Combination Therapeutic Effect of Asiatic Acid & Curcumin-Loaded Liposomes Modified by Neutrophil Extracellular Traps for the Treatment of Oral Squamous Cell Carcinoma

Abstract

Background

Tumor metastasis has been proven to represent the predominant cause of cancer-related mortality; therefore, the strategies simultaneously eradicating primary lesions and blocking dissemination cascades hold revolutionary potential in curative oral squamous cell carcinoma (OSCC) therapy.

Methods

In this study, we designed novel neutrophil extracellular traps (NETs)-coating liposomes (CUR@AA-NLIP) for co-delivery of curcumin (CUR) and asiatic acid (AA), in which the introduction of NETs was expected to improve tumor-targeting ability, and the combination of CUR and AA was used to synergistically inhibit tumor development.

Results

The characteristics analysis showed that CUR@AA-NLIP exhibited uniform size, phospholipid bilayer structure, and sustained drug release. In vitro studies demonstrated that CUR@AA-NLIP enhanced tumor cell uptake and macrophage escape, inhibited fibroblast activation and vessel tube formation, showing enhanced anti-proliferation and anti-migration capacities. In vivo results confirmed that CUR@AA-NLIP increased drug accumulation in the tumor region, showed enhanced anti-tumor efficacy (> 80% tumor suppression), and reduced lung metastasis.

Conclusion

CUR@AA-NLIP holds great potentials in OSCC-targeting delivery of synergistic natural compounds and can achieve anti-proliferative and anti-metastatic effects by simultaneously enhancing tumor accumulation, reducing immune system clearance, and inhibiting of cancer-associated fibroblast (CAF) activation and angiogenesis, therefore exhibiting enhanced anti-OSCC efficacy.

Introduction

Oral squamous cell carcinoma (OSCC) represents over 90% of head and neck malignancies, with approximately 650,000 new cases diagnosed annually worldwide.^1–3 Despite the implementation of multimodal therapeutic strategies encompassing surgical resection, radiotherapy, and conventional chemotherapy, the 5-year survival rate has remained disappointingly stagnant at 55–60%.⁴ A major obstacle to successful treatment is the inherent or acquired drug resistance of tumor cells, coupled with the propensity for distant metastasis,⁵ both of which significantly contribute to treatment failure and poor prognosis.^6–9 Mounting evidence indicates that traditional therapies often neglect the critical role of the tumor microenvironment (TME) in promoting tumor progression, metastasis, and drug resistance.^10–13 There is, therefore, an urgent imperative to develop innovative therapeutic strategies that not only potentiate pro-apoptotic effects but also inhibit drug resistance and metastasis of tumor cells by systematically remodeling TME.

To address these challenges, we have designed a combination therapeutic strategy integrating two natural bioactive compounds with complementary mechanisms of action: curcumin (CUR) and asiatic acid (AA). CUR, a polyphenolic compound derived from Curcuma longa, exhibits multi-targeted antitumor activities including inhibition of proliferation, suppression of invasion, reversal of drug resistance, and induction of apoptosis.^14–16 AA, a pentacyclic triterpenoid extracted from Centella asiatica, demonstrates potent antitumor effects through blockade of STAT3 and NF-κB signaling pathways, thereby suppressing tumor cell survival and proliferation.^17–19 Importantly, both compounds have been documented to modulate the TME through inhibition of cancer-associated fibroblast (CAF) activation and suppression of tumor angiogenesis.^20,21 The combination of CUR and AA was expected to inhibit OSCC development by regulating TME.

Notwithstanding their promising pharmacological profiles, the clinical translation of CUR and AA has been severely impeded by shared physicochemical limitations, including extremely poor aqueous solubility, short plasma half-lives, and inadequate tumor accumulation.^22–25 To circumvent these barriers, liposomal nanocarriers have emerged as a mainstream delivery platform for antitumor therapy, leveraging their enhanced biocompatibility, low immunogenicity, and high encapsulation efficiency for both hydrophobic and hydrophilic drugs.^26–28 However, conventional liposomes exhibit inherent limitations, including large particle sizes and reliance on passive targeting through the enhanced permeability and retention (EPR) effect, which often results in suboptimal drug delivery efficiency.^29–31 Recent innovations in biomimetic nanotechnology have demonstrated that cell-derived membrane coatings can endow nanoparticles with the surface chemistry and targeting capabilities of their source cells, simultaneously prolonging circulation time and enabling active recognition of diseased tissue.^32–34 Neutrophil Extracellular Traps (NETs) are a fibrous network structure composed of deoxyribonucleic acid (DNA), histones, and various granular proteins (such as neutrophil elastase (NE), matrix metalloproteinase 9 (MMP9)) released after neutrophil activation.^35,36 Given that NETs are inherently capable of capturing circulating tumor cells (CTCs) and facilitating their immune evasion and metastatic colonization, they possess intrinsic tumor-homing properties that can be exploited for targeted drug delivery.^37–39 Furthermore, crystallographic analyses have revealed that the sterol scaffold of AA is virtually superimposable on that of cholesterol, enabling AA to function as a structural substitute within lipid bilayers.^40,41 By partially replacing cholesterol, AA condenses membrane fluidity, thereby reducing particle size while preserving high drug loading capacity and exerting intrinsic antitumor activity.

In this study, we engineered a novel NETs-coated liposomal system (CUR@AA-NLIP) that integrates these strategic innovations for synergistic anti-OSCC therapy. The incorporation of AA serves a dual function: as a membrane-stabilizing component that optimizes nanocarrier physicochemical properties, and as a bioactive therapeutic agent contributing to antitumor efficacy. The NETs membrane coating confers active tumor-targeting capability and immune evasion properties, while the liposomal core enables sustained co-delivery of CUR and AA. This biomimetic nanoplatform would enhance drug accumulation at tumor sites, overcome TME-mediated drug resistance, and produce enhanced therapeutic outcomes through the combined effects of enhanced apoptosis induction and microenvironment modulation. To rigorously evaluate this system, we established co-culture and co-implantation models comprising cancer-associated fibroblasts and tumor cells to recapitulate the TME, and systematically investigated the antiproliferative, anti-migratory, and anti-metastatic efficacy of CUR@AA-NLIP both in vitro and in vivo.

Materials and Methods

Materials

Egg yolk phospholipids were purchased from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). Near-infrared fluorescent membrane dyes (DID and DIR) were obtained from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Fetal bovine serum (FBS), curcumin (CUR), asiatic acid (AA), DMEM high-glucose medium, 4′,6-diamidino-2-phenylindole (DAPI), Triton X-100, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). The SDS-PAGE reagent kit was provided by Beyotime Biotechnology (Shanghai, China). The CFSE cell labeling kit, anti-CD31, anti-α-SMA, and other antibodies were sourced from Abcam (Cambridge, MA, USA). All other reagents were of analytical grade.

Cell Lines and Animals

OSCC cell line (CAL27), human fetal lung fibroblasts (MRC5), and mouse embryonic fibroblasts (NIH/3T3) were obtained from the Institute of Biological Sciences (Beijing, China). CAL27, MRC5, and NIH/3T3 cells were maintained in DMEM at 37°C in a humidified atmosphere containing 5% CO₂. Female nude mice (6–8 weeks old) were purchased from Pengyue Laboratory Animal Technology Co., Ltd. All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of China (Document No. 55, 2001) and were approved by the Animal Ethics Committee of Shandong Second Medical University (Approval No. 2019–045). Anesthetic administration and euthanasia procedures in animal experiments adhered to the Guidelines for the Euthanasia of Animals published by the American Veterinary Medical Association (AVMA, 2020 Edition).

Preparation of NETs-Coating CUR@AA-NLIP

Neutrophils were isolated from heparin sodium-anticoagulated whole blood using a neutrophil separation kit (cat. no. P2091, Solarbio). Isolated neutrophils were resuspended in RPMI-1640 medium supplemented with 50 nM phorbol 12-myristate 13-acetate (PMA) and incubated in 6-well plates for 4 hours under light-protected conditions. The NETs-containing supernatant was centrifuged and subjected to three freeze-thaw cycles (−80°C) to obtain NETs.Egg yolk lecithin, cholesterol, AA, and CUR were dissolved in 2 mL ethanol at a mass ratio of 24:5:3:1. The lipid mixture was then injected dropwise into 8 mL PBS under stirring (20 rpm). Subsequently, ethanol was evaporated using a water bath at 55°C to form liposomes. The resulting suspension was sonicated (120 W, 10 min), successively filtered through 0.45 μm and 0.22 μm membranes, and stored at 4°C. For NETs coating, CUR@AA-LIP and NETs fragments were sonicated (1 min, 120 W) and extruded six times through 200-nm polycarbonate membranes to form CUR@AA-NLIP (Figure 1).

Figure 1.

Schematic Diagram of Preparation, Uptake, and Release of CUR@AA-NLIP.

Characterization of CUR@AA-NLIP

CUR@AA-NLIP was diluted in PBS, and particle size distribution, polydispersity index (PDI), and Zeta potential were measured using a Malvern particle size analyzer. Morphology was observed via transmission electron microscopy (TEM). For stability assessment, 10-fold diluted samples in PBS were stored at 4°C, and particle size and PDI were monitored every other day for 14 days. The loading efficiency (LE) and encapsulation efficiency (EE) were important evaluation indexes for nano-sized drug delivery system. In this study, the content of CUR was detected at the absorption wavelength of 425 nm using an ultraviolet (UV) spectrophotometer, while AA was determined by high-performance liquid chromatography (HPLC) at 210nm. LE and EE of were calculated by the following equations:

Protein Detection in NETs and CUR@AA-NLIP

The protein profiles of NETs, CUR@AA-LIP, and CUR@AA-NLIP were analyzed by SDS-PAGE. Proteins were extracted from NETs, CUR@AA-LIP, and CUR@AA-NLIP using RIPA lysis buffer, and their concentrations were quantified with a BCA protein assay kit. The resulting gels were stained with Coomassie Brilliant Blue for 2 hours and destained overnight. The processed gels were imaged using an Amersham ImageQuant 800 multifunctional imaging system. Citrullinated histone H3 (CITH3), a specific marker of neutrophil extracellular traps (NETs), was detected to verify the presence of NETs proteins in the formulations. To further confirm the successful encapsulation of NETs, the gels were transferred onto polyvinylidene difluoride (PVDF) membranes for Western blot analysis. The PVDF membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) and developed using an enhanced chemiluminescence (ECL) detection kit. Protein signals were captured with the Amersham ImageQuant 800 system.

In vitro Drug Release

The drug release profile from CUR-loaded liposomes (CUR@AA-LIP and CUR@AA-NLIP) was assessed by dialysis. Briefly, 1 mL of the sample was loaded into a dialysis bag and dialyzed against 20 mL of PBS containing 1% Tween 80 at 37°C for 48 h. At fixed time intervals, 2 mL of the dialysate was collected and replaced with an equal volume of fresh dialysate. The cumulative release of CUR was quantified by UV spectrophotometry.

Hemolysis Assay

Murine blood (1 mL) was collected in heparinized tubes and centrifuged at 3500 rpm for 10 min. Erythrocytes were collected and washed with PBS until the supernatant became colorless, then resuspended to a 2% (v/v) suspension. CUR@AA-NLIP was mixed separately with the erythrocyte suspension to final concentrations of 1, 5, 10, 50, and 100 μg/mL (n = 3). Normal saline and Triton X-100 served as negative and positive controls, respectively. All samples were incubated at 37°C for 2 h, followed by centrifugation (3500 rpm, 10 min). The supernatant absorbance was measured at 545 nm, and the hemolysis rate was calculated using the formula:

where A, A₁, and A₂ represent the absorbance values of the positive control, test group, and negative control, respectively.

Cellular Uptake Assay

Coumarin-6 (C6, a fluorescent dye) was used instead of curcumin (CUR) for fluorescent labeling of the formulations. CAL27 cells (5 × 10⁴ cells/dish) were seeded in confocal dishes and cultured for 12 h. Subsequently, the cells were incubated with serum-free medium containing Free C6, C6-labeled liposomes (C6@AA-LIP), and C6-labeled NETs-coating liposomes (C6@AA-NLIP) for 30 min (C6 = 10 µg/mL). After incubation, the cells were rinsed with PBS, fixed in 4% paraformaldehyde, and stained with DAPI (1 µg/mL) for 8 min prior to confocal laser scanning microscopy (CLSM). For quantitative analysis, the cells were detached with EDTA-free trypsin, washed thoroughly, and subjected to flow cytometry (FCM) to determine fluorescence intensity.

Drug Retention Assay

Similarly, the retention of C6-labeled liposomes was evaluated via confocal microscopy. Free C6, C6@AA-LIP, and C6@AA-NLIP (2 µg/mL) were incubated with “CAL27 + MRC-5” co-cultured cells for 4, 12, and 24 h to simulate drug retention in the tumor microenvironment. After each incubation period, the cells were sequentially fixed, stained, and washed. Intracellular drug retention was analyzed via CLSM for morphological observation and FCM for quantitative assessment.

Immune Escape Characteristics

To evaluate the in vitro immune escape capability of the liposomes, macrophage uptake was performed using CLSM. RAW264.7 cells were seeded in 8-well chamber slides at 1.0×10⁵ cells/well and incubated at 37°C for 24 h. The medium was replaced with fresh medium supplemented with free C6, C6@AA-LIP, or C6@AA-NLIP (10 µg/mL), followed by incubation for 1, 3, or 5 h. Cells were then washed with PBS, fixed in 4% paraformaldehyde (10 min), stained with DAPI (8 min), and rinsed three times with PBS. Cellular uptake was visualized by CLSM, and fluorescence intensity was quantified using Image Pro Plus software.

Cytotoxicity Assay

The in vitro cytotoxicity of CUR@AA-NLIP was evaluated against CAL27 cells and “CAL27 + MRC-5” co-cultured cells via MTT assay. Cells were seeded in 96-well plates at 5.0 × 10³ cells/well and cultured for 24 h. Cells were then treated with free CUR, free AA, CUR + AA, CUR@AA-LIP, or CUR@AA-NLIP (0.01–10 μg/mL), using untreated cells as negative controls. After 48 h incubation, MTT solution was added for 4 h. The medium was removed, and DMSO was added to dissolve formazan crystals. Absorbance at 570 nm was measured using a microplate reader (ELX800; BioTek Instruments, Winooski, VT), and cell viability was calculated.

Inhibition of CAFs Activation in vitro

The inhibitory and reversal effects on CAFs activation were assessed by immunofluorescence staining of α-SMA. CAFs were induced by incubating MRC-5 cells with CAL27 supernatant. CAFs were seeded in chamber slides, treated with free CUR, CUR + AA, CUR@AA-LIP, or CUR@AA-NLIP (1 µg/mL) for 24 h, then fixed, blocked, and stained with anti-α-SMA antibody and Alexa Fluor 488-conjugated secondary antibody, followed by DAPI staining. Fluorescence was visualized by confocal microscopy and quantified using Image-Pro Plus software.

The in vitro Tube Formation Assay

The anti-angiogenic effect was evaluated using a Matrigel tube formation assay. Briefly, 20 μL Matrigel was coated in 24-well plates and polymerized at 37°C for 30 min. HUVEC were trypsinized, washed with PBS to remove serum residues, and labeled with 5 μM CFSE at 37°C in the dark for 30 min. Labeled cells were resuspended in medium containing free CUR, CUR + AA, CUR@AA-LIP, or CUR@AA-NLIP (1 µg/mL), with DMEM as a negative control. Cells (8.0×10⁴/well) were seeded onto Matrigel-coated plates and incubated for 4 h. Vascular networks were imaged using an inverted microscope, and nodes were quantified with ImageJ.

Cell Migration Assays

A scratch wound healing assay was carried out to evaluate cell migration. CAL27 cells and “CAL27 + MRC-5” co-cultured cells in logarithmic growth phase were seeded in 6-well plates and cultured at 37°C/5% CO₂ for 24 h until reaching full confluency. After aspirating the medium and washing with PBS, a linear scratch was created using a sterile 200 μL pipette tip. Following PBS washes, baseline images (0 h) were captured. Free CUR, CUR + AA, CUR@AA-LIP, and CUR@AA-NLIP (1 µg/mL) were diluted in serum-free medium and added to designated wells, with serum-free medium as negative controls. After 24-h incubation, cells were washed with PBS, and 24-h endpoint images were acquired at the same locations. Scratch closure was quantified using ImageJ 2.3.0. The migration rate was calculated as:

S0 h denotes the scratch wound area at 0 h, and S24 h denotes the scratch wound area at 24 h.

A Transwell assay was performed. Serum-starved CAL27 cells and “CAL27 + MRC-5” co-cultured cells (5.0×10⁴ cells/well) pretreated for 8 h were seeded in the upper chamber and treated with free CUR, CUR + AA, CUR@AA-LIP, or CUR@AA-NLIP, while the lower chamber contained 2% serum-supplemented medium. After 16 h, the medium was aspirated, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with crystal violet. Non-migrated cells were removed with a cotton swab. Migrated cells were observed under an optical microscope and quantified using ImageJ 2.3.0.

In vivo Drug Biodistribution Experiments

To investigate in vivo drug biodistribution, a “CAL27 + NIH/3T3” tumor model was established by injecting CAL27 and NIH/3T3 cells (5:1 ratio, 1.0×10⁶ cells/mouse) into the right flank of nude mice. When tumor volume reached 120 mm³, mice were randomly assigned to three groups for intravenous injection of free DID, DID@AA-LIP, or DID@AA-NLIP (4 μg per mouse). Whole-body fluorescence imaging was performed at 2, 6, 12, 24, and 48 h post-injection. Tumor-bearing mice were anaesthetized by intraperitoneal injection of a mixture of ketamine (60 mg/kg) and xylazine (5 mg/kg). Subsequently, mice were humanely euthanized by CO₂ inhalation to harvest tumors and organs, and their fluorescence intensities were measured.

In vivo Anti-Tumor Efficacy

The “CAL27 + NIH/3T3”-bearing mice were used to evaluate the antitumor efficacy of different formulations. Tumor-bearing mice were randomly divided into five groups (n = 5 per group): saline (negative control), free CUR, CUR + AA, CUR@AA-LIP, and CUR@AA-NLIP (5 mg/kg). Treatments were administered via tail vein injection every two days for 14 days (7 doses). Body weight and tumor volume (calculated as V = 0.5 × length × width²) were measured every other day. After two weeks of treatment, the animals were humanely euthanized by CO₂ inhalation, and their organs and tumors were collected for subsequent H and E staining and immunohistochemical (IHC) analysis.

Histological Staining Techniques for Evaluating Anti-Tumor Efficacy of Formulations

This study systematically assessed the anti-tumor effects of formulations using hematoxylin and eosin (H and E) staining, Masson’s trichrome staining, immunohistochemistry (IHC), and immunofluorescence (IF). Key procedures included: fixation of tumors and major organs in 4% paraformaldehyde for 1 week, paraffin embedding, sectioning at 5 μm thickness, deparaffinization in xylene, rehydration through graded ethanol, staining following manufacturer protocols, and observation under a light microscope (H and E/Masson/IHC) or fluorescence microscope (IF) (IX51; Olympus, Tokyo, Japan).

Lung Metastasis Assays

A lung metastasis mouse model was established to investigate the anti-metastasis effect of CUR@AA-NLIP In brief, the mice were randomly divided into five groups (n = 5 per group). Treatment was initiated one week post-CAL27 cell injection, and the intravenous injections were administered every other day for 14 days. Lung tissues were harvested post-treatment for macroscopic imaging and metastatic nodule enumeration. Additionally, hematoxylin and eosin (H and E) staining was performed on lung sections to histologically analyze metastatic lesions.

Statistical Analysis

Statistical analysis was performed using Prism 8.0 (GraphPad). Data were presented as Mean ± SD. Differences between groups were analyzed using Student’s t-test and one-way analysis of variance. P<0.05 was considered statistically significant.

Results and Discussion

Physicochemical Characterization of CUR@AA-NLIP

The synthesis route of CUR@AA-NLIP was described in Figure 2A. The average particle size of CUR@AA-NLIP was approximately 113.53 nm, with a PDI of 0.14 and a negative zeta potential (Figures 2B, C and S1A). In CUR@AA-LIP (CUR: AA = 1:6), the encapsulation efficiency (EE) and loading efficiency (LE) of CUR were 89.77% and 2.77%, respectively, whereas in CUR@AA-LIP (CUR: AA = 1:3), the EE and LE of CUR reached 94.07% and 2.87%, respectively (Table S1). The 1:3 ratio showed optimal EE and LE, likely due to the appropriate size of CUR@AA-LIP. Additionally, CUR and AA exhibited synergistic effects at the 1:3 ratio (Figure S1B). As shown in Figure 2D and E, TEM images revealed that both CUR-LIP and CUR@AA-NLIP were spherical. Neutrophils were successfully isolated, and NETs were induced (Figure S2A–B). TEM images showed that a membrane-like layer was observed on the CUR@AA-NLIP. Furthermore, SDS-PAGE and Western blot results also confirmed the presence of NETs-derived proteins (Figure 2H and S2C), suggesting that NETs were successfully coated on the surface of liposomes. The particle size of CUR@AA-NLIP showed no significant change in PBS or DMEM over 14 days (Figure 2F), indicating good stability. The in vitro drug release profiles of CUR@AA-LIP and CUR@AA-NLIP were presented in Figure 2G. Free CUR exceeded 80% release within 10 h, whereas CUR@AA-NLIP released less than 80% even at 48 h. Additionally, in vivo pharmacokinetic studies demonstrated that CUR@AA-NLIP exhibited prolonged plasma circulation time and enhanced drug retention compared to free CUR (Figure S3). These results demonstrated that CUR@AA-NLIP could serve as a drug delivery platform for sustained release.

Figure 2.

Characterization of CUR@AA-LIP and CUR@AA-NLIP. (A) Preparation schematic of CUR@AA-NLIP; (B and C) Particle-size distribution of CUR-LIP and CUR@AA-NLIP; (D and E) Representative TEM (Red boxes indicate magnified liposome details) photographs of CUR-LIP and CUR@AA-NLIP (scale bars: 200 nm); (F) Stability in PBS/DMEM (14 days, n = 3); (G) In vitro CUR release kinetics (n = 3); (H) Western blot confirming the presence of NETs protein citH3 in CUR@AA-NLIP.

Cell Uptake and Drug Retention

NETs avidly bind to tumor cells, enabling NETs-coated nanocarriers to home selectively to the tumor niche. We quantified this tropism by tracking the cellular uptake of coumarin-6 (C6)-loaded liposomes in CAL27 cells (Figure 3A). As shown in Figure 3B, blue and green fluorescence signals represented nuclei and liposomes, respectively. Notably, C6@AA-NLIP exhibited significantly stronger green fluorescence than C6@AA-LIP, indicating that NETs coating promoted liposome internalization. Quantitative analysis (Figure 3C) revealed that the mean fluorescence intensity of C6@AA-NLIP was 1.91-fold and 1.40-fold higher than that of free C6 and C6@AA-LIP, respectively, demonstrating that NETs coating effectively enhances cellular uptake.

Figure 3.

Cellular uptake and drug retention. (A) Schematic of the cell uptake and drug retention; Cellular uptake by TEM (B) and flow cytometry (C); Drug retention by TME (D) and flow cytometry (E and F); Immune escape behavior (G) and fluorescence quantification (H) on RAW264.7 cells (scale bars: 50 μm). *P < 0.05, **P < 0.01, ***P < 0.001.

To evaluate drug retention capacity, a “CAL27 + MRC-5” co-culture model was established. As shown in Figure 3D, green fluorescence signals were observed in all drug-treated groups after 4, 12, and 24 h, with intensity increasing over time. Flow cytometry quantification showed the same trend (Figure 3E and F). The free C6 population always displayed the dimmest fluorescence, consistent with rapid drug expulsion. After 24 h, C6@AA-NLIP yielded substantially brighter intracellular fluorescence than C6@AA-LIP, demonstrating that NETs-derived surface fragments markedly prolong intracellular drug retention.

To determine whether NETs-mediated camouflage enables liposomes to evade innate immune surveillance, the uptake of free C6, C6@AA-LIP, and C6@AA-NLIP by RAW264.7 was examined. In Figure 3G, the C6@AA-NLIP-treated group exhibited weaker green fluorescence than C6@AA-LIP. Quantitative results (Figure 3H) showed that the fluorescent signals in C6@AA-NLIP were less than those in free C6 and C6@AA-LIP. These findings suggested that NETs modification might confer immune escape properties to liposomes, thereby reducing phagocytosis by macrophages.

Cell Viability Inhibition Assay

In this study, the in vitro cytotoxicity of various formulations was evaluated by MTT assay (Figure 4A). As shown in Figure 4B and C, all drug formulations exhibited concentration-dependent cytotoxicity. Interestingly, free CUR exhibited significantly lower cytotoxicity on “CAL27 + MRC-5” co-cultured cells than CAL27 cells alone, indicating that the addition of MRC-5 cells reduced the cytotoxicity of CUR on CAL27 cells. This result is consistent with recent studies, in which CAFs induce drug resistance of tumor cells.^42–44 The combination of AA and CUR exerted a stronger anti-proliferation effect on co-cultured cells compared to CUR alone, suggesting a synergistic effect between AA and CUR, where AA might act as an adjuvant to enhance CUR’s cytotoxicity. Notably, the cell viability of NETs-coated CUR@AA-NLIP (CUR at 10 μg/mL) was 19.19%, which was significantly lower than that of CUR@AA-LIP. This might be attributed to its tumor-targeting capability, which facilitates drug internalization in tumor cells, thereby enhancing the inhibitory effect.

Figure 4.

In vitro Cytotoxicity evaluation. (A) Pattern diagram of MTT assay; Cell viability for CAL27 cells (B) and the co-culture model (C); (D and E) The α-SMA expression and quantitative analysis (scale bars: 50 μm) and (F and G) HUVEC tube formation and quantitative analysis (scale bars: 100 μm). *P < 0.05, **P < 0.01, ***P < 0.001.

Considering that CAFs play a pivotal role in tumorigenesis, progression, metastasis, and drug resistance through dynamic interactions with tumor cells, the activation of fibroblasts was examined through detecting the expression of α-SMA (a biomarker of activated fibroblasts) in Figure 4D and E. The results showed that more green fluorescent signals were observed in the NIH/3T3 cells treated with condition medium of tumor cells (Enriched with tumor cell-secreted factors), suggesting NIH/3T3 cells were activated to become a CAF-like phenotype. Compared with free CUR, the combined drug groups showed weaker fluorescence, indicating that the combination of CUR and AA obviously inhibited the activation of fibroblasts. Interestingly, the fluorescent intensity of CUR@AA-NLIP was lower than that of CUR + AA, suggesting that the nano-sized carrier based on NETs-coated liposomes enhanced the anti-CAFs effect of drugs.

Recent research revealed that tumor growth is always accompanied by the formation of new blood vessels.⁴⁵ The HUVEC tube formation assay is a rapid in vitro method to evaluate the effects of drugs on the angiogenic capacity of vascular endothelial cells. As shown in Figure 4F and G, CUR + AA exhibited stronger inhibitory effects on tube formation in comparison with free CUR, indicating a synergistic anti-vascular effect. CUR@AA-NLIP exhibited an 80% suppression rate in vascular node formation, surpassing that of other groups, indicating its potent inhibition of the angiogenic capacity of vascular endothelial cells.

In vitro Anti-Migration Analysis

To evaluate the inhibitory effects of different drug formulations on the cell migration of oral squamous carcinoma cells in vitro, wound healing assays were performed using both CAL27 cells and “CAL27 + MRC-5” co-culture model (Figure 5A). As shown in Figure 5B–E, the cell migration rate in the co-culture model was significantly higher than that in the CAL27 cells alone, demonstrating that MRC-5 cells enhanced the migratory capacity of CAL27 cells. After drug treatment, all therapeutic groups exhibited migration inhibition. The residual wound area in the combination groups was markedly larger than that in the free CUR group, indicating an enhanced anti-migration effect. Notably, in the co-culture model, the CUR@AA-NLIP group showed the lowest migration rate among all treatment groups, which was substantially reduced compared to both the CUR + AA group and the CUR@AA-LIP group. This indicated that NETs-coated liposomes significantly potentiated the anti-migratory effect of the drugs. This finding was corroborated by the transwell assay (Figure 5F and G), in which fewer migrating cells were observed in the CUR@AA-NLIP group compared to other groups, confirming that CUR@AA-NLIP effectively suppressed tumor cell migration.

Figure 5.

In vitro anti-migratory effects. (A) The diagram of wound-healing assays; (B) Representative scratch wound images and (C) migration rate on CAL27 cells; (D) Representative scratch wound images and (E) migration rate on “CAL27 + MRC-5” co-culture model (MRC-5 cells labeled with green fluorescence) (scale bars: 200 μm). (F) Transwell assay (scale bars: 500 μm) and (G) quantitative results. *p<0.05, **p<0.01, ***P < 0.001.

In vivo Biodistribution Assays

To further evaluate the in vivo targeting capability of the formulations, near-infrared fluorescence (NIRF) imaging was performed in “CAL27+NIH/3T3” mice models (Figure 6A). Compared with free DID, there were fluorescence signals in the two liposomal formulations (DID@AA-LIP and DID@AA-NLIP) in the tumor site (Figure 6B). This might be attributed to the fact that the nano-sized liposomes could improve drug accumulation in tumor cells through EPR-mediated passive-targeting drug delivery, leading to stronger fluorescence in the tumor region. After 48 h, the tumors and major organs (heart, liver, spleen, lung, kidney) were harvested for the ex vivo imaging (Figure 6C). The results showed that DID@AA-NLIP groups exhibited higher fluorescence intensity in the tumor than DID@AA-LIP, suggesting that the coating of NETs improved tumor-targeting capacities of liposomes. This was consistent with the cellular uptake assay.

Figure 6.

In vivo biodistribution analysis. (A) Model establishment and drug administration; (B) DID fluorescence imaging of DID, DID@AA-LIP, and DID@AA-NLIP in 0–48 h; (C) Ex vivo fluorescence images of heart, liver, spleen, lungs, kidneys, and tumors harvested at the endpoint (48 h).

In vivo Anti-Tumor Assays

A “CAL27 + NIH/3T3” subcutaneous xenograft model was used to evaluate the anti-tumor efficacy in vivo (Figure 7A). Results showed that there was no significant weight loss in the CUR@AA-LIP and CUR@AA-NLIP groups compared to the saline group (Figure 7B), indicating good biocompatibility of these formulations. As shown in Figure 7C and D, tumor growth was inhibited to varying degrees in all drug-treated groups. The free CUR + AA group had significantly smaller tumors than the CUR group, indicating that combination therapy enhanced anti-OSCC efficacy. Notably, the CUR@AA-NLIP group showed the most potent tumor growth inhibition among all treatment groups (Figure S4), suggesting that CUR@AA-NLIP exerted enhanced in vivo anti-proliferation effects. This therapeutic effect might be attributed to NETs-mediated active targeting, which enhances drug accumulation in the tumor region.

Figure 7.

In vivo anti-tumor assays. (A) Schematic illustration of drug treatment; (B) Mouse body weight curves; (C) Photos of tumor tissues; (D) Tumor volume growth curves during treatment; (E) H and E staining of tumor tissues; (F) Immunohistochemistry of Ki67 protein; (G) Masson trichrome staining; (H) Immunofluorescence of α-SMA; (I) Immunohistochemistry of CD31 protein. (Scale bar: 50 µm),***P<0.001.

Histological analysis was used to further detect the anti-tumor effect of the different formulations. Hematoxylin and eosin (H and E) staining in Figures 7E and S5 revealed that the treatment of CUR or CUR + AA induced slight nuclear pyknosis and cytoplasmic vacuolization in tumor tissues. In contrast, the liposome preparation groups, especially the CUR@AA-NLIP group, had a lower tumor cell density and a larger area of necrosis, showing a stronger pro-apoptotic effect. This result was consistent with the cell proliferation assay, in which CUR@AA-NLIP demonstrated a lower level of Ki67 expression, indicating a significant Inhibitory effect on tumor cell proliferation (Figures 7F and S6).

More and more evidences reveal that CAFs activation and ECM formation in TME promote drug resistance and metastasis of tumors.^46–48 Masson trichrome staining was used to investigate the deposition of ECM. Abundant blue-stained collagen fibers appeared in the saline group (Figure 7G and S7). While almost no blue signals were observed in the CUR@AA-NLIP group, indicating its enhanced inhibitory effect on ECM deposition.

Considering that CAFs were the primary source of ECM components, the immunofluorescence staining for α-SMA (a marker of CAFs) was examined (Figure 7H and S8). Compared with other drug-treated groups, fewer fluorescent signals were observed in the CUR@AA-NLIP group, indicating that the combination therapy based on CUR@AA-NLIP effectively inhibited the activation of CAFs.

Results of immunohistochemistry demonstrated extensive tumor angiogenesis in the Saline group with extensive CD31 expression (Figure 7I and S9), whereas neovascularization was significantly reduced in the CUR@AA-NLIP group, indicating effective inhibition of tumor blood vessel formation.

Lung Metastasis Assays

Distal metastasis is a major reason accounting for the poor prognosis of OSCC. To investigate the anti-metastasis effect of CUR@AA-NLIP, CAL27 cells were injected intravenously to establish a lung-metastasis mouse model (Figure 8A). Results showed that there was no significant weight loss in the CUR@AA-NLIP group compared to that of the control group (Figure 8B), indicating its good biosafety. In Figure 8C, many lung metastatic nodules were distributed in thecontrol group, and decreased in all drug-treated groups. As expected, CUR@AA-NLIP had fewer metastatic nodules than other groups, demonstrating enhanced anti-metastasis effect. This might be due to two reasons: (1) the nano-sized carrier could accumulate in the lung metastatic region by the EPR effect,^49,50 improving the bioavailability of drugs, (2) the coating of NETs on liposomes could bind to tumor cells in the lung and eliminate them.

Figure 8.

Lung metastasis assays. (A) Schematic of lung metastasis establishment and treatment; (B) Mouse body weight curves; (C) Photos of lung tissues and H and E staining of lung tissues (Scale bar: 50 µm) (Yellow circles indicate metastatic lesions).

Biosafety Tests

Biosafety tests were performed to evaluate the biocompatibility of nano-sized formulations. The results of H and E staining of major organs showed that no significant pathological damages were detected in the CUR@AA-NLIP group in comparison to the Saline group (Figure S10A), indicating its good biocompatibility and safety. Additionally, hemolysis assays showed that there was no obvious hemolysis in the CUR@AA-NLIP group at concentrations ranging from to (Figure S10B), further confirming its suitability as a safe drug delivery carrier. Coagulation tests (PT, APTT, FIB, TT) and serum inflammatory cytokine levels (TNF-α, IL-6, IL-1β) showed no significant differences between the CUR@AA-NLIP and control groups (Figure S11–S12), supporting the absence of thrombotic and inflammatory risks.

Conclusion

A novel NETs-coating liposome (CUR@AA-NLIP) was successfully prepared for co-delivery of CUR and AA for anti-OSCC therapy. The introduction of AA not only optimized the physicochemical properties of the liposomes but also achieved synergistic antitumor effects of AA and CUR. The coating of NETs on liposomes improved immune evasion and active tumor-targeting capabilities. Compared with CAL-27 cells alone, the addition of MRC-5 cells increased drug resistance and migration of tumor cells. CUR@AA-NLIP could improve drug internalization in tumor cells, block the cross-talk between CAFs and tumor cells, decrease ECM deposition and tumor angiogenesis, and exhibit enhanced anti-proliferation and anti-metastasis efficacy. In summary, CUR@AA-NLIP effectively inhibits OSCC development by combining proapoptotic and TME-regulating activities, and is a potential formulation for anti-OSCC treatment.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.