Experimental evaluation of Nilotinib and Paclitaxel co-delivered via albumin nanoparticles as a therapeutic strategy for lung squamous cell carcinoma

Abstract

Chemotherapy remains the primary treatment for advanced lung squamous cell carcinoma (LUSC); however, its severe toxic side effects significantly limit therapeutic efficacy. There is an urgent clinical need to integrate molecular targeting with chemotherapy to enhance treatment outcomes. Consequently, this study developed a co-loaded albumin-based nanodelivery system (NLB/PTX-NPs) for targeted delivery of nilotinib alongside the chemotherapeutic agent paclitaxel. We systematically evaluated its in vitro and in vivo anti-tumor activity, safety, and underlying molecular mechanisms. NLB/PTX-NPs were synthesized via the solvent displacement method, followed by characterization of their physicochemical properties, drug-loading performance, and biosafety. Using NCI-H520 cells as a model, the in vitro efficacy and molecular mechanisms were evaluated through cellular uptake studies, cytotoxicity assessments, in vitro cellular experiments, and Western Blot assays. Additionally, subcutaneous tumor and lung metastasis models were established in nude mice to evaluate in vivo targeted distribution, as well as anti-tumor and anti-metastatic activities. NLB/PTX-NPs exhibited a uniform spherical morphology, demonstrating excellent dispersibility, stability, and biosafety. In vitro experiments revealed that the nanoparticles significantly enhanced cellular uptake and inhibited the proliferation, migration, and invasion of NCI-H520 cells. Western blot analysis confirmed their ability to regulate the expression of proteins associated with apoptosis and epithelial-mesenchymal transition (EMT). Furthermore, in vivo experiments indicated that the nanoparticles significantly accumulated in tumor tissues, synergistically inhibiting tumor growth (P < 0.001) and lung metastasis (P < 0.05) through the downregulation of NF-κB and IKBα phosphorylation levels, as well as a reduction in the Ki67 index, without any apparent systemic toxicity. This study successfully developed NLB/PTX-NPs, achieving effective co-loading of dual drugs, efficient enrichment, and synergistic delivery at tumor sites. The system demonstrated promising preclinical anti-tumor and anti-metastatic effects in the NCI-H520 models by synergistically inhibiting the NF-κB pathway, thereby providing a novel strategy for the treatment of lung squamous cell carcinoma.

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) representing over 80% of all cases [1]. Lung squamous cell carcinoma (LUSC) accounts for approximately 15% to 30% of NSCLC instances [2]. Current standard treatments for LUSC encompass surgery, chemoradiotherapy, targeted therapy, and immunotherapy [3, 4]. In contrast to the rapid advancements seen in targeted therapies for lung adenocarcinoma, LUSC treatment predominantly relies on conventional chemotherapy agents, such as paclitaxel, due to the absence of effective therapeutic targets [5]. Nevertheless, the emergence of acquired multidrug resistance (MDR) and the presence of severe toxic side effects substantially hinder treatment efficacy [6, 7]. Furthermore, single-agent chemotherapy is often insufficient to combat LUSC’s aggressive invasiveness and propensity for early distant metastasis, as residual tumor cells activate various survival and escape signalling pathways [8, 9].

Our research group identified Nilotinib, a novel ILK inhibitor, through screening based on protein structure and receptor-ligand affinity experiments. Both in vitro and in vivo studies demonstrated its significant ability to inhibit the proliferation, invasion, and metastasis of esophageal squamous cell carcinoma (ESCC) while promoting apoptosis. Additionally, it enhances mitochondrial oxidative stress levels in cells. These findings indicate that Nilotinib exerts its anti-ESCC effects by targeting ILK [10–12]. Nilotinib is a second-generation tyrosine kinase inhibitor originally developed for treating hematologic malignancies [13–15]. Beyond inhibiting BCR-ABL, Nilotinib also exhibits multi-target kinase inhibitory activity against c-Kit, PDGFR, SRC, and others, broadly interfering with tumor proliferation, metastasis, and microenvironment remodeling [16–18]. This pleiotropic effect provides a theoretical basis for repurposing Nilotinib in solid tumors. Recent studies have demonstrated its significant anti-tumor activity in various solid tumors, including gastrointestinal stromal tumors, colorectal cancer, breast cancer, and thyroid cancer [19–22]. Clinical studies on the combination of Nilotinib and paclitaxel have confirmed that this regimen is safe, manageable, and well-tolerated in some advanced solid tumors, with potential tumor-suppressive effects. Sustained objective responses were observed even in refractory tumors such as ovarian granulosa cell tumors [23, 24]. Our research findings and reports from domestic and international studies confirm that Nilotinib is a multi-target anticancer drug.

This study investigates the use of human serum albumin (HSA) as a nanocarrier platform to develop an albumin nanoparticle system (NLB/PTX-NPs) that co-loads Nilotinib and Paclitaxel. The design leverages HSA to overcome the solubility challenges of the dual drugs and to facilitate tumor targeting, thereby enhancing the delivery efficiency of both agents in lung squamous cell carcinoma lesions while minimizing nonspecific distribution in normal tissues. Through an examination of formulation characterization, cellular and molecular mechanisms, and in vivo efficacy, this study evaluates the potential of this nano co-delivery system to inhibit the growth and metastasis of lung squamous cell carcinoma, offering a promising clinical treatment strategy for LUSC.

Experimental method

Materials, cell line and animals

Nilotinib, paclitaxel, and human serum albumin (HSA) were purchased from Macklin, China. The human lung squamous cell line (NCI-H520) was obtained from procell, China. All male BALB/c Nude mice utilized in the experiments were sourced from Nanjing MouseBorn Biotechnology Co., Ltd. (Nanjing, China). All animal experimental procedures complied with the established guidelines for the care and use of laboratory animals. The experimental protocol was approved by the Animal Ethics Committee of Xinjiang Medical University (Approval No.: IACUC-JT-20251015-40).

Synthesis of NLB/PTX-NPs

The nano-drug was synthesized using the solvent displacement method. Nilotinib and paclitaxel were dissolved in anhydrous ethanol in a specific ratio, and 1 mL of deionized water was prepared. Under magnetic stirring at 37 °C, the NLB/PTX mixed solution was rapidly introduced into 200 mg/mL HSA and incubated for 60 s. Subsequently, 1 mL of PBS was added to stabilize the system, leading to the formation of NLB/PTX-NPs. High-temperature rotary evaporation was conducted for 10 min to eliminate the anhydrous ethanol, followed by ultrafiltration centrifugation using a 100 kDa cutoff membrane for 10 min to remove unencapsulated drugs and phosphates from PBS, ultimately yielding NLB/PTX-NPs. The resulting nanoparticles were lyophilized and stored at room temperature, protected from light, for subsequent experiments.

Characterization

The particle size and polydispersity index (PDI) of the nanoparticles were measured at a scattering angle of 90° using dynamic light scattering (DLS). The zeta potential of the nanoparticles was determined using a zeta potential analyzer. The structure and morphology of the nanoparticles were characterized through transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Fourier transform infrared spectroscopy (FTIR) was utilized to analyze the chemical modifications of human serum albumin (HSA) nanoparticles. The synthesized nanoparticles were lyophilized into a freeze-dried powder, and their physical changes were observed after long-term storage. The lyophilized powder was dissolved in purified water, and the particle size and polydispersity index of NLB/PTX-NPs in purified water were measured, with changes recorded over one week.

The contents of NLB and PTX in nanoparticles were simultaneously quantified using high-performance liquid chromatography (HPLC). The chromatographic conditions employed an Agilent 1260 HPLC system equipped with a C18 column (4.6 mm × 250 mm, 5 μm). The mobile phase comprised acetonitrile and water (containing 0.1% phosphoric acid) in a ratio of 60:40 (v/v), with a flow rate of 1.0 mL/min and a column temperature maintained at 30 °C. A series of mixed standard solutions of NLB and PTX, ranging from 1 to 80 µg/mL, was accurately prepared and injected for detection. A standard curve was constructed by plotting drug concentration (C) on the x-axis against peak area (A) on the y-axis, allowing for the calculation of the regression equation. For drug content determination, an appropriate volume of the NLB/PTX-NPs solution was mixed with ten times its volume of methanol/acetonitrile for ultrasonic demulsification, followed by centrifugation to collect the supernatant, which was then injected into the HPLC system.

The calculation formula is as follows:

Cellular uptake assay

In vitro uptake experiments were conducted using NCI-H520 cells, employing coumarin-6 (C6) as the fluorescent probe. The differences in cellular uptake between C6/NLB/PTX-NPs and free C6 were investigated using confocal laser scanning microscopy (CLSM). NCI-H520 cells were seeded in confocal culture dishes and treated with coumarin-6 and coumarin-6-labeled NLB/PTX-NPs following cell attachment. The cells were incubated for 2, 6, and 8 h, respectively, washed with PBS, fixed with 4% paraformaldehyde, and subsequently stained with DAPI after another PBS wash. Finally, cellular uptake was observed using a confocal laser scanning microscope.

CCK-8 assay

The in vitro cytotoxicity of NLB, PTX, the NLB/PTX mixture, and NLB/PTX-NPs was evaluated using the CCK-8 assay. NCI-H520 cells (1 × 10⁴ cells/well) were seeded in 96-well plates and treated according to the designated experimental groups after cell attachment. Once the cell confluence reached approximately 70%, the medium was replaced with fresh medium containing varying concentrations of NLB, PTX, the NLB/PTX mixture, and NLB/PTX-NPs, followed by incubation for 24 and 48 h. After incubation, 10 µL of CCK-8 reagent was added to each well and incubated for an additional 2 h. Finally, the absorbance of each well was measured at 450 nm using a microplate reader, and the half-maximal inhibitory concentration (IC₅₀) was calculated.

Edu proliferation assay

Cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single-cell suspension, which was then seeded at a density of 3 × 10⁴ cells per well in 24-well plates and incubated overnight at 37 °C in a 5% CO₂ atmosphere. After 24 h of treatment for each group, the prepared EdU reagent was added to the culture medium, followed by an additional 2-hour incubation. The cells were subsequently fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Following this, the Click reaction solution was added for a 30-minute incubation at room temperature in the dark, and nuclear staining was performed using Hoechst dye. Fluorescence images of the same field of view under blue and green fluorescence were captured with an inverted fluorescence microscope, and the analysis was conducted using ImageJ software.

Plate cloning assay

Cells from each group were seeded in 6-well plates at a density of 1 × 10³ cells per well. After adherence, they were treated according to their respective groups and cultured in an incubator until visible cell clusters formed. The cultures were then terminated, fixed with 4% paraformaldehyde, stained with crystal violet, washed, air-dried, and images were captured.

Transwell assay

NCI-H520 cells were divided into five groups: a control group, an NLB group, a PTX group, an NLB/PTX mixture group, and an NLB/PTX-NPs group. Except for the control group, the other four groups were treated for 24 h with culture media containing NLB (1 µg/mL), PTX (4 µg/mL), NLB/PTX mixture (NLB at 1 µg/mL and PTX at 4 µg/mL), and NLB/PTX-NPs (with NLB at 1 µg/mL), based on the IC50 design. The control group was incubated with regular culture medium for 24 h. Following incubation, cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare single-cell suspensions. For the invasion assay, 50 µL of diluted Matrigel matrix was uniformly added to the upper chamber, and the uncoagulated Matrigel matrix solution was removed after 4 h. The cell concentration was adjusted to 5 × 10^5 cells/mL. An equal volume of cell suspension was added to the upper chamber of the Transwell, while the lower chamber was filled with culture medium containing 20% FBS. After 48 h, the upper chamber was rinsed with PBS, and the cells that had migrated through the upper chamber were fixed with 4% paraformaldehyde. The cells were stained with 1% crystal violet for 20 min, washed with PBS, inverted, and air-dried. Finally, the cells were observed, and images were captured using an inverted microscope.

Western blot analysis

Total protein samples were extracted from each group of cells and tumor tissues using RIPA lysis buffer containing protease and phosphatase inhibitors on ice. The intervention method for NCI-H520 cells followed the protocol outlined in Sect.  2.7, while tumor tissues were obtained as described in Sect.  2.13. Following protein extraction, the protein concentration was determined using the BCA method. Proteins were separated via 10% SDS-PAGE gel electrophoresis and subsequently transferred onto PVDF membranes. After blocking with skim milk, primary antibody working solutions were added (Bcl2: 1:1000; BAX: 1:2000; β-actin: 1:50,000; GAPDH: 1:50,000; E-cadherin: 1:1000; Vimentin: 1:2000; β-catenin: 1:1000; p-NF-κB: 1:1000; NF-κB: 1:1000; p-IKBα: 1:1000; IKBα: 1:1000) and incubated overnight at 4 °C with shaking. After washing three times with TBST, the membranes were incubated with an HRP-conjugated secondary antibody working solution for 1 h at room temperature. Protein bands were visualized using a chemiluminescence imaging system, and quantitative analysis was performed using ImageJ software. The antibodies were sourced from Proteintech (Wuhan, China), Zenbio (Chengdu, China), and Affinity Biosciences (USA).

Quantitative Real-Time PCR (qRT-PCR) analysis

To validate the functional blockade of the NF-κB signaling pathway at the transcriptional level, qRT-PCR was performed using the excised tumor tissues. Total RNA was extracted from the frozen tumor tissues using a Total RNA Isolation Kit (Foregene, China) according to the manufacturer’s protocol. The concentration and purity of the isolated RNA were determined using a NanoDrop spectrophotometer. Subsequently, the RNA was reverse-transcribed into complementary DNA (cDNA) using a PrimeScript RT reagent Kit (Takara, Japan). Quantitative real-time PCR was performed on a Real-Time PCR System (Sichuan Jelaimei Technology, China) using SYBR Green qPCR Master Mix (Vazyme, China). The thermocycling conditions were set as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative mRNA expression levels of the target genes were calculated using the 2^−ΔΔCt method, with GAPDH serving as the internal reference gene for normalization. The specific primer sequences (synthesized by Sangon Biotech, China) used in this study were as follows: GAPDH (Forward: 5’-GGAGCGAGATCCCTCCAAAAT-3’, Reverse: 5’-GGCTGTTGTCATACTTCTCATGG-3’); RELA (Forward: 5’-ATGTGGAGATCATTGAGCAGC-3’, Reverse: 5’-CCTGGTCCTGTGTAGCCATT-3’); NFKBIA (Forward: 5’-CTCCGAGACTTTCGAGGAAATAC-3’, Reverse: 5’-GCCATTGTAGTTGGTAGCCTTCA-3’); BCL2 (Forward: 5’-CGACGACTTCTCCCGCCGCTACCGC-3’, Reverse: 5’-CCGCATGCTGGGGCCGTACAGTTCC-3’); and MMP9 (Forward: 5’-TGTACCGCTATGGTTACACTCG-3’, Reverse: 5’-GGCAGGGACAGTTGCTTCT-3’).

Biosafety evaluation

The pharmacological safety of NLB, PTX, HSA, and NLB/PTX-NPs was assessed using a hemolysis assay. A 1 mL blood sample was collected from euthanized mice, and the blood was transferred into a centrifuge tube containing ethylenediaminetetraacetic acid disodium salt (EDTA-Na2). The sample was then centrifuged at 3000 rpm for 10 min to separate the red blood cells (RBCs). The RBC pellet at the bottom of the tube was collected, resuspended in phosphate-buffered saline (PBS), and centrifuged at 1200 rpm for 15 min. This washing process was repeated three times until the supernatant became colorless. Pure water served as the positive control, while PBS was used as the negative control. The test drugs were added to the RBC suspension and incubated at 37 ± 0.5 °C for 3 h. After incubation, the sample was centrifuged at 3000 rpm for 10 min to separate the intact red blood cells, and the supernatant was collected to measure the absorbance at 542 nm. The hemolysis rate was calculated according to the formula to analyze the amount of hemoglobin released.

To evaluate the systemic biosafety of each formulation in vivo, nude mice were sacrificed, and major organs (heart, liver, spleen, lung, and kidney) were harvested following the treatment cycle. The tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was performed to observe histopathological changes in the major organs of mice from each group under a microscope. Meanwhile, whole blood was collected from the mice, and after centrifugation to separate the serum, relevant biochemical indicators were measured using an automated biochemical analyzer. These indicators included liver function markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST), renal function markers creatinine (CREA) and urea (UREA), as well as myocardial injury and routine metabolic markers creatine kinase (CK) and lactate dehydrogenase (LDH), to comprehensively evaluate the potential toxicity of the nano-drug delivery system.

In vivo distribution and accumulation of drugs at tumor sites

To investigate the fluorescence distribution of indocyanine green (ICG) and ICG-loaded albumin nanoparticles containing paclitaxel (ICG-NLB/PTX-NPs) in biological systems, a tumor model was established using male BALB/c nude mice (6–8 weeks old, weighing 18–22 g) implanted with NCI-H520 cells. In this study, each group of NCI-H520 tumor-bearing mice consisted of three male mice (n = 3), resulting in a total of twelve male mice (n = 12). To ensure statistical independence, each mouse was treated as an individual experimental unit. When the tumor volume reached approximately 150 mm³, the NCI-H520 tumor-bearing mice were randomly divided into four groups. A 200 µL PBS solution containing free ICG, ICG-NLB, ICG-PTX, and ICG-NLB/PTX-NPs was prepared and injected into the mice. The distribution of ICG and ICG-NLB/PTX-NPs was monitored 24 h post-injection using the Pearl Trilogy small animal fluorescence imaging system (LI-COR Biosciences, USA), with excitation and emission wavelengths set at 785 nm and 820 nm, respectively. At 24 h following intravenous injection, the mice were euthanized, and tumor tissues along with major organs (heart, liver, spleen, lung, and kidney) were dissected for in vitro fluorescence intensity measurement.

In vivo anti-tumor activity efficacy

A tumor model was established using male BALB/c Nude mice inoculated with NCI-H520 cells. In this study, each group of NCI-H520 tumor-bearing mice consisted of 5 male mice, totaling 25 male mice. A suspension of NCI-H520 cells was subcutaneously injected into the right lower limb of the nude mice at a dose of 3 × 10^6 cells per mouse. When the tumor volume reached between 50 and 70 mm³, the mice were randomly divided into five groups, each containing five mice. The treatment regimen included NLB, PTX, NLB/PTX mix, NLB/PTX-NPs, and saline (NLB: 3 mg/kg, PTX: 12 mg/kg), administered every three days for a total of four doses. Body weight of the mice was measured and recorded every three days, while tumor length (L) and width (W) were measured once using a vernier caliper. Tumor volume was calculated using the formula V = L × W² / 2. All animal studies were conducted in accordance with the approval granted by the Institutional Animal Care and Use Committee. After three weeks of continuous treatment, the mice in each group were euthanized, and the tumors were excised for volume and weight measurement. Some specimens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for immunofluorescence staining with Ki67 and DAPI. Observations were made under a microscope, and photographs were taken for documentation. The remaining tissues were stored at -80 °C for future analysis.

In vivo anti-tumor metastasis research

Male BALB/c nude mice were utilized to establish a lung metastasis model. NCI-H520 cells (1 × 10^6 cells per mouse) were injected into the mice via the tail vein to create an intrapulmonary metastasis model of lung squamous cell carcinoma. All animal experiments were conducted in accordance with the experimental guidelines set forth by the Animal Ethics Committee of Xinjiang Medical University. On day 15 post NCI-H520 cell inoculation, mice exhibiting lung metastases were randomly divided into five groups (n = 5 per group). The mice received treatments of NLB, PTX, NLB/PTX mix, NLB/PTX-NPs, or normal saline at doses of NLB: 3 mg/kg and PTX: 12 mg/kg. Treatments were administered every three days for a total of four doses. Mice were euthanized in accordance with ethical principles 30 days following the initial drug intervention, and the number of metastatic nodules on the lung surface was counted, while lung weight was measured. After fixing lung tissues in 4% paraformaldehyde for 48 h, the samples were embedded, sectioned, and stained with hematoxylin-eosin (HE).

Statistical analysis

All results are expressed as mean ± standard deviation. Data were analyzed using GraphPad Prism 10.0 and SPSS 19.0. For normally distributed measurement data, comparisons between two groups were performed using t-tests, while one-way ANOVA was utilized for comparisons among three or more groups, with subsequent multiple comparisons conducted using the LSD-t method. Significance was denoted as * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001.

Result

Characterization of NLB/PTX-NPs

The size of nanoparticles significantly influences their pharmacokinetics, transport, and cellular uptake, thereby directly affecting therapeutic efficacy. The particle sizes of NLB/PTX-NPs in pure water and PBS were measured at 137.2 ± 0.95 nm and 128.0 ± 1.15 nm, respectively, with polydispersity indices (PDI) of 0.22 ± 0.03 and 0.16 ± 0.01, and zeta potentials of -22.53 ± 1.18 mV and − 21.35 ± 4.08 mV, indicating good stability (Fig. 1A, B). To evaluate the microstructure of the nano-delivery system, we observed NLB/PTX-NPs using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). As shown in Figs. 1C and D, the NLB/PTX-NPs exhibited regular spherical or near-spherical shapes with smooth edges and good dispersion, showing no significant aggregation or adhesion. FTIR spectroscopy (Fig. 1E) confirmed the binding between the drug and the carrier, retaining the characteristic peaks of HSA at 1650 cm⁻¹ (amide I band) and 1546 cm⁻¹ (amide II band). Additionally, the characteristic peak attributed to PTX (689 cm⁻¹) and the C-F stretching vibration peak of NLB (1050–1150 cm⁻¹) appeared in the spectrum, confirming the successful complexation of both drugs with the albumin carrier. Furthermore, stability tests demonstrated that the reconstituted NIL/PTX-NPs lyophilized powder exhibited good dispersibility. During the 7-day continuous monitoring, DLS results verified its physical stability (Fig. 1F), indicating that this nano-system possesses ideal monodisperse characteristics and storage stability. The encapsulation efficiencies and drug loading capacities (DLC) of NLB and PTX were determined via HPLC. It is important to note that the drug loading level of Nilotinib is consistent with reported literature values for human serum albumin nanoparticles [25, 26] and, coupled with the high encapsulation efficiency and rapid cellular internalization (as shown in Fig. 2), ensures the delivery of an effective therapeutic dose to the NCI-H520 cells. These results provide a robust foundation for the cellular and molecular responses characterized in subsequent sections. The encapsulation efficiencies of NLB and PTX in the nanoparticles were measured at (76.92 ± 0.63)% and (87.49 ± 0.91)%, respectively, with drug loading capacities of (3.61 ± 0.54)% and (15.12 ± 0.76)%. The HSA carrier demonstrated an excellent loading capacity for both hydrophobic drugs. The high encapsulation efficiency helps reduce drug waste. It facilitates the delivery of sufficient drug doses to target tissues, laying a foundation for subsequent in vitro and in vivo anti-tumor activity studies.

Fig. 1.

Characterization of NLB/PTX-NPs. (A, B) Size distribution and zeta potential of NLB/PTX-NPs measured by dynamic light scattering (DLS) in deionized water (A) and PBS (B). Insets show the particle size, PDI, and zeta potential values. (C) Representative transmission electron microscopy (TEM) images of NLB/PTX-NPs. (D) Representative scanning electron microscopy (SEM) images of NLB/PTX-NPs. (E) Fourier transform infrared (FT-IR) spectra of free NLB, free PTX, HSA, and NLB/PTX-NPs. Characteristic peaks (Amide I and Amide II bands) are highlighted. (F) Stability of NLB/PTX-NPs in deionized water over 7 days at room temperature, monitoring changes in particle size and PDI. Data are presented as mean ± SD (n = 3)

Cellular uptake of nanomaterials

Comparing the C6/NLB/PTX-NPs group with the Free C6 group (Fig. 2A), it was observed that, under the same intervention duration, the C6/NLB/PTX-NPs group exhibited a higher fluorescence signal intensity than the Free C6 group. Semi-quantitative analysis using ImageJ confirmed that NLB/PTX-HSA NPs achieved significantly higher intracellular accumulation than free C6 at all evaluated time points (Fig. 2B, **P < 0.01 at 2 h and 6 h, and **P < 0.0001 at 8 h). Furthermore, with increased intervention time, the C6/NLB/PTX-NPs group showed an even stronger fluorescence signal, indicating that the nanocarrier facilitated more efficient cellular uptake of hydrophobic drugs. Typically, free small molecules enter cells through passive diffusion. In contrast, albumin-based nanoparticles are primarily internalized via specific receptor-mediated endocytotic pathways. Tumor cells frequently overexpress Secreted Protein Acidic and Rich in Cysteine (SPARC) and glycoprotein 60 (gp60) receptors. The HSA carrier actively binds to these receptors, triggering caveolae- or clathrin-mediated endocytosis, which significantly enhances the intracellular accumulation of the loaded drugs compared to passive diffusion. Notably, the green fluorescence in the nanoparticle group was predominantly distributed around the blue-stained cells, suggesting that the nanoparticles were endocytosed by tumor cells and could exert their effects within the cytoplasm. While nanoparticles were successfully internalized by nearly 100% of visible cells within 8 h, minor variations in fluorescence intensity were observed among individual cells. This likely reflects the inherent biological heterogeneity of the population, where differences in cell cycle stages (e.g., S or G2/M phases) and the heterogeneous expression levels of SPARC/gp60 receptors modulate the specific rate of receptor-mediated endocytosis in individual cells.

Fig. 2.

Cellular uptake and semi-quantitative analysis. (A) Confocal laser scanning microscopy (CLSM) images of NCI-H520 cells treated with free Coumarin-6 (C6) and C6-loaded NLB/PTX-NPs for 2 h, 6 h, and 8 h. Blue fluorescence represents nuclei stained with DAPI, and green fluorescence represents C6. (B) Semi-quantitative analysis of mean fluorescence intensity (MFI) of Coumarin-6 signals using ImageJ. Data are presented as mean ± SD  n=3. P < 0.01, ****P < 0.0001 compared to the free C6 group at the corresponding time points

In vitro anti-tumor activity of NLB/PTX-NPs nanoparticles

In this study, we successfully synthesized human serum albumin nanoparticles (NLB/PTX-NPs) for the delivery of NLB and PTX and evaluated their in vitro anti-tumor effects on the lung squamous cell carcinoma line NCI-H520. We compared these effects with those of individually administered NLB, PTX, and a drug mixture (NLB/PTXmix). The cytotoxicity of each formulation on NCI-H520 cells was assessed using the CCK-8 assay. As shown in Fig. 3A and B, all treatment groups exhibited dose-dependent inhibition of cell growth, with the NLB/PTX-NPs group demonstrating the strongest cytotoxicity. After 24 h of treatment, the IC50 value of NLB/PTX-NPs (0.99 µg/mL) was lower than that of free NLB (25.70 µg/mL), free PTX (5.04 µg/mL), and NLB/PTX mix (3.44 µg/mL). After 48 h of treatment, the IC50 value of NLB/PTX-NPs was 0.08 µg/mL, which remained significantly lower than that of free NLB (4.41 µg/mL), free PTX (1.58 µg/mL), and NLB/PTX mix (0.90 µg/mL). This indicates that the nano-drug NLB/PTX-NPs significantly enhance the drug’s cytotoxicity. Furthermore, this study investigated the effect of NLB/PTX-NPs on the proliferative capacity of lung squamous cell carcinoma using EdU and colony-forming assays. The EdU assay results (Fig. 3C, D) indicated that the NLB/PTX-NPs group exhibited the most significant inhibition of cell proliferation. The colony formation assay (Fig. 3E, F) demonstrated that the colony formation rate was significantly lower in this group compared to the physical mixture group (NLB/PTXmix, P < 0.001). Both the NLB/PTX-NPs and NLB/PTXmix groups exhibited stronger inhibition of lung squamous cell carcinoma proliferation than the single-drug groups (NLB and PTX groups, P < 0.05). The Transwell migration assay results (Fig. 3G, H) demonstrated that NLB/PTX-NPs more effectively blocked the transmembrane migration ability of NCI-H520 cells compared to the NLB, PTX, and NLB/PTXmix groups (P < 0.01), which may be attributed to the nanoparticles’ higher cellular uptake efficiency and the synergistic effect of the dual drugs. Additionally, the Transwell assay (with Matrigel matrix) was used to evaluate the impact of each treatment group on the invasive behavior of lung squamous cell carcinoma cells (Fig. 3I). Quantitative analysis (Fig. 3J) revealed that the inhibitory effect of the NLB/PTX-NPs group on cell transmembrane behavior was significantly superior to that of the NLB/PTXmix group (P < 0.05).Consistent with the observed enhancement in cellular uptake, NLB/PTX-NPs demonstrated a significantly superior capacity to suppress tumor cell viability compared to both the physical mixture and monotherapies. This potent anti-proliferative effect was consistently validated across three dimensions: immediate metabolic inhibition (), DNA synthesis blockade (EdU assay, P < 0.01), and long-term survival suppression (Colony formation, P < 0.001). Furthermore, the Transwell assays provided compelling evidence that the synergistic delivery of Nilotinib and Paclitaxel through HSA nanoparticles effectively crippled the migratory and invasive behaviors of lung squamous carcinoma cells (P < 0.01), collectively confirming that the nanodelivery system significantly inhibits the key drivers of tumor progression and metastasis.

Fig. 3.

In vitro anti-tumor effects of NLB/PTX-NPs on NCI-H520 lung squamous carcinoma cells. (A, B) Cell viability of NCI-H520 cells after treatment with free NLB, free PTX, NLB/PTXmix, and NLB/PTX-NPs at various concentrations for 24 h (A) and 48 h (B), assessed by CCK-8 assay. (C) Representative EdU (green) and Hoechst 33,342 (blue) fluorescence staining images of NCI-H520 cells following different treatments for 24 h. (D) Statistical analysis of the percentage of EdU-positive cells. (E) Representative images of the colony formation assay. (F) Quantitative analysis of the number of colonies per well. (G, H) Transwell migration assay showing representative images of migrated cells (G) and quantitative analysis of the number of migrated cells per field (H). (I, J) Transwell invasion assay showing representative images of invaded cells through Matrigel (I) and statistical analysis of the number of invaded cells per field (J). Data are presented as mean ± SD (n = 3). Statistical significance is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Effects of NLB/PTX-NPs on apoptosis and epithelial-mesenchymal transition-related proteins in lung squamous cell carcinoma

To further elucidate the molecular mechanisms underlying the inhibition of cell proliferation and invasion by NLB/PTX-NPs, we examined the expression levels of apoptosis- and epithelial-mesenchymal transition (EMT)-related proteins in NCI-H520 cells through Western blot analysis. As demonstrated in Fig. 4C and D, treatment with NLB/PTX-NPs significantly upregulated the expression of the pro-apoptotic protein Bax while markedly downregulating the expression of the anti-apoptotic protein Bcl-2 (P < 0.05) compared to both the single-drug and free drug combination groups. This resulted in a significant increase in the Bax/Bcl-2 ratio, suggesting the activation of apoptotic signaling cascades at the protein level. Regarding the EMT process (Fig. 4A, B), treatment with NLB/PTX-NPs significantly upregulated the expression of E-cadherin while downregulating the expression of Vimentin and β-catenin (P < 0.05). Compared with the physical mixture group (NLB/PTXmix), the nanoparticle group showed more pronounced regulatory effects on these proteins, confirming that co-delivery via nanocarriers can enhance intracellular synergistic drug effects.

Fig. 4.

Regulatory effects of NLB/PTX-NPs on apoptosis and epithelial-mesenchymal transition (EMT)-related proteins in NCI-H520 cells in vitro. (A) Representative Western blot images showing the protein expression levels of EMT markers (E-cadherin, Vimentin, and β-catenin) in NCI-H520 cells after various treatments. GAPDH was used as the loading control. (B) Quantitative densitometric analysis of relative protein expression levels of E-cadherin, Vimentin, and β-catenin. (C) Representative Western blot images showing the protein expression levels of apoptosis-related markers (Bax and Bcl-2). GAPDH and β-actin were used as internal controls for Bax and Bcl-2, respectively. (D) Quantitative analysis of relative Bax and Bcl-2 protein expression. Data are presented as mean ± SD (n = 3). Statistical significance compared to the NLB/PTX-NPs group is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Biosafety evaluation

In vivo biosafety evaluation is a critical factor that determines the clinical applicability of nanomedicines. Consequently, we conducted an in-depth investigation into the biosafety of NLB/PTX-NPs.

Good blood compatibility is a prerequisite for the intravenous administration of nanomedicines. This study preliminarily evaluated the biosafety of NLB/PTX-NPs through hemolysis assays. As illustrated in Fig. 5A, deionized water (DI), used as the positive control, induced complete hemolysis, while no significant hemolysis was observed in the PBS negative control group. The free drug groups (NLB and PTX) exhibited substantial hemolytic activity, suggesting that their direct contact with blood may lead to erythrocyte membrane rupture. In contrast, both the HSA carrier and NLB/PTX-NPs groups demonstrated extremely low hemolysis rates, with the hemolysis rate of NLB/PTX-NPs maintained below 1% (Fig. 5B), significantly lower than the internationally recognized safety threshold for hemolysis (5%). These results strongly indicate that encapsulating the drug within nano-carriers effectively shields it from direct contact with red blood cells, thereby significantly reducing the hemolytic toxicity of the drug and greatly enhancing its biosafety.

Fig. 5.

Biocompatibility and safety evaluation of NLB/PTX-NPs. (A) Photographs of hemolysis assay tubes showing red blood cells incubated with deionized water (DI, positive control), PBS (negative control), free NLB, free PTX, HSA, and NLB/PTX-NPs. (B) Quantitative analysis of the hemolysis ratio. (C) Representative H&E staining images of major organs (heart, liver, kidney, spleen, and lung) from tumor-bearing mice after various treatments. Scale bar = 100 μm. (D) Serum biochemical analysis of liver function markers (ALT, AST), kidney function markers (UREA, CREA), and tissue damage markers (CK, LDH) in mice after treatment. Data are presented as mean ± SD (n = 3). No significant pathological changes or biochemical abnormalities were observed in the NLB/PTX-NPs group

At the end of the administration cycle, major organs (heart, liver, kidney, spleen, lung) and serum samples were collected from mice for detailed histopathological and blood biochemical analyses. H&E staining results of tissue sections (Fig. 5C) revealed that, compared to the control group, mice in the NLB/PTX-NPs group exhibited intact organ structures, normal cellular morphology, and no significant pathological changes, such as necrotic lesions, congestion, edema, or inflammatory cell infiltration in any major organs. These findings confirm, at the histomorphological level, that the nanoformulation did not cause significant organ damage. Further serum biochemical analysis (Fig. 5D) provided quantitative functional evidence. The results indicated that the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), reflecting liver function; creatinine (CREA) and urea (UREA), reflecting kidney function; as well as creatine kinase (CK) and lactate dehydrogenase (LDH), indicating myocardial injury, all remained within normal reference ranges without pathological elevation following NLB/PTX-NPs treatment. Combined histopathological and serum biochemical verification strongly demonstrates that NLB/PTX-NPs exhibit excellent in vivo biocompatibility, and the therapeutic dose does not cause significant toxic effects in critical organs such as the heart, liver, and kidneys in mice.

In vivo distribution and accumulation of drugs at tumor sites

To further investigate the in vivo distribution of NLB/PTX-NPs, NCI-H520 tumor-bearing nude mice were intravenously injected with ICG-labeled NLB/PTX-NPs via the tail vein. At 24 h post-injection, fluorescence distribution in various tissues and organs of the nude mice was detected using an in vivo imaging system (Fig. 6A). Analysis of fluorescence intensity across these tissues and organs (Fig. 6B and C) revealed that 24 h post-administration, the control group (free ICG) exhibited rapid systemic clearance, with nearly undetectable fluorescence signals in all organs except for a minimal presence in the liver. In contrast, the ICG-NLB/PTX-NPs group demonstrated prolonged retention and significantly higher accumulation in the kidneys, lungs, liver, and tumor tissues compared to the free ICG group (Fig. 6C, P < 0.05), while no significant differences were observed in the heart and spleen (ns). While minor variations in tumor fluorescence intensity were observed in Fig. 6A—specifically, the tumors of Mouse 2 and Mouse 3 displayed minimal signal due to biological heterogeneity in microvascular density—the overall mean accumulation in the tumor was significantly enhanced by the nanoparticles (Fig. 6B, P < 0.05). According to the established Enhanced Permeability and Retention (EPR) effect, nanoparticle accumulation is highly dependent on tumor development [27]. Preclinical models generally demonstrate optimal nanoparticle accumulation in medium-sized tumors (typically 100–300 mm³), where functional angiogenesis is highly active. In contrast, very small tumors lack mature leaky vessels, whereas overly large tumors develop high interstitial fluid pressure and necrotic cores that impede uniform nanoparticle perfusion [28].

Fig. 6.

In vivo biodistribution and organ targeting. (A) Ex vivo fluorescence images of major organs (heart, liver, spleen, lung, kidney) and tumors excised 24 h post-injection. (B) Quantitative analysis of total cumulative signal in tumor tissues (P < 0.05). (C) Grouped quantitative comparison of radiant efficiency across all individual organs and tumors between the Free ICG and ICG-NLB/PTX-NPs groups. Data are presented as mean ± SD  n=5. P < 0.05, ns: no statistical significance

Study on the Anti-Tumor Effects and Mechanisms of NLB/PTX-NPs in Vivo

Preliminary in vitro experimental results indicated that NLB/PTX-NPs exhibited enhanced anti-tumor activity compared to NLB, PTX, and NLB/PTXmix. To assess the in vivo anti-tumor efficacy of the nanodrug, this study established tumor-bearing mouse models and monitored tumor growth in real-time during drug administration. Tumor volume (Fig. 7A, B) and final tumor weight (Fig. 7C) demonstrated that, compared with the control group (Saline), both the free drug groups (NLB, PTX) and the physical mixture group (NLB/PTXmix) delayed tumor growth to some extent (P < 0.05). The NLB/PTX-NPs group exhibited the most pronounced tumor-inhibiting effect, with significantly smaller tumor volume and lower tumor weight compared to the physical mixture group (P < 0.05), thereby confirming that the nano-delivery system significantly enhanced the in vivo anti-tumor efficacy of the dual drugs.

Fig. 7.

In vivo anti-tumor efficacy and mechanistic evaluation of NLB/PTX-NPs in an NCI-H520 subcutaneous xenograft mouse model. (A) Representative photographs of excised tumors from different treatment groups at the end of the experiment (day 24). (B) Tumor growth curves monitoring changes in tumor volume over the 24-day treatment period (n = 5). (C) Average tumor weights measured at the endpoint (n = 5). (D) Representative Western blot images showing the expression levels of NF-κB p65, p-NF-κB p65, IκBα, and p-IκBα in the excised tumor tissues. GAPDH and β-actin were used as loading controls. (E) Quantitative densitometric analysis of relative protein expression levels from the Western blot assay (n = 3). (F) Quantitative real-time PCR (qRT-PCR) analysis of the relative mRNA expression levels of RELA, NFKBIA, BCL2, and MMP9 in the excised tumor tissues. GAPDH was used as the internal reference gene (n = 3). Data are presented as mean ± SD. Statistical significance compared to the NLB/PTX-NPs group is indicated as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

The Ki67 immunofluorescence staining results of tumor tissues  indicated that the control group exhibited high-intensity Ki67 fluorescence signals, reflecting a highly active proliferative state of the cells. In comparison to the control and monotherapy groups, the red fluorescence intensity was diminished in the NLB/PTXmix group, while the NLB/PTX-NPs group displayed the weakest fluorescence signals. Furthermore, the cell proliferation rate in the NLB/PTX-NPs group was significantly lower than that in both the NLB/PTXmix group (P < 0.05) and the other groups (P < 0.0001), thereby demonstrating that NLB/PTX-NPs can effectively inhibit the malignant proliferation of tumor cells in vivo.

The NF-κB signaling pathway serves as a critical hub regulating tumor cell proliferation, survival, and chemoresistance, with NF-κB being constitutively activated in most malignant tumors. To elucidate the molecular mechanism underlying the potent anti-tumor effects of NLB/PTX-NPs, this study extracted tumor tissue proteins from each group and analyzed the expression of the core effector protein p65 and its inhibitory protein IκBα in this pathway using Western blot analysis. The results (Fig. 7D, E) revealed significant differences in phosphorylation levels among the groups, contrasting sharply with the stable expression of total proteins. Compared with the control group, free-drug group, and physical-mixture group, NLB/PTX-NPs treatment significantly reduced the protein expression levels of p-NF-κB p65 and p-IκBα (P < 0.05). This finding suggests that the nanoformulation may inhibit the phosphorylation of IκBα, thereby preventing its subsequent proteasomal degradation and allowing it to continuously bind to the NF-κB p65 dimer, anchoring it in the cytoplasm. This mechanism further hinders the phosphorylation activation of p65, ultimately suppressing the transcription of NF-κB downstream pro-proliferative and anti-apoptotic target genes.

Furthermore, to further validate the functional blockade of the NF-κB pathway at the transcriptional level, qRT-PCR analysis was conducted on the tumor tissues (Fig. 7F). Notably, the relative mRNA expression levels of RELA (encoding the p65 subunit) and NFKBIA (encoding IκBα) remained stable across all treatment groups with no significant differences. This robustly aligns with our Western blot findings (Fig. 7D), where the total protein pools of p65 and IκBα were unaltered. These data collectively indicate that NLB/PTX-NPs do not disrupt the basal transcription machinery of the core pathway components, but rather act precisely at the post-translational level by markedly inhibiting their phosphorylation activation. Crucially, as a direct consequence of this phosphorylation blockade and halted nuclear translocation of NF-κB, the transcription of its classical downstream effector genes was significantly suppressed. Specifically, the mRNA levels of the anti-apoptotic gene BCL2 and the metastasis-promoting gene MMP9 were significantly downregulated in the NLB/PTX-NPs group compared to the control and physical mixture groups. This provides compelling transcriptional evidence that the nanodelivery system effectively silences the NF-κB signaling cascade to inhibit tumor progression.

The anti-tumor metastasis effect of NLB/PTX-NPs in vivo

To evaluate the inhibitory effect of NLB/PTX-NPs on tumor metastasis, this study established a mouse lung metastasis model. Following the completion of the treatment cycle, the lung tissues were dissected for observation. Gross examination of the lungs (Fig. 8A) and statistical analysis of nodule counts and lung weights (Fig. 8B, C) post-treatment revealed that the lungs of the control group mice were densely covered with milky-white metastatic nodules. The average number of nodules in the control group was 23, whereas the NLB and PTX monotherapy groups had averages of 17 and 16, respectively. The NLB/PTXmix group had an average of 13 nodules, which still differed from the NLB/PTX-NPs group. Notably, the average number of nodules in the NLB/PTX-NPs group decreased to fewer than 8, which was significantly lower than that of the control group and demonstrated a statistically significant difference compared to the NLB/PTXmix group (P < 0.01). These data strongly indicate that the nano-formulation can effectively inhibit the metastasis and growth of tumor cells in the lungs.

Fig. 8.

In vivo anti-metastatic efficacy of NLB/PTX-NPs in a lung metastasis mouse model. (A) Representative macroscopic photographs of excised lungs from mice in different treatment groups. Tumor nodules appear as white, raised spots on the lung surface. (B) Quantitative analysis of the number of metastatic tumor nodules on the lung surfaces (n = 5). (C) Statistical analysis of the excised lung weights from each group, serving as an indicator of metastatic tumor burden (n = 5). (D) Representative hematoxylin and eosin (H&E) staining images of lung tissue sections. Upper panels show low-magnification images (100×), and lower panels show high-magnification views (200×) of the corresponding boxed areas, illustrating the preservation of alveolar structures in the NLB/PTX-NPs group versus extensive tumor infiltration in the control group. Data are presented as mean ± SD. Statistical significance compared to the NLB/PTX-NPs group is indicated as *P < 0.05, **P < 0.01, and ****P < 0.0001

To further validate therapeutic efficacy at the microscopic level, we performed HE staining of lung tissues (Fig. 8D). Sections from both the control and monotherapy groups exhibited extensive solid tumor tissue (indicated by darkly stained areas) within the pulmonary parenchyma, characterized by tightly packed tumor cells with high nuclear-to-cytoplasmic ratios. The normal alveolar architecture was severely disrupted or compressed, indicating a high degree of invasiveness. Although the NLB/PTXmix group demonstrated a reduction in the tumor foci area, distinct micro-metastases and inflammatory infiltration were still observed. In contrast, the NLB/PTX-NPs group showed that most fields of view retained clear and intact alveolar structures (reticular patterns), with only occasional small clusters of tumor cells visible and no significant tissue necrosis or hemorrhage detected. This histopathological analysis further confirms the superior inhibitory effect of NLB/PTX-NPs on lung metastasis. NLB/PTX-NPs exhibit excellent anti-metastatic activity by reducing the number of pulmonary metastatic nodules, inhibiting tumor lesion growth, and preserving normal alveolar structure.

Discussion

In recent years, lung cancer has emerged as one of the leading causes of mortality worldwide [29]. Non-small cell lung cancer (NSCLC) constitutes approximately 80% of all lung cancer cases, which includes lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), large cell lung cancer, and various rare tumors. Among these, LUSC is recognized as one of the most aggressive and metastatic malignancies [30]. In contrast to the rapid advancement of targeted therapies for LUAD, the treatment of LUSC continues to rely predominantly on conventional chemotherapy agents, such as paclitaxel, due to the absence of effective therapeutic targets. Paclitaxel, a non-selective anticancer agent, faces significant limitations in clinical application due to its low solubility and considerable toxic side effects. Furthermore, residual tumor cells activate multiple survival and escape signaling pathways, complicating the efficacy of single-agent chemotherapy in addressing the high invasiveness and propensity for early distant metastasis associated with LUSC. Previous investigations conducted by our research group have demonstrated that Nilotinib can effectively inhibit the proliferation and metastasis of esophageal squamous cell carcinoma (ESCC) by targeting integrin-linked kinase (ILK) and inducing oxidative stress. Beyond its established target, BCR-ABL, Nilotinib exhibits broad-spectrum inhibitory effects against kinases such as DDR1 and c-Kit, highlighting its significant anti-tumor potential in treating solid tumors, including gastrointestinal stromal tumors (GIST), colorectal cancer, and breast cancer [31]. In this study, we employed human serum albumin (HAS) to encapsulate two hydrophobic drugs, Nilotinib and paclitaxel, thereby enhancing drug accumulation in tumor tissues through binding with gp60 and SPARC proteins. This strategy improves therapeutic efficacy while mitigating toxicity, presenting a potential preclinical strategy for LUSC treatment.

This study demonstrates that the self-assembled nanoparticles NLB/PTX-NPs exhibit a high drug-loading capacity and excellent biocompatibility, characterized by their small size, good dispersibility, and stability. The stability of these nanoparticles is attributed to the strong amphiphilic encapsulation capacity of human serum albumin (HSA), suggesting that the formulation has good storage stability and meets stringent requirements for clinical translation regarding preparation, storage, and transportation [32]. Uptake experiments indicate that the intracellular fluorescence intensity in the C6/NLB/PTX-NPs group was significantly higher than that in the free control group, while the cytotoxicity of NLB/PTX-NPs surpassed that of the physical mixture group. Our nanocarrier notably enhances the uptake efficiency of hydrophobic drugs in lung squamous cell carcinoma cells. The EdU and plate clone assays demonstrated that NLB/PTX-NPs exert the strongest inhibitory effect on cell proliferation. Furthermore, the Transwell migration assay confirmed that this group most effectively obstructed transmembrane cell migration. This finding indicates that NLB/PTX-NPs fully exploit the synergistic advantages of the dual-drug combination, comprehensively suppressing the malignant biological behaviors associated with lung squamous cell carcinoma. Previous research has shown that HSA nanocarriers can overcome intracellular barriers to deliver encapsulated drugs to target cells, thereby achieving favourable therapeutic outcomes [33]. As demonstrated by Tatyana Kovshova et al., the co-delivery of paclitaxel and etoposide prodrugs using HSA and polylactic-co-glycolic acid nanoparticles exhibited synergistic cytotoxicity against brain tumor cells [34]. Inhibiting the proliferation and migration of tumor cells is a crucial strategy for controlling cancer progression [35, 36].

The nano co-delivery system can simultaneously intervene in multiple signaling pathways, thereby more effectively inhibiting tumor cell colony formation and invasive behavior compared to single-drug administration [37, 38]. Constructing a co-loaded drug system enables synergistic regulation of cell cycle and apoptotic proteins, thereby significantly attenuating the malignant phenotypes of cancer cells [39]. In this study, the NLB/PTX-NPs group markedly upregulated the pro-apoptotic protein Bax while simultaneously suppressing the anti-apoptotic protein Bcl-2, resulting in a significant increase in the Bax/Bcl-2 ratio, which exhibited superior effects compared to the physical mixture group. These biochemical changes suggest that NLB/PTX-NPs could modulate key regulators of the mitochondria-mediated apoptotic pathway, providing potential molecular evidence for the cytotoxicity observed in the CCK-8 assay. Although the significant modulation of the Bax/Bcl-2 ratio strongly suggests the activation of apoptotic pathways, a direct quantification of the apoptotic cell percentage via Annexin V/PI flow cytometry was not performed in this study due to current instrument limitations. Future investigations will incorporate flow-cytometric analyses to further substantiate these biochemical observations. Consistent with the findings of Melayshia McFadden et al., dual-drug-loaded nanoparticles demonstrated superior inhibition of cell proliferation and potential activation of cell death pathways compared to single-drug formulations. This effect was achieved through enhanced intracellular drug concentrations, significantly upregulating Bax while downregulating Bcl-2, thereby contributing to the promotion of tumor cell death [40]. Epithelial-mesenchymal transition (EMT) is a critical process that enables tumor cells to acquire motility and invasive capabilities, leading to distant metastasis and chemotherapy resistance [41, 42]. EMT endows cancer cells with mesenchymal characteristics, resulting in insensitivity to apoptosis and triggering drug resistance mechanisms such as drug efflux. For instance, EMT has been associated with EGFR-TKI resistance in lung adenocarcinoma [43]. The findings of this study indicate that NLB/PTX-NPs could modulate the EMT process, thereby suppressing the invasion and metastatic potential of lung squamous cell carcinoma cells.

Our findings suggest that NLB/PTX-NPs nanoparticles exhibit favorable biosafety and biocompatibility in the tested models. By establishing subcutaneous xenograft tumor models and lung squamous cell carcinoma metastasis models in nude mice, the research examined the in vivo anti-tumor growth and metastasis capabilities of NLB/PTX-NPs. Fluorescence in vivo imaging demonstrated that, compared to free ICG, NLB/PTX-NPs showed more effective accumulation at tumor sites. Utilizing the EPR effect and an active targeting strategy, the nanocarrier enhanced drug accumulation in tumor tissues, resulting in superior tumor inhibition effects compared to free drugs, consistent with multiple previous studies [44, 45]. Results from the subcutaneous xenograft model indicated that the NLB/PTX-NPs group exhibited the most significant tumor growth inhibition capability. After treatment, the tumor volume and mass in this group were significantly smaller than those in the physical mixture group and the single-drug group. The co-delivery system ensures that multiple drugs reach tumor tissue synchronously at the optimal ratio, contributing to enhanced therapeutic efficacy in vivo [46]. Effectively blocking distant metastasis is crucial for improving the prognosis of lung squamous cell carcinoma [47]. The results of this study revealed that the number of metastatic nodules on the lung surface and lung weight in the NLB/PTX-NPs group were considerably lower than those in the physical mixture group, with HE staining showing the most intact preserved alveolar structure in this group. This outcome is attributed to nanotechnology’s capability to simultaneously deliver multiple drugs, inhibiting the colonization of circulating tumor cells and thereby reducing the formation of pulmonary metastatic foci [48, 49].

The NF-κB signaling pathway serves as a critical hub regulating tumor cell proliferation, survival, inflammation, and chemoresistance [50]. This study investigated the regulatory effects of NLB/PTX-NPs on this pathway. Western blot results demonstrated that the NLB/PTX-NPs group significantly inhibited the phosphorylation level of IκBα, an upstream inhibitory protein of NF-κB, compared to the physical mixture group, along with a marked reduction in p65 phosphorylation levels. The NLB/PTX-NPs group exhibited a stronger capability to inhibit the pathway compared to the NLB/PTXmix group, which may be attributed to the nanocarrier’s role in promoting drug accumulation in tumor tissues, enhancing cellular uptake, and achieving synchronized intracellular release of both drugs, thereby generating a synergistic blocking effect on the NF-κB signaling cascade. The nano co-delivery system simultaneously administers chemotherapeutic drugs and molecular targeted agents, potentially modulating downstream NF-κB signal transduction and contributing to therapeutic efficacy. In non-small cell lung cancer (NSCLC), NF-κB is constitutively activated, and this aberrant activation drives malignant tumor progression while counteracting chemotherapy-induced cell death through the upregulation of anti-apoptotic genes [51]. Previous studies have confirmed that blocking the NF-κB pathway is an effective strategy to enhance chemotherapy sensitivity and inhibit tumor growth [52, 53]. Research has found that activation of the NF-κB pathway drives lymphatic metastasis in hepatocellular carcinoma patients [54]. Another study reported that the TNF-α/NF-κB pathway promotes T-cell activation in the tumor microenvironment and liver metastasis in colorectal cancer [55]. Additionally, Huang S.L. et al. demonstrated that inhibiting NF-κB activity reduces paclitaxel resistance in ovarian cancer cells [56].

The HSA-based nano co-delivery system developed in this study effectively integrates biocompatible carriers, synergizes chemotherapy, inhibits tumor growth and metastasis, and enhances biosafety, thereby providing a promising preclinical strategy to address the therapeutic bottleneck associated with lung squamous cell carcinoma (LUSC). However, despite these encouraging outcomes, certain limitations of the current work must be objectively acknowledged. First, it must be acknowledged that our in vitro evaluations were limited to the NCI-H520 cell line. While NCI-H520 is a well-established and representative model for human LUSC [57, 58], utilizing a single cell line in vitro restricts the generalizability of our findings. Future investigations incorporating multiple LUSC cell lines (such as H226 and SK-MES-1) and patient-derived primary tumor cells are warranted to validate the broad applicability of this co-delivery system. Second, the existing subcutaneous and tail-vein injection xenograft models do not fully replicate the complex immune microenvironment of human LUSC, highlighting the need for the establishment of orthotopic or humanized models to investigate the system’s role in reshaping the tumor immune microenvironment. Third, the pharmacokinetics (PK) of the two drugs in vivo and their optimal drug-loading ratio require further optimization through comprehensive pharmacokinetic studies. Finally, while preliminary biosafety was confirmed, long-term systemic toxicity and potential off-target effects must be rigorously evaluated in larger animal models before any clinical translation. In future research, a thorough exploration of these avenues will facilitate the advancement of this nano-system, ultimately offering more effective treatment options for patients with LUSC.

Author contributions

XW and LC contributed equally to this work and share first authorship. XW and LC conceived and designed the study. XW performed the in vitro and in vivo experiments, analyzed the data, and drafted the manuscript. LC focused on the synthesis and characterization of nanoparticles and assisted with animal modeling. YG provided technical support for imaging (TEM/CLSM) and cell culture. CQ assisted with statistical analysis and figure preparation. LZ and ZF provided laboratory resources, supervised the project, and critically revised the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.

Funding

This research was supported by Key Project of the Natural Science Foundation of Xinjiang Uygur Autonomous Region (Project No. 2023D01D15).

Data availability

All data generated or analysed during this study are included in this article. Any additional data related to this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.