMed]
29. Hao Y, Chen Y, He X, et al. RGD peptide modified platinum nanozyme Co-loaded glutathione-responsive prodrug nanoparticles for enhanced chemo-photodynamic bladder cancer therapy. Biomaterials 2023;293:121975. [Crossref] [PubMed]
30. Liebmann JE, Fisher J, Teague D, et al. Sequence dependence of paclitaxel (Taxol) combined with cisplatin or alkylators in human cancer cells. Oncol Res 1994;6:25-31. [PubMed]
31. Bi YY, Chen Q, Yang MY, et al. Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer. Nat Commun 2024;15:2759. [Crossref] [PubMed]
32. Wang X, Liu G, Pu X, et al. Combating cisplatin-resistant lung cancer using a coiled-coil lipopeptides modified membrane fused drug delivery system. J Control Release 2025;379:45-58. [Crossref] [PubMed]
33. Sarıçam M, Ercan Ayra M, Culha M. Cellular Response to RGD Peptide Configuration on Gold Nanoparticles: A Surface Chemistry Investigation. ACS Omega 2025;10:20487-96. [Crossref] [PubMed]
34. Yang GG, Pan ZY, Zhang DY, et al. Precisely Assembled Nanoparticles against Cisplatin Resistance via Cancer-Specific Targeting of Mitochondria and Imaging-Guided Chemo-Photothermal Therapy. ACS Appl Mater Interfaces 2020;12:43444-55. [Crossref] [PubMed]
35. Wang DR, Wu XL, Sun YL. Therapeutic targets and biomarkers of tumor immunotherapy: response versus non-response. Signal Transduct Target Ther 2022;7:331. [Crossref] [PubMed]
36. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol 2000;182:311-22. [Crossref] [PubMed]
37. Wang B, Hu W, Yan H, et al. Lung cancer chemotherapy using nanoparticles: Enhanced target ability of redox-responsive and pH-sensitive cisplatin prodrug and paclitaxel. Biomed Pharmacother 2021;136:111249. [Crossref] [PubMed]
38. Li S, Wang X, Han H, et al. Metallic nanomedicine in cancer immunotherapy. Acta Pharm Sin B 2025;15:4614-43. [Crossref] [PubMed]
39. Mordant P, Loriot Y, Lahon B, et al. Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells. PLoS One 2011;6:e26073. [Crossref] [PubMed]
40. Hendriks LE, Kerr KM, Menis J, et al. Non-oncogene-addicted metastatic non-small-cell lung cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Ann Oncol 2023;34:358-76. [Crossref] [PubMed]

(English Language Editor: J. Gray)