Inhalable Herbal Nano-Liposomes Co-Delivering Astragaloside IV and Polyphyllin VII Reprogram Immunosuppressive Microenvironment To Overcome Anti-PD-1 Resistance in NSCLC

Abstract

Non-small cell lung cancer (NSCLC) frequently develops acquired resistance to immune checkpoint inhibitors (ICIs), necessitating innovative strategies to remodel the immunosuppressive tumor microenvironment (TME). This study engineered an inhalable pH-responsive nano-liposome co-delivering Astragaloside IV (AS-IV) and Polyphyllin VII (Pol VII) (AS-IV/Pol VII-Lipo) to overcome anti-PD-1 resistance via spatiotemporal-controlled dual-drug delivery. AS-IV/Pol VII-Lipo (1:1 mass ratio) exhibited optimal physicochemical properties: high drug loading and pH-triggered release. Nebulized inhalation achieved 3.4-fold higher lung accumulation than oral administration. Suppressed orthotopic LLC-Luc tumor growth by 54% and reduced exhausted CD8⁺ T cells while increasing cytotoxic CD8⁺Granzyme B⁺ T cells. Combination therapy further inhibited tumor metastasis and elevated survival. Transcriptomics (RNA-seq) identified suppression of IL-2/STAT5/BLIMP1 pathway and T-cell exhaustion genes. AS-IV/Pol VII-Lipo reprograms the immunosuppressive TME through three synergistic mechanisms: (1) enhanced lung-targeted drug delivery via inhalation; (2) reversal T-cell exhaustion through IL-2/STAT5/BLIMP1 pathway inhibition; (3) synergizing with αPD-1 therapy to overcome ICI resistance. This inhalable nanoplatform presents a promising clinical strategy for NSCLC patients with acquired immunotherapyresistance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12575-025-00302-4.

Background

Non-small cell lung cancer (NSCLC) constitutes 85% of global lung cancer diagnoses, positioning it as the dominant histological subtype [1, 2]. Despite substantial advances in the treatment of NSCLC in recent years, particularly through the application of targeted therapies and immunotherapies, the overall survival prognosis for NSCLC patients remains poor [3]. The therapeutic benefits of conventional chemo-radiotherapy in NSCLC remain constrained by their narrow therapeutic indices, where significant toxicities frequently offset survival gains [4, 5]. The recent emergence of immune checkpoint inhibitors (ICIs), particularly programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors, has heralded a paradigm shift in the therapeutic landscape of NSCLC [2]. These immunotherapies activate host immunity to selectively target tumor cells, demonstrating remarkable clinical efficacy in multiple pivotal trials with durable objective responses [6, 7]. Despite their efficacy, ICIs demonstrate clinical activity largely confined to tumors with high immunogenic potential [8]. Only approximately 20% of NSCLC patients achieve initial treatment response, and 61% of responders ultimately progress due to acquired resistance (AR) [9]. Furthermore, despite the paradigm-shifting success of chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies, its therapeutic efficacy against solid tumors remains suboptimal. This limitation stems principally from the absence of truly tumor-specific antigens compounded by profoundly immunosuppressive barriers within the tumor microenvironment (TME), which collectively dampen CAR-T cell trafficking, infiltration, and cytotoxic functionality [10]. Consequently, devising actionable strategies to surmount acquired resistance to immunotherapy has emerged as a pivotal research imperative in contemporary lung cancer investigation.

B lymphocyte induced maturation protein 1 (BLIMP1) determines the fate of CD8⁺ T cells through dual regulation: ① binding to the regulatory regions of Tcf7/Cxcr5 to block self-renewal of Stem-like/Progenitor exhausted T cells (Tprog cells); ② promoting the expression of effector molecules such as Granzyme B and exhaustion markers (e.g., TIM-3). The CD8αα⁺/αβ⁺ heterodimer is one of the characteristics of CD8⁺ T cells. B-cell lymphoma 6 protein (BCL6) deficiency leads to impaired differentiation and maturation of CD8⁺ T cells, accompanied by increased apoptosis [11], including inhibition of CD8⁺ T cell glycolysis, proliferation, and production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BLIMP1 competitively binds to the enhancer region of BCL6 through its zinc finger domain, blocking STAT5/SMAD2-mediated transcriptional activation of BCL6 [12]. TGF-β-SMAD2 signaling promotes BCL6 expression to maintain Tprog cells, while IL-2-STAT5 signaling induces BLIMP1 to drive terminally differentiated exhausted T cells (Tterm cells) differentiation. These two pathways antagonize each other to regulate T cell fate [13]. Preclinical studies have shown that BLIMP1 knockout or BCL6 overexpression can significantly enhance the efficacy of ICIs [12], but the risk of autoimmunity caused by systemic BLIMP1 inhibition requires caution. Clinical trials of TGF-β inhibitors combined with ICIs have preliminarily validated the regulatory value of this pathway. However, antitumor drugs specifically targeting BLIMP1 remain unavailable, urgently calling for the development of precise intervention strategies.

Small molecules derived from natural products constitute a pivotal source of innovative therapeutics. Traditional Chinese Medicine (TCM) holds immense potential for drug discovery, exemplified by artemisinin—a crown jewel of TCM development that has been honored with the Nobel Prize [14]. Fortifying host defense (Fu Zheng) and combating tumor pathogenesis (Qu Xie) constitute a fundamental therapeutic paradigm for NSCLC [15]. Polyphyllum rhizome (Chong Lou) and Astragalus root (Huang Qi) represent not only a clinically validated herb pair for fortifying host defense and combating tumor pathogenesis, but also serve as the primary active constituents of Jin Fu Kang (JFK) Oral Liquid—a China Food and Drug Administration (CFDA)-approved TCM formula for NSCLC adjuvant therapy [16]. In prior investigations, JFK has demonstrated significant therapeutic efficacy against NSCLC, consistently augmenting antitumor immunity—whether administered as monotherapy or integrated with standard chemotherapy regimens [17]. Polyphyllin VII (Pol VII) and Astragaloside IV (AS-IV), the principal bioactive constituents derived from Paris polyphylla and Astragalus membranaceus respectively, are recognized for their significant antitumor immunomodulatory potential. Consequently, Pol VII and AS-IV emerge as promising candidate agents for NSCLC therapeutics [18–20]. However, the therapeutic utility of AS-IV and Pol VII is severely compromised by their intrinsically poor aqueous solubility, suboptimal bioavailability, and inadequate lung-targeting specificity [21]. To address these limitations, we engineered a spatiotemporally synchronized co-delivery platform designed to potentiate NSCLC management—constituting the primary objective of this investigation [22, 23].

A pivotal challenge in NSCLC therapeutics lies in the suboptimal efficiency of conventional drug delivery routes, such as oral administration or intravenous infusion, are inefficient in delivering drugs to lung parenchyma and metastatic sites [24]. This inefficiency not only compromises therapeutic outcomes but also elevates risks of systemic adverse effects. Non-invasive pulmonary inhalation offers a direct, localized pathway to augment drug concentration within target tissues [25]. Nevertheless, this approach still faces significant hurdles, including the requirements for drug particle size and clearance mechanisms of pulmonary [24]. Optimal aerodynamic diameter is critical for maximizing the potential of lung deposition, with deposition diameters of less than 5 μm for adults and less than 3 μm for children [26]. Pulmonary clearance mechanisms, including mucus, cilia, and local macrophages, are the main factors affecting the deposition rate of aerosols in the lungs [27]. Therefore, designing drugs with appropriate aerodynamic diameters is necessary to achieve the best intrapulmonary deposition rate and improve the efficacy of inhalation therapy.

In this study, we developed a spatiotemporally controllable strategy to achieve combined therapy for lung cancer and AR through non-invasive inhalation of AS-IV/Pol VII-Lipo (Scheme 1). AS-IV/Pol VII-Lipo was prepared on a large scale under mild conditions. The non-invasive inhalation of AS-IV/Pol VII-Lipo offers a unique advantage, allowing liposomes to effectively accumulate in lung and tumor tissues. AS-IV/Pol VII-Lipo can sensitizing immunotherapy by inhibiting T cell exhaustion through a pH “trigger effect”. Meanwhile, the excellent antitumor effect of Pol VII enables it to effectively kill tumor cells. Tumor destruction releases tumor antigens, which are then processed by antigen-presenting cells and presented to activated CD8⁺ T cells. This process promotes the reuse of these activated CD8⁺ T cells, enhancing their ability to target and eliminate residual tumor cells. This dual-function strategy achieves spatiotemporal controlled combined therapy for lung cancer and AR, providing new hope for patients who have to discontinue immunotherapy due to AR.

Scheme 1.

Schematic depiction of utilizing inhalable AS-IV/Pol VII-Lipo to improve lung cancer immunotherapy. (A) Schematic diagram of the preparation of AS-IV/Pol VII-Lipo. (B) The pH-responsive AS-IV/Pol VII-Lipo, administered via nebulized inhalation, effectively accumulates in primary lung tumors. By suppressing T-cell exhaustion, it potently inhibits tumor growth and enhances the efficacy of lung cancer immunotherapy

Materials and Methods

Reagents, Cells Lines, and Animals

Some of the reagents, brands, and product numbers used in this study are as follows: AS-IV (Macklin, A928102), Pol VII (Chengdu Push Bio-Technology, PU0811-0025), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Avanti Polar Lipids, 850365P), cholesterol (Sigma-Aldrich, C8667), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-carboxylic acid (DSPE-PEG2000-COOH, Avanti, 880130P), 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA, Sigma-Aldrich, D6883), sodium dodecyl sulfate (SDS, Sigma-Aldrich, 71729), acetate-sodium acetate buffer (Biogradetech, A-CSH571), phosphate buffered saline (PBS, pH 7.4, Gibco, 10010023), 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine,4-Chlorobenzenesulfonate (DiD, Beyotine, C1039), cell counting kit-8 (CCK-8, Biosharp, BS350B).

Lewis lung cancer cells (LLC cells), Luciferase stable transfection cell line of mouse Lewis lung cancer cells (LLC-luc cells), mouse lung epithelial cells (TC-1 cell) and human bronchial epithelial cells (BEAS-2B cells) were provided by Cancer Institute, Shanghai Hospital of Traditional Chinese Medicine (Shanghai, China), and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Peiyuan Biological Technology Co., Ltd) medium, which was supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, USA) and 1% penicillin − streptomycin (Gibco, Thermo Fisher Scientific, USA). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂.

C57BL/6J and Balb/c mice (male, 6 − 8 weeks old) were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China). All animal experiments were evaluated and approved by the Experimental Animal Ethics Committee of Fudan University.

Preparation of AS-IV/Pol VII-Lipo

The pH-responsive AS-IV - Pol VII atomizable inhalable nano-liposomes (AS-IV/Pol VII-Lipo) were prepared by the thin-film hydration method according to the reported method with some modification [28]: (1) Dissolve DSPC (60 mg), cholesterol (30 mg), and DSPE-PEG2000-COOH (10 mg) in a chloroform-methanol mixture (3:1, v/v), and sonicate until completely dissolved. (2) Perform rotary evaporation (40 °C, 150 rpm, 30 min) to form a uniform lipid film, then vacuum-dry overnight. (3) Add the dried lipid film to a PBS buffer solution (pH 7.4, 10 mL) containing AS-IV and Pol VII (3:1, 2:1, 1:1, 1:2, 1:3, w/w, with a total mass of 8 mg), incubate in a 60 °C water bath for 1 h, and perform intermittent sonication (power: 200 W, pulse: 5 s on/5 seconds off). (4) Control the particle size using a microfluidic system (flow rate ratio: aqueous phase: organic phase = 3:1, total flow rate: 12 mL/minute) to obtain uniform liposomes. (5) React the liposome suspension with EDC/NHS (molar ratio 1:1.5) at 4 °C for 2 h to activate the carboxylic acid groups. (6) Add chitosan (0.1% w/v, pH 5.0) for cross-linking over 30 min to form a pH-sensitive surface coating. (7) Remove unencapsulated drugs by ultracentrifugation (100,000×g, 4 °C, 1 h), and resuspend in PBS to a final volume of 5 mL.

Characterization of AS-IV/Pol VII-Lipo

Encapsulation efficiency (EE%) and drug loading (DL%) of AS-IV/Pol VII-Lipo with varying component ratios were quantified by high-performance liquid chromatography (HPLC). The pH-responsive drug release behavior of AS-IV/Pol VII-Lipo was evaluated in vitro using pH 7.4 (0.01 M PBS containing 0.1% SDS, simulating physiological blood conditions) and pH 5.5 (0.01 M acetate-sodium acetate buffer with 0.1% SDS, mimicking the acidic tumor microenvironment) release media. The particle size and stability of the AS-IV/Pol VII-Lipo were measured by DLS (Nano ZS, Malvern Instruments, Ltd.).

The micromorphologies of the AS-IV/Pol VII-Lipo were characterized by TEM (JEM-1400 plus, JEOL). Detailed TEM samples were prepared as follows: First of all, dilute AS-IV/Pol VII-Lipo suspension to 0.1 mg/mL using ultrapure water to prevent particle aggregation. Next, Place 10 µL of diluted sample onto a carbon-coated copper grid (300 mesh, Ted Pella Inc.) and incubate for 1 min to allow liposome adsorption. Third, Wick away excess liquid with filter paper. Add 10 µL of 2% phosphotungstic acid (PTA, pH 7.0) for 30 s. Repeat staining twice for enhanced contrast. Finally, Air-dry grids for 10 min in a desiccator at 25 °C. Acquire images at 80 kV accelerating voltage.

Cell Viability Assay

To assess the cytotoxicity of AS-IV/Pol VII-Lipo with varying component ratios, TC-1 cells and LLC cells were seeded into a 96-well plate at a density of 5 × 10⁴/well. After culturing for 12 h, the culture medium was removed, and various ratios of AS-IV/Pol VII-Lipo (AS-IV: Pol VII (w/w) = 3:1, 2:1, 1:1, 1:2, 1:3) were added for culture for another 24 h. Afterwards, the cells were treated with CCK-8/culture medium (10 mL/100 mL) for an additional 1.5 h of incubation. The absorbance value at 450 nm (OD 450) of each well was measured by multifunctional microplate reader (Synergy H1, BioTek, USA).

In Vitro Safety Profiling of AS-IV/Pol VII-Lipo

The effect of liposomes on respiratory barrier function was evaluated using normal lung epithelial cells BEAS-2B. BEAS-2B cells were seeded into a 96-well plate at a density of 5 × 10⁴/well and divide them into the following 5 groups (n = 6): G1 (PBS), G2 (blank liposome, equivalent lipid concentration), G3 (AS-IV/Pol VII-Lipo : 50 µg/mL AS-IV + 40 µg/mL Pol VII), G4: free drug cocktail (50 µg/mL AS-IV + 40 µg/mL Pol VII in solution), G5: LPS-stimulated positive control (100 ng/mL). After culturing for 12 h, the culture medium was removed, add the corresponding drugs according to the above grouping, and incubate again for 24 h. Afterwards, the cells were treated with CCK-8/culture medium (10 mL/100 mL) for an additional 1.5 h of incubation. The absorbance value at 450 nm (OD 450) of each well was measured by multifunctional microplate reader.

Using the same grouping method, a lactate dehydrogenase (LDH) kit (Beyotime, C0016) was used to detect LDH activity in the supernatant to evaluate the degree of damage to cell membrane integrity. All experimental methods were performed according to the manufacturer’s protocol. Cytokines related to inflammation, including interleukin-6 (IL-6, MultiSciences Biotech, EK206HS), tumor necrosis factor α (TNF-α, MultiSciences Biotech, EK282HS) and interleukin-1β (IL-1β, MultiSciences Biotech, EK201BHS), were measured by enzyme-linked immunosorbent assay (ELISA) kits.

Intracellular ROS Generation by DCFH-DA Staining

BEAS-2B cells were co-incubated with blank liposome (equivalent lipid concentration), AS-IV/Pol VII-Lipo (50 µg/mL AS-IV + 40 µg/mL Pol VII), free drug cocktail (50 µg/mL AS-IV + 40 µg/mL Pol VII in solution), LPS-stimulated positive control (100 ng/mL) for 4 h, and PBS was used as a control. Afterwards, all the cells were stained with DCFH-DA (10 μm, 1 mL) for 30 min and with DAPI for 15 min. Confocal laser scanning microscopy (CLSM, Carl Zeiss LSM710 Zeiss, Germany) was used to capture fluorescence images of the cells.

In Vitro BMDMs Stimulation

Bone Marrow-Derived Macrophages (BMDM) were generated by culturing bone marrow cells flushed from the femurs of C57BL/6J mice in DMEM medium added with granulocyte-macrophage colony stimulating factor (GM-CSF) (10 ng/mL). The medium was half replaced every 2 days. After 7 days of culture, BMDMs were suspended in fresh medium in 12-well plates and stimulated with the corresponding drugs according to the pre-established groups. The cells were harvested after 24 h, and resuspended cells at 10⁶ cells/100 µL PBS. To reduce nonspecific Fc receptor binding, cells were blocked by anti-mouse CD16/CD32 (Biolgend, 101301) for 10 min. To analyze the polarization degree of BMDMs, an antibody cocktail was prepared in dark (per test): BV510 anti-mouse CD45 (Biolegend, 103137) 0.25 µg, FITC anti-mouse CD80 (Biolegend, 100705) 0.5 µg, PerCP-Cy5.5 anti-mouse CD206 (Biolegend, 141716) 1 µg. Added antibody cocktail and incubated at 4 °C for 30 min (light-protected). FlowJo software was used to analysis results.

In Vivo Safety Evaluation of Aerosol Inhalation of AS-IV/Pol VII-Lipo

BALB/c mice are sensitive to respiratory tract irritation, so they were selected for the safety evaluation of inhaled formulations. Thirty Balb/c mice were randomly divided into 5 groups to evaluate the in vivo safety of aerosol inhalation of AS-IV/Pol VII-Lipo (n = 6): G1: aerosolization with PBS; G2: Aerosolization with blank liposomes (blank liposomes at equivalent concentration); G3: AS-IV/Pol VII-Lipo (inh, AS-IV 5 mg/kg + Pol VII 4 mg/kg); G4: mixture of AS-IV 5 mg/kg + Pol VII 4 mg/kg (inh); G5: AS-IV/Pol VII-Lipo (iv, AS-IV 5 mg/kg + Pol VII 4 mg/kg). Hematological examinations including routine blood and serum biochemistry assays were conducted to make certain of the safety of AS-IV/Pol VII-Lipo.

For the acute toxicity test, 3 mice were randomly selected from each group. After being administered once according to the above protocol, 50 µL of blood was collected at specified time points (before administration, and on the 1 st, 3rd, and 7th days after administration) for the detection of white blood cells (WBC), red blood cells (RBC), platelets (PLT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN).

For the long-term local toxicity test of the respiratory tract, the remaining 3 mice in each group were given aerosol inhalation of drugs for 20 min daily according to the above grouping (aerosol particle size: 150 nm, flow rate: 0.3 mL/minutes) for 14 consecutive days; the intravenous group received tail vein injection once every 3 days. Body weight, activity status and mortality were monitored daily; after 28 consecutive days of monitoring, the mouse lung tissues, nasal mucosa, oral mucosa and tracheal mucosa were collected for H&E histopathological analysis.

Comparative Analysis of Tissue Distribution between Aerosol Inhalation and Oral Administration of AS-IV/Pol VII-Lipo

Twelve C57BL/6J mice were randomly divided into 3 groups to evaluate the in vivo drug distribution of aerosol inhalation versus oral administration of AS-IV/Pol VII-Lipo (n = 4): G1: aerosolization with PBS; G2: AS-IV/Pol VII-Lipo (i.g, AS-IV 5 mg/kg + PolVII 4 mg/kg); G3: AS-IV/Pol VII-Lipo (inh, AS-IV 5 mg/kg + PolVII 4 mg/kg). After aerosolization/oral administration of DiD-labeled liposomes, the fluorescence intensity in the lungs was detected using a small animal in vivo imaging system at 2 and 24 h (Ex/Em = 644/665 nm). Moreover, major organs (lung, heart, liver, spleen, kidney) harvested from the remaining three mice per group at 24 h post-administration were homogenized and subjected to HPLC analysis for quantification of AS-IV and Pol VII concentrations.

Comparative Analysis of Antitumor Immunity between Aerosol Inhalation and Oral Administration of AS-IV/Pol VII-Lipo

The orthotopic LLC-Luc lung cancer mouse model was established as follows: LLC-Luc at logarithmic growth phase were digested with 0.25% trypsin for 1 min, washed three times with PBS, and resuspended to form a single-cell suspension. After confirming > 95% viability via trypan blue exclusion assay, cells were adjusted to 1 × 10⁵ cells/100 µL. C57BL/6J mice were placed on a 45 °C heating pad for 5 min to induce tail vein dilation, restrained in a dedicated holder, and the tail surface disinfected with 75% ethanol. Using an insulin syringe with the bevel facing upward, the needle was inserted into the distal one-third of the tail vein at a 15° angle. The cell suspension (100 µL) was slowly infused over 60 s, followed by needle withdrawal and immediate hemostatic compression with sterile gauze for 30 s. They were randomly divided into 3 groups (n = 3): G1: aerosolization with PBS; G2: AS-IV/Pol VII-Lipo (i.g, AS-IV 5 mg/kg + PolVII 4 mg/kg); G3: AS-IV/Pol VII-Lipo (inh, AS-IV 5 mg/kg + PolVII 4 mg/kg). Starting from day 3 post-tumor inoculation, treatments were administered every 3 days. Tumor progression in the lungs was monitored via in vivo bioluminescent imaging on days 4 and 14. Concurrently, body weights were recorded every other day until day 24, when tumor-bearing mice were euthanized under anesthesia. Lung tissues were harvested for immunofluorescence co-staining of CD3, CD8 and Granzyme B to quantify tumor-infiltrating T cell proportions and activation status.

Aerosol Inhalation of AS-IV/Pol VII-Lipo Combined with αPD-1mAb Immunotherapy

Twelve C57BL/6J mice with orthotopic LLC-Luc lung cancer were randomly divided into 4 groups: G1: aerosolization with PBS; G2: αPD-1mAb (Leinco P504) (i.p, 10 mg/kg, Q3D); G3: AS-IV/Pol VII-Lipo (inh, AS-IV 5 mg/kg + PolVII 4 mg/kg, QD); G4: AS-IV/Pol VII-Lipo (inh, AS-IV 5 mg/kg + PolVII 4 mg/kg, QD) + αPD-1mAb (i.p, 10 mg/kg, Q3). Tumor progression in the lungs was monitored via in vivo bioluminescent imaging on days 4 and 24. Concurrently, body weights were recorded every other day until day 24, when tumor-bearing mice were euthanized under anesthesia. Lung tissues were harvested for immunofluorescence co-staining of CD3, CD8 and Granzyme B to quantify tumor-infiltrating T cell proportions and activation status. Lung tissues were collected for photographic documentation, followed by flow cytometric analysis of the following T-cell subsets: CD3⁺CD8⁺TIM-3⁺PD-1⁺ T cells, CD3⁺CD4⁺Foxp3⁺ T cells (regulatory T cells, Tregs), CD3⁺CD8⁺Granzyme B⁺ T cells (cytotoxic T lymphocytes, CTLs). All of the antibody, brands, and product numbers used in this study are as follows: BV510 anti-mouse CD3 (Biolegend, 100214), APC anti-mouse CD8 (eBioscience, 25-0081-82), PE-Cy7 anti-mouse TIM-3 (BioLegend, 119716), PE anti-mouse PD-1 (BioLegend, 135218), FITC anti-mouse CD4 (BioLegend, 100406), PerCP-e710 anti-mouse Foxp3 (eBioscience, 12-5773-82), AF647 anti-mouse Granzyme B (eBioscience, 12-8898-82). To further investigate the therapeutic mechanism of AS-IV/Pol VII-Lipo to the tumor, the transcriptome sequencing and 16 s rDNA sequencing of above-mentioned tumor tissue were performed.

Statistical Analysis

All results were shown as mean ± SD. Analyses were performed by using GraphPad Prism version 9.5.1 software. Comparisons of parameters among three or more groups were made using one-way ANOVA for single-factor variables followed by Tukey’s multiple comparisons test. Tumor growth between different groups was analyzed by two-way ANOVA, followed by Dunnett’s multiple comparisons test. Statistical significance was accepted at a p-value of < 0.05.

Results and Discussion

Preparation and Characterization of AS-IV/Pol VII-Lipo

In view of the poor water solubility and easy off-target effect of single drugs, we constructed a novel inhalable pH-responsive AS-IV/Pol VII-Lipo liposome. The applicant prepared pH-responsive dual-drug co-loaded liposomes with different ratios of AS-IV: Pol-VII (3:1, 2:1, 1:1, 1:2, 1:3, w/w, total mass 8 mg) by the thin-film hydration method. The detection results of particle size, potential, polydispersity index (PDI), dynamic light scattering (DLS), entrapment efficiency (EE%) and drug loading (DL%) of different liposomes are shown in Table 1. The PDI of all ratios of liposomes was less than 0.2, indicating good uniformity. However, when the particle size decreased, the PDI decreased accordingly, and the liposomes tended to have higher uniformity.

Subsequently, we applied LLC and TC-1 cells to perform toxicity assays on liposomes with different ratios. CCK-8 results showed that at the same liposome concentration (Fig. 1A), cell viability decreased as the AS-IV: Pol-VII ratio increased, which is attributed to the specific cytotoxicity of Pol VII against lung cancer cells. The 1:1 AS-IV: Pol VII ratio yielded liposomes with optimal aerodynamic diameter (176.6 ± 2.01 nm, PDI 0.155) for alveolar deposition, near-neutral surface charge (−15.1 ± 1.5 mV) to evade macrophage clearance, and high dual-drug loading (AS-IV DL% 12.6 ± 0.02, Pol VII DL% 11.9 ± 0.51); This ratio demonstrated > 79% viability in normal lung cells. The mass ratio corresponds to a charge-balanced molar ratio, enabling pH-triggered co-release in tumor microenvironments. Thus, the 1:1 formulation was selected for research object to ensure minimal damage to the body while activating anti-tumor immunity. The nebulized inhalable AS-IV/Pol VII-Lipo prepared with AS-IV: Pol-VII (1:1) showed a light blue opalescence and a translucent appearance without visible precipitation (Fig. 1B). Transmission electron microscopy (TEM) observation (Fig. 1C) revealed that AS-IV/Pol VII-Lipo exhibited a spherical morphology with a smooth surface and intact bilayer structure, consistent with the typical morphology of pH-responsive liposomes reported in the literature, without obvious rupture. The particle size under the microscope was approximately 150 nm, close to the results detected by the particle size analyzer, indicating successful preparation of AS-IV/Pol VII-Lipo.

Fig. 1.

Characterization of pH-responsive AS-IV/Pol VII-Lipo. (A) The in vitro safety of AS-IV/Pol VII-Lipo prepared at varying mass ratios (AS-IV: Pol VII = 3:1, 2:1, 1:1, 1:2, 1:3) was evaluated using TC-1 and LLC cell models (n = 6). (B) Successfully prepared pH-responsive AS-IV/Pol VII-Lipo suspension. (C) Representative TEM image of pH-responsive AS-IV/Pol VII-Lipo (bar = 100 nm). (D) Monitoring of the hydrodynamic diameter of pH-responsive AS-IV/Pol VII-Lipo at 4 °C and 25 °C for 21 consecutive days (n = 6). (E) The cumulative release profile of AS-IV from pH-responsive AS-IV/Pol VII-Lipo was measured under pH 5.0 and pH 7.4 conditions. (F) The cumulative release profile of Pol VII from pH-responsive AS-IV/Pol VII-Lipo was measured under pH 5.0 and pH 7.4 conditions

Subsequently, the nebulized inhalable AS-IV/Pol VII-Lipo prepared with AS-IV: Pol-VII (1:1) was stored at room temperature (25 °C) and 4 °C, and its storage stability was evaluated by detecting particle size changes. As shown in Fig. 1D, the particle size of AS-IV/Pol VII-Lipo remained stable at approximately 176 nm within 21 days, indicating no aggregation and good stability under both 25 °C and 4 °C conditions. Finally, in the simulated tumor microenvironment (pH 5.0), the cumulative release rates of AS-IV and Pol VII reached 67.6% and 63.7% within 24 h, respectively, while under physiological conditions (pH 7.4), the release rates were only 47.8% and 41.1% (Figs. 2E, F), confirming the pH responsiveness of the system. The similarity of the release profiles of the two drugs (f2 factor = 75.8) indicated significant synchronous release characteristics, which may enhance the anti-tumor efficacy through synergistic effects.

Fig. 2.

In vivo biocompatibility assessment of pH-responsive AS-IV/Pol VII-Lipo. Analysis of WBC (A), RBC (B), PLT (C), AST(D), ALT (E), BUN (F), and body weight (G) in mice under different treatment conditions (n = 6). (H) Histopathological analysis with H&E staining of murine lung (bar = 500 μm), nasal cavity (bar = 50 μm), trachea (bar = 100 μm), and oral mucosa (bar = 200 μm) under different treatment conditions. *p < 0.05, **p < 0.01

In conclusion, this study successfully constructed pH-responsive AS-IV/Pol VII-Lipo liposomes. Their high entrapment efficiency, controlled release, and excellent stability lay the foundation for subsequent in vivo anti-tumor research. The rapid release characteristics triggered by the acidic microenvironment can precisely target tumor tissues and reduce systemic toxicity. In the future, it is necessary to further verify the synergistic efficacy and targeting efficiency in animal models, and optimize the freeze-drying process to extend the storage period.

Safety Assessment of Nebulized AS-IV/Pol VII-Lipo

This study systematically evaluated the local and systemic toxicity risks of nebulized inhalable AS-IV/Pol VII-Lipo liposomes through comprehensive in vitro and in vivo experiments. As shown in Figure S1A, after treating BEAS-2B cells with AS-IV/Pol VII-Lipo in Group G3 for 24 h, the cell viability was 92.3% ± 3.1%, significantly higher than that of the free drug group (61.2% ± 4.5%, p < 0.01), indicating that liposomal encapsulation effectively reduced the direct toxicity of the drug to normal lung epithelial cells. Similarly, the LDH release rate in the AS-IV/Pol VII-Lipo liposome group was 42.7% ± 1.2%, that in the blank liposome group was 30.5% ± 1.0%, while that in the free drug group reached 62.4% ± 2.1% (Figure S1B), suggesting that the liposomes reduced cell membrane damage through their sustained-release properties.

Subsequently, macrophages were used to evaluate the regulation of inflammatory and immune responses by drugs in each group. The levels of IL-6, TNF-α, and IL-1β in the liposome group were 30.2 ± 4.1 pg/mL, 35.6 ± 3.2 pg/mL, and 221.6 ± 2.8 pg/mL, respectively, significantly lower than those in the free drug group (IL-6: 52.5 ± 6.3 pg/mL, TNF-α: 61.4 ± 5.7 pg/mL, IL-1β: 356.7 ± 2.8 pg/mL, p < 0.01), demonstrating that liposomal delivery could reduce the risk of excessive immune activation (Figures S1C-E). Reactive oxygen species (ROS) detection showed that the DCFH-DA fluorescence intensity in the liposome group was significantly lower than that in the positive control group (Figure S1F), confirming that liposomal delivery did not induce a strong oxidative stress response. In addition, flow cytometry showed that the M1/M2 ratio (CD80⁺CD206⁻/CD206⁺CD80⁻) of macrophages in the liposome group was 2.88 ± 0.3, significantly lower than that in the LPS positive control group (7.45 ± 0.5, p < 0.001) (Figure S1G), indicating that liposomal delivery had the ability to inhibit excessive inflammatory responses.

Subsequently, 30 Balb/c mice were randomly divided into 5 groups to evaluate the in vivo safety of aerosol inhalation of AS-IV/Pol VII-Lipo: G1: normal saline atomization; G2: blank liposome atomization (equivalent concentration of blank liposomes); G3: AS-IV/Pol VII-Lipo atomization (AS-IV 10 µg/mL + Pol VII 8 µg/mL); G4: free drug atomization group (AS-IV 50 µg/mL + Pol VII 40 µg/mL mixture); G5: AS-IV/Pol VII-Lipo intravenous injection group: AS-IV 5 mg/kg + PolVII 4 mg/kg). As shown in Fig. 2A-C, the white blood cell count and red blood cell count in the aerosol inhalation AS-IV/Pol VII-Lipo group had no significant difference from the control group, and no thrombocytopenia (PLT > 800 × 10^9/L) occurred, suggesting no risk of bone marrow suppression. The levels of ALT, AST, and BUN (Fig. 2D-F) in the intravenous injection group showed a small peak on the 1 st day after administration, while ALT, AST, and BUN in the atomization group were all within the normal range, confirming that aerosol inhalation can reduce systemic toxicity. In addition, the 28-day survival rate of all groups was 100%. Only the intravenous injection group showed a slight weight loss one week after administration (Fig. 2G), and the difference was not statistically significant. In addition, during the entire administration process, the mice in all groups had normal food intake and activities, without wheezing or hunchback, indicating no obvious acute toxicity within the therapeutic dose. Among them, only the naked drug atomization group showed a transient increase in respiratory rate (days 3–5), suggesting that liposome encapsulation can reduce the direct stimulation of the drug to the airway. H&E staining (Fig. 2H) showed that there was no inflammatory cell infiltration in the alveoli, nasal cavity, oral mucosa, and bronchi of the AS-IV/Pol VII-Lipo atomization group, which was attributed to the slow-release property of liposomes to reduce the transient impact of the drug on the mucosa.

In summary, AS-IV/Pol VII-Lipo showed excellent local and systemic safety compared with free drugs. Its mechanism involves the slow-release properties and the restriction of the lung-blood barrier. In the future, it is necessary to further verify the long-term safety by combining dynamic monitoring of lung function.

Aerosol Inhalation of AS-IV/Pol VII-Lipo Demonstrates Better Antitumor Effect than Oral Administration

To more comprehensively reveal how changing the administration route and dosage form can enhance the anti-lung cancer effect, we first compared the pulmonary tissue enrichment effect of aerosol inhalation versus oral administration. As shown in Fig. 3A, after fluorescent labeling of the drug, the fluorescence intensity in the lung tumor area of the aerosol group was significantly higher than that of the oral group 2 h after administration, and this remained true at 24 h. Additionally, HPLC detection of drug concentrations in lung tumor tissues 24 h after aerosol inhalation of AS-IV/Pol VII-Lipo showed AS-IV at 12.3 ± 1.8 µg/g and Pol VII at 9.7 ± 1.2 µg/g in the aerosol group, both higher than those in the oral group (Fig. 3B). This difference stems from aerosol inhalation directly delivering the drug through the alveolar-capillary barrier, avoiding gastrointestinal degradation and the first-pass effect of the oral route, which increases bioavailability by approximately 3–4 times. This is consistent with literature reporting the high permeability and retention of aerosolized drugs in tumor neovessels.

Fig. 3.

Comparative analysis of tissue distribution and antitumor immunity between nebulized inhalation and oral administration of AS-IV/Pol VII-Lipo. (A) Drug distribution fluorescence imaging of pulmonary, cardiac, hepatic, splenic, and renal tissues at predetermined time points following PBS, oral gavage, or nebulized administration of DiD-labeled pH-responsive AS-IV/Pol VII-Lipo. (B) Quantitative analysis of AS-IV and Pol VII concentrations in pulmonary tissue at 24 h post-administration by HPLC. (C) Schematic diagram of experimental design: Orthotopic lung cancer model was established by tail vein injection of LLC-Luc cells, followed by group-based treatments administered every 3 days. (D) Bioluminescence imaging of pulmonary metastatic tumors in mice under different treatments at days 4 and 14 post-inoculation. (E) Body weight in different treatment groups over time. (F) Representative DAPI (blue), CD3 (purple), CD8 (green), Granzyme B (red) expressions were detected in LLC-Luc tumor tissue by CLSM; bar = 100 μm. ***p < 0.001; ns means no statistical difference

Subsequently, we conducted experiments on the anticancer effects of different administration routes. First, an orthotopic lung cancer animal model was successfully established by intravenous injection of LLC-luc lung cancer cells into C57BL/6J mice, which were then randomly divided into three groups: control group, gavage group, and aerosol inhalation group, with administration performed according to the grouping (Fig. 3C). A comprehensive analysis was performed on the differences in antitumor effects between aerosol inhalation and oral administration of AS-IV/Pol VII-Lipo. In vivo imaging showed that the fluorescence intensity of chest tumor tissues in mice of the aerosol inhalation AS-IV/Pol VII-Lipo group was significantly lower than that in the oral group, indicating a stronger antitumor effect of aerosol inhalation (Fig. 3D). During the experiment, mouse body weight was monitored, and AS-IV/Pol VII-Lipo was found to have no effect on mouse body weight (Fig. 3E). Furthermore, immunofluorescence staining of lung tissues (Fig. 3F) showed higher immunofluorescence enrichment of CD3⁺, CD8⁺, and Granzyme B⁺ in the aerosol inhalation group, suggesting that this group better improved the immunosuppressive state of mice. Representative CLSM images acquired using a Zeiss LSM 710 microscope with 20× objective (scale bar: 100 μm).

Aerosol Inhalation of AS-IV/Pol VII-Lipo Synergizes with αPD-1 mAb Immunotherapy To Enhance Efficacy

To comprehensively reveal the anti-tumor immune effect of Fuzheng Juandu Chinese medicine, we investigated the synergistic anti-tumor effect of aerosol inhalation of AS-IV/Pol VII-Lipo and αPD-1 mAb. An orthotopic tumor animal model was established by tail vein injection of LLC-luc cells into mice. According to previous studies, the mice were randomly divided into four groups: control group (normal saline atomization), aerosol inhalation AS-IV/Pol VII-Lipo group, αPD-1 mAb group (i.p.), and aerosol inhalation AS-IV/Pol VII-Lipo + αPD-1 mAb combination therapy group, with administration performed according to the grouping (Fig. 4A).

Fig. 4.

Aerosol inhalation of AS-IV/Pol VII-Lipo synergizes with αPD-1 mAb immunotherapy to enhance efficacy. (A) Schematic diagram of experimental design: Orthotopic lung cancer model was established by tail vein injection of LLC-Luc cells, followed by group-based treatments administered. Flow cytometry analysis of lung tissues infiltrating CD3⁺CD8⁺TIM-3⁺PD-1⁺ T cells (B), CD3⁺CD4⁺Foxp3⁺ T cells (C), CD3⁺CD8⁺Granzyme B⁺ T cells (D). (E) Bioluminescence imaging of pulmonary metastatic tumors in mice under different treatments at days 4 and 24 post-inoculation. (F) Gross specimens of pulmonary tissues and quantitative statistical analysis of lung metastatic tumors. (G) Body weight in different treatment groups over time. Aerosol inhalation of enhances the anti-lung cancer effect of αPD-1 mAb. *p < 0.05, **p < 0.01, and ***p < 0.001; ns means no statistical difference

Fourteen days after administration, flow cytometry was used to detect changes in immune cells in mouse lung tissues. It was found that aerosol inhalation of AS-IV/Pol VII-Lipo similarly inhibited the infiltration proportions of exhausted CD8⁺ T cells (PD-1⁺Tim-3⁺) and Treg cells (CD4⁺Foxp3⁺), while increasing the infiltration proportion of CD8⁺Granzyme B⁺ T cells in the tumor microenvironment (Fig. 4B, C, D). In vivo imaging during the experiment showed that the aerosol inhalation AS-IV/Pol VII-Lipo group could inhibit tumor formation in mice, and this effect was amplified after combined αPD-1 mAb treatment (Fig. 4E). When the survival viability of mice in the control group was observed to weaken, the mice were anesthetized and euthanized. Lung tissues from each group were photographed (Fig. 4F), and lung metastatic tumors were counted (Fig. 4G). The results showed that the combination of aerosol inhalation AS-IV/Pol VII-Lipo and αPD-1 mAb had the best effect in inhibiting lung tumor formation in mice, indicating that AS-IV/Pol VII-Lipo could also improve the immunosuppressive state of mice and sensitize lung cancer immunotherapy. Mouse body weight was monitored during the experiment, and no significant effect on body weight was observed in any group (Fig. 4H).

These results suggest that aerosol inhalation of AS-IV/Pol VII-Lipo can enhance the immune status of CD8⁺ T cells in the tumor immune microenvironment of mice, thereby sensitizing αPD-1 mAb immunotherapy.

Nebulized AS-IV/Pol VII-Lipo Remodels metabolic-immunological Crosstalk in Lung Cancer

The above results suggest that the mechanism by which AS-IV/Pol VII-Lipo sensitizes lung cancer immunotherapy is related to the activation and infiltration of T cells. Using RNA-seq high-throughput sequencing technology, we detected genome-wide changes in gene transcription levels in tumor-bearing lung tissues of mice in the control group and those treated with AS-IV/Pol VII-Lipo. Analysis of the sequencing results was performed by screening genes with a fold change of differential gene expression ≥ 1.5 or ≤ 0.67. Compared with the control group, 2360 genes changed after AS-IV/Pol VII-Lipo treatment, among which 1120 genes were upregulated and 1240 genes were downregulated (Figure S2A). Subsequently, the 20 genes with the most significant differences were screened out as shown in Figure S2B. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted on the genes with altered expression in lung tissues after AS-IV/Pol VII-Lipo treatment. The results indicated that the enriched pathways were related to metabolism-immune response pathways (p < 0.001), including T cell-related signaling pathways, cytokine-cytokine receptor interaction pathways, TGF signaling pathways, IL-2 signaling pathways, etc. (Figure S2 B). Through KEGG pathway enrichment analysis, we clustered the differential genes into transcription factors. The results suggested that AS-IV/Pol VII-Lipo treatment significantly inhibited the expression of BLIMP1 and the transcription levels of various proteins related to the IL-2 signaling pathway axis (Figures S2C, D). Western blot results showed that AS-IV/Pol VII-Lipo significantly inhibited the expression of BLIMP1 in T cells and also inhibited the phosphorylation of pSTAT5 in T cells (Figure S2E). The qPCR technique was used to verify the above analysis results, and the same results were obtained (Figure S2 F). This phenomenon was not verified in lung cancer cells. The above results suggest that AS-IV/Pol VII-Lipo mainly acts on T cells.

Conclusion

Based on the results presented above, we have successfully prepared pH-responsive AS-IV/Pol VII-Lipo. While acute biosafety is confirmed (Figure S1 and Fig. 2), chronic exposure implications require further validation. The DSPC/cholesterol matrix degrades efficiently in lysosomal conditions (t₁/₂ = 9.2 days) [29, 30], and hepatic clearance predominates for 150–200 nm nanoparticles [31]. Potential anti-PEG immunity remains a concern for repeat dosing; future formulations may incorporate cleavable PEG or reduce density to < 5 mol% [32]. Our planned 90-day GLP study will track serum lathosterol (endogenous cholesterol synthesis marker) and TGF-β to preempt fibrotic risks. Furthermore, we can preliminarily draw the following conclusions: AS-IV/Pol VII-Lipo, by modulating the IL-2/STAT5/BLIMP1 signaling pathway and inducing reshaping of the immune microenvironment, effectively reversed acquired resistance to immunotherapy in NSCLC, demonstrating its potential in anticancer therapy. Specifically, AS-IV/Pol VII-Lipo suppresses IL-2/STAT5/BLIMP1 signaling, enhances T cell proliferation and cytotoxicity, thereby restoring anti-tumor immune responses and reversing immune resistance. These findings are important for developing new immunotherapy strategies, particularly for patients resistant to current treatment methods. The development of AS-IV/Pol VII-Lipo offers a possible solution to overcoming resistance to immunotherapy, especially in complex cases of NSCLC resistance caused by T cell.

Although our findings hold potential clinical application value, this study has several limitations. Firstly, the limited number of transcriptome sequencing samples used may affect the statistical robustness and generalizability of the conclusions. Secondly, despite the promising treatment prospects in in vivo and in vitro experiments, these results need validation in larger-scale clinical studies. Furthermore, the long-term effects and safety of AS-IV/Pol VII-Lipo require further confirmation in broader pre-clinical studies. Additionally, the complexity of the IL-2/STAT5/BLIMP1 pathway and its roles in various cell types and physiological processes necessitate deeper investigation into the specific mechanisms and impacts of AS-IV/Pol VII-Lipo in future studies.

In summary, this study showcases the potential of AS-IV/Pol VII-Lipo in treating NSCLC and underscores the importance of in-depth research into drug delivery systems in modulating the immune microenvironment. These achievements contribute to understanding the mechanisms of action of AS-IV/Pol VII-Lipo and offer crucial biological insights for future drug development and treatment strategies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

Yao Liu performed experiments. Zujun Que and Wenfei Shiperformed the analysis. Qitian An and Bin Luo prepared figures. Yao Liu wrote the manuscript, which was reviewed by all the authors. Jianhui Tian acquired funding for this study and together with Yiyang Zhou supervised the work. All authors made substantial, direct and intellectual contribution to the research. All authors read and approved the final manuscript.

Funding

This study was supported by the Shanghai 2024 “Science and Technology Innovation Action Plan” kick-off star cultivation (24YF2741300).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics Approval and Consent to Participate

All animal experiments were approved by the Animal Experimentation Ethics Committee of Shanghai Traditional Chinese Medicine Hospital and conducted by the Guide for the Care and Use of Laboratory Animals (approval number: SYXK2020-0014).

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.