A Quercetin/Rg3-Loaded Calcium Phosphate Nanoplatform Alleviates Breast Cancer-Induced Peripheral Hyperalgesia and Enhances Antitumor Immunity

Abstract

Purpose

This study aimed to develop a multifunctional nanoplatform, Rg3-Lip-CaPO₄/Quercetin (R-LCP/Q), for integrating antitumor therapy, immune modulation, and cancer pain relief.

Methods

R-LCP/Q was constructed by incorporating ginsenoside Rg3 into a lipid-coated calcium phosphate nanoplatform loaded with quercetin (Que). Its physicochemical properties, pH-responsive release, cellular uptake, antitumor activity, Ca²⁺-associated intracellular effects, immunogenic cell death (ICD), immune modulation, biodistribution, biosafety, and analgesic efficacy were evaluated in vitro and in a 4T1 breast cancer-associated pain model. The involvement of transient receptor potential vanilloid 1 (TRPV1) signalling in analgesia was further assessed using the selective TRPV1 antagonist AMG9810.

Results

R-LCP/Q exhibited pH-responsive degradation and release of Ca²⁺, Que, and Rg3 under acidic conditions, together with enhanced cellular uptake and tumor-associated accumulation following Rg3 functionalization. R-LCP/Q induced intracellular Ca²⁺ accumulation, mitochondrial depolarization, ROS generation, and tumor-cell apoptosis; these effects were markedly attenuated by the intracellular Ca²⁺ chelator BAPTA, supporting a substantial Ca²⁺-dependent contribution to the cytotoxic response. R-LCP/Q also increased ICD-associated hallmarks and promoted dendritic cells (DCs) maturation. In vivo, R-LCP/Q suppressed tumor growth and was associated with enhanced DCs maturation, increased CD8⁺T-cell responses, a shift towards an M1-like macrophage phenotype, reduced regulatory T-cell frequencies, and decreased tumor-associated TNF-α and IL-6 levels. R-LCP/Q also alleviated mechanical and thermal hyperalgesia, accompanied by reduced TRPV1 expression in dorsal root ganglia and decreased serum substance P levels; pharmacological TRPV1 blockade with AMG9810 also produced an analgesic response. Short-term haematological, biochemical, histological, and haemocompatibility assessments indicated favourable tolerability.

Conclusion

R-LCP/Q provides preclinical proof of concept for a multifunctional nanotherapeutic strategy integrating tumor suppression, antitumor immune modulation, and cancer pain relief. The findings support further investigation of coordinated tumor-immune-nociceptive regulation as a therapeutic approach for cancer treatment.

Introduction

Cancer remains a major global public health challenge because of its high incidence and mortality.^1,2 In addition to tumor progression itself, cancer-associated pain substantially impairs quality of life, particularly in patients with advanced disease.^3,4 Cancer pain arises from multiple interacting mechanisms, including tumor-induced tissue compression, local inflammation and ischaemia, as well as aberrant activation of peripheral nociceptive pathways.^5–7 However, therapeutic development has traditionally focused predominantly on tumor control, whereas the simultaneous management of tumor progression and cancer-associated pain remains insufficiently addressed.^2,8 Increasing evidence indicates that bidirectional interactions between tumors and peripheral sensory nerves contribute to both cancer progression and nociceptive sensitization.^9–12 Among the molecular mediators involved, transient receptor potential vanilloid 1 (TRPV1), a nociceptive ion channel highly expressed in primary afferent sensory neurons, can be activated by thermal, chemical, and inflammatory stimuli.^13–19 TRPV1 activation promotes the release of neuropeptides, including substance P (SP),^20,21 which contributes not only to nociceptive sensitization but also to tumor-associated inflammation, angiogenesis, and metastatic progression.^22–26 Thus, modulation of the TRPV1–SP axis represents a potentially valuable strategy for integrating cancer pain control with tumor-directed therapy.

Quercetin (Que), a naturally occurring flavonoid, has attracted considerable interest because of its antitumor, anti-inflammatory, and analgesic properties.^27–31 Previous studies have implicated modulation of TRPV1-related signalling in the analgesic effects of Que in experimental models of bone cancer pain and chemotherapy-induced neuropathic pain, suggesting that Que may provide a pharmacological link between tumor control and nociceptive regulation.^28,32 However, the therapeutic application of Que is limited by its poor aqueous solubility, low bioavailability, and limited tumor-selective accumulation.^33–35 These limitations highlight the need for an efficient delivery system capable of improving Que availability while integrating complementary antitumor mechanisms. Calcium phosphate nanoparticles (CaPO₄ NPs) provide an attractive delivery platform because of their biocompatibility, biodegradability, and acid-sensitive dissolution.³⁶ Under acidic conditions, degradation of CaPO₄ can release Ca²⁺, thereby perturbing intracellular calcium homeostasis and contributing to tumor-cell injury.³⁷ Lipid coating and PEGylation can further improve the colloidal stability and biological performance of CaPO₄-based nanocarriers.^38,39 These properties make lipid-coated CaPO₄ nanoparticles suitable for integrating Que delivery with calcium-associated tumor-cell stress.

Ginsenoside Rg3, a bioactive constituent of Panax ginseng, has demonstrated multiple antitumor and immunomodulatory activities, including inhibition of tumor-cell proliferation, invasion, and angiogenesis, as well as modulation of tumor-associated immune cells.^40–44 Previous studies have shown that Rg3-containing delivery systems can enhance antitumor activity and modulate tumor-associated immune responses.^45–47 Co-delivery of Rg3 and Que has also demonstrated complementary therapeutic potential in preclinical tumor models. In addition, the amphiphilic structure of Rg3 enables its incorporation into lipid membranes, allowing it to serve as both a bioactive component and a structural substitute for cholesterol in liposomal formulations.^40,48,49 These properties provided the rationale for incorporating Rg3 into the lipid layer of the present CaPO₄-based nanoplatform.

Tumor cells frequently upregulate nutrient transporters to sustain their increased metabolic demands.⁵⁰ Glucose transporter 1 (GLUT-1) is highly expressed in many malignant cells, including breast cancer cells, and has therefore attracted interest as a potential route for enhancing tumor-associated uptake of glucose-related or glycosylated molecules.^51–53 Rg3 possesses an amphiphilic steroid-like backbone together with glycosidic moieties, and previous Rg3-functionalized liposomal systems have demonstrated enhanced tumor-associated delivery.⁴⁸ On this basis, we hypothesized that incorporation of Rg3 into the lipid layer might enhance the cellular uptake and tumor-associated accumulation of R-LCP/Q. This possibility was evaluated using cellular uptake, GLUT-1 colocalization, glucose-competition, and in vivo fluorescence-distribution experiments.

In this study, we developed a multifunctional Rg3-functionalized lipid–calcium phosphate nanoplatform loaded with Que (Rg3-Lip-CaPO₄/Quercetin, R-LCP/Q) to integrate tumor suppression, immune modulation, and cancer pain relief. Rg3 was incorporated into the lipid layer as a bioactive membrane component, whereas Que was loaded within the CaPO₄-based formulation. We hypothesized that acid-responsive degradation of the CaPO₄ component would facilitate intracellular Ca²⁺ and drug release, thereby promoting calcium-associated mitochondrial stress, reactive oxygen species generation, tumor-cell death, and immunogenic cell death.^45–47 We further investigated whether Rg3 functionalization enhanced tumor-associated uptake and whether treatment with R-LCP/Q was accompanied by dendritic cells (DCs) maturation, T-cell activation, and modulation of immunosuppressive immune-cell populations.^31,54–56 Finally, using a 4T1 breast cancer-associated pain model, we evaluated whether R-LCP/Q could alleviate mechanical and thermal hyperalgesia in association with modulation of the TRPV1–SP axis. Key mechanistic links were further examined using glucose competition, intracellular calcium chelation, in vivo fluorescence imaging, and pharmacological TRPV1 blockade. Collectively, this study was designed as a preclinical proof-of-concept evaluation of a nanotherapeutic strategy that simultaneously addresses tumor progression, antitumor immunity, and cancer-associated pain.

Experimental Section

Materials and Reagents

Detailed information regarding the materials, reagents, antibodies, fluorescent probes, assay kits, and pharmacological agents used in this study, including their suppliers and catalogue numbers, is provided in the Supporting Information.

Synthesis of R-LCP/Q Nanoparticles

CaPO₄ nanoparticles were synthesized using a reverse microemulsion method. Briefly, two organic phases were prepared: phase A, consisting of cyclohexane/Igepal CO-520 (70:30, v/v), and phase B, consisting of cyclohexane/Triton X-100/hexanol (75:15:10, v/v). After 15 mL of each phase had been mixed separately for 15 min, the two phases were combined and divided into two equal portions. For preparation of the calcium microemulsion, 600 μL of CaCl₂ solution (500 mM, pH 9.0) was added to 15 mL of the mixed organic phase. The phosphate microemulsion was prepared by adding 600 μL of Na₃PO₄ solution (25 mM) and 400 μL of L-DOPA (20 mg/mL in chloroform) to another 15 mL aliquot of the mixed organic phase. CaPO₄ core formation was initiated by dropwise addition of the calcium microemulsion to the phosphate microemulsion under vigorous stirring. After 12 h, the microemulsion was disrupted by addition of 30 mL of absolute ethanol. The resulting CaPO₄ cores were collected by centrifugation at 10,000 × g for 15 min, washed three times with ethanol, and redispersed in 1 mL of chloroform.

To prepare Que-loaded LCP/Q nanoparticles, 10 mg of Que was co-dissolved with DPPC, cholesterol, and DSPE-PEG_5k at a molar ratio of 2:2:1 and a total lipid concentration of 20 mM in 5 mL of chloroform. Subsequently, 1 mL of the prefabricated CaPO₄ cores suspension were added dropwise under ultrasonication. After stirring at 20°C for 24 h, chloroform was removed using a rotary evaporator. The resulting lipid–drug film was hydrated with 2 mL of deionized water under gentle sonication to promote assembly of the lipid layer around the CaPO₄ cores. The nanoparticles were purified by centrifugation at 1000 × g for 3 min and lyophilized for storage.

R-LCP/Q nanoparticles were prepared using the same procedure, except that ginsenoside Rg3 was incorporated into the lipid formulation in place of cholesterol. R-LCP nanoparticles were prepared using the same Rg3-containing lipid–CaPO₄ formulation but without the addition of Que.

Fluorescently labelled formulations were prepared using the same procedure by incorporating rhodamine B (RhB) or IR-780 into the corresponding lipid formulation during nanoparticle preparation.

Characterization of R-LCP/Q Nanoparticles

The hydrodynamic size, size distribution, and zeta potential of LCP/Q and R-LCP/Q nanoparticles were determined by dynamic light scattering (DLS), and their morphology was examined by transmission electron microscopy (TEM). The elemental distribution and surface chemical composition of R-LCP/Q were further characterized by elemental mapping and X-ray photoelectron spectroscopy (XPS), respectively.

The colloidal stability of R-LCP/Q was evaluated by monitoring changes in hydrodynamic size during incubation in PBS and PBS containing 10% foetal bovine serum (FBS).

The drug-loading content and encapsulation efficiency of Que were determined using UV-Vis spectroscopy and high-performance liquid chromatography (HPLC). Calcium content was quantified by inductively coupled plasma-mass spectrometry (ICP-MS).

pH-Responsive Release from R-LCP/Q Nanoparticles

The release profiles of Que, Rg3, and Ca²⁺ from R-LCP/Q nanoparticles were evaluated at pH 7.4, 6.5, and 5.5 to represent physiological, mildly acidic, and more strongly acidic conditions, respectively. R-LCP/Q nanoparticles corresponding to 1.0 mg of Que were dispersed in 10 mL of PBS at the indicated pH values and incubated at 37°C under continuous agitation. At predetermined time points, the supernatants were collected for analysis, and the release medium was replenished with an equal volume of fresh PBS at the corresponding pH.

The concentrations of released Que, Rg3, and Ca²⁺ were quantified using UV–Vis spectroscopy, HPLC, and ICP–MS, respectively. In addition, R-LCP/Q nanoparticles incubated in PBS at pH 5.5 or 7.4 for 24 h were examined by TEM to evaluate pH-dependent structural changes.

Cell Culture

HEK-293T human embryonic kidney cells, MCF-10A non-malignant mammary epithelial cells, and 4T1 murine breast cancer cells were obtained from Sunncell (Wuhan, China). HEK-293T cells were cultured in DMEM (Gibco), whereas 4T1 cells were maintained in RPMI-1640 medium (Gibco). Both media were supplemented with 10% foetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. MCF-10A cells were cultured in DMEM supplemented with 5% foetal bovine serum, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, 1% non-essential amino acids, and 1% penicillin–streptomycin. All cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Cellular Uptake

To evaluate cellular uptake, 4T1 cells were seeded onto 15-mm glass coverslips at a density of 1×10⁵ cells per well and incubated with RhB-labelled LCP/Q or R-LCP/Q at a nanoparticle concentration of 8 μg/mL for 4 h. For the glucose-competition experiment, cells were pretreated with D-glucose (50 nM) for 2 h, followed by incubation with RhB-labelled R-LCP/Q (8 μg/mL) for an additional 4 h in the continued presence of D-glucose. Cells were subsequently washed with PBS, fixed with 4% paraformaldehyde for 15 min, and counterstained with DAPI for 10 min. Fluorescence images were acquired using a Leica DM IL LED fluorescence microscope, and cellular uptake was evaluated based on RhB fluorescence intensity.

To examine the spatial association between R-LCP/Q and GLUT-1, 4T1 cells were seeded onto glass coverslips at a density of 5×10⁴ cells per well and cultured for 24 h. Cells were then incubated with RhB-labelled R-LCP/Q nanoparticles at 4 °C for 4 h and washed with PBS. After blocking with 2% BSA in PBS for 60 min at 4 °C, the cells were incubated with an anti-GLUT-1 primary antibody overnight at 4 °C, followed by incubation with an appropriate fluorescent secondary antibody and DAPI. Fluorescence images were acquired using a Leica DM IL LED fluorescence microscope, and the spatial colocalization between R-LCP/Q-associated RhB fluorescence and GLUT-1-associated fluorescence was evaluated.

In vivo Fluorescence Distribution of R-LCP/Q in 4T1 Tumor-Bearing Mice

When the tumor volume reached approximately 100 mm³, 4T1 tumor-bearing mice were randomly assigned to receive IR-780-labelled LCP/Q or IR-780-labelled R-LCP/Q by intravenous injection via the tail vein (n = 5 per group). Whole-body fluorescence images were acquired using an IVIS Spectrum imaging system at 1, 4, 8, 12, and 24 h after administration. Fluorescence intensity within the tumor region of interest was quantified to compare the temporal tumor-associated accumulation of the two formulations.

At 24 h, mice in the R-LCP/Q group were euthanized, and the major organs were harvested for ex vivo fluorescence imaging and quantitative analysis.

Intracellular Ca²⁺ Detection in 4T1 Cells

Intracellular Ca²⁺ levels in 4T1 cells were assessed using the fluorescent probe Fluo-4 AM. Briefly, cells were treated with PBS, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q. The nanoparticle formulations were applied at a total formulation concentration of 8 μg/mL for the Que-containing formulations, this corresponded to a Que concentration of 2.57 μg/mL.

For the BAPTA + R-LCP/Q group, cells were pretreated with BAPTA (1 μM) for 1 h at 37°C before addition of R-LCP/Q.⁵⁷ After 4 h of treatment, cells were washed three times with PBS and stained with Fluo-4 AM (1.0 μM). Fluorescence images were acquired using a Leica DM IL LED fluorescence microscope, and intracellular Ca²⁺ levels were quantified according to the mean fluorescence intensity of Fluo-4 AM.

Assessment of Mitochondrial Membrane Potential

Mitochondrial membrane potential (ΔΨm) in 4T1 cells were assessed using JC-1 staining. Cells were treated with PBS, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q at a nanoparticle formulation concentration of 8 μg/mL in RPMI-1640 medium supplemented with 10% foetal bovine serum. For the BAPTA + R-LCP/Q group, cells were pretreated with BAPTA (1 μM) for 1 h before addition of R-LCP/Q.

After 12 h of treatment, cells were washed with PBS and stained with JC-1 (10 μg/mL) at 37°C for 30 min in the dark. Fluorescence images were acquired using a fluorescence microscope, and mitochondrial membrane-potential changes were quantified according to the relative JC-1 monomer and aggregate fluorescence signals.

Intracellular ROS Generation

Intracellular reactive oxygen species (ROS) generation in 4T1 cells was assessed using DCFH-DA. Cells were treated with PBS, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q at a nanoparticle formulation concentration of 8 μg/mL for 12 h. BAPTA pretreatment was performed as described in Intracellular Ca²⁺ Detection in 4T1 Cells.

After treatment, the medium was replaced with fresh RPMI-1640 containing DCFH-DA (10 μM), and the cells were incubated at 37°C for 20 min in the dark. Cells were subsequently washed three times with PBS and fluorescence images were acquired using a fluorescence microscope. Intracellular ROS levels were quantified according to DCF fluorescence intensity.

In vitro Antitumor Activity and Cytocompatibility

The effects of the different formulations on 4T1 cell viability were evaluated using a CCK-8 assay. Cells were treated with increasing concentrations of LCP/Q, R-LCP or R-LCP/Q for 24 h to assess concentration-dependent cytotoxicity. For comparison among different treatments, 4T1 cells were treated with PBS, free Que, free Rg3, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q, and cell viability was determined using the CCK-8 assay according to the manufacturer’s instructions.

Cytocompatibility was assessed in HEK-293T, MCF-10A, RAW264.7 and DCs following exposure to R-LCP/Q for 24 h.

For live/dead staining, 4T1 cells were treated with PBS, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q for 24 h, followed by staining with calcein-AM and propidium iodide. Apoptosis was evaluated after the same treatments using Annexin V-FITC/PI flow cytometry. Detailed treatment concentrations and experimental procedures are provided in the Supporting Information.

Assessment of ICD and DCs Maturation in vitro

Immunogenic cell death (ICD) was evaluated by assessing cell-surface calreticulin (CRT) exposure and the release of damage-associated molecular patterns (DAMPs), including ATP and HMGB1. 4T1 cells were treated with PBS, LCP/Q, R-LCP or R-LCP/Q for 24 h. For CRT detection, cells were washed with cold PBS, fixed with 4% paraformaldehyde, blocked with 5% foetal bovine serum in PBS and incubated with a rabbit anti-CRT primary antibody overnight at 4°C, followed by staining with a fluorescent secondary antibody and DAPI. To specifically detect surface-exposed CRT, cells were not permeabilized before antibody incubation. Fluorescence images were acquired by fluorescence microscopy, and CRT exposure was quantified based on fluorescence intensity.

For DAMPs analysis, culture supernatants were collected after treatment. Extracellular ATP and HMGB1 levels were quantified using a chemiluminescence detection kit and a commercial ELISA kit, respectively, according to the manufacturers’ instructions.

DCs maturation was evaluated using a conditioned-medium assay. Supernatants collected from treated 4T1 cells were applied to bone marrow-derived DCs for an additional 24 h. The DCs were subsequently stained with fluorescent antibodies against CD11c, CD80, and CD86 and analysed by flow cytometry. Mature DCs were defined as CD11c⁺CD80⁺CD86⁺ cells.

In vivo Antitumor Efficacy and Immune Profiling

A 4T1 tumor-associated pain model was established in 4–5-week-old female BALB/c mice by intraplantar injection of 4T1 cells into the left hind paw. When the tumor volume reached approximately 100 mm³, the mice were randomly assigned to four groups (PBS, LCP/Q, R-LCP and R-LCP/Q. n = 5 per group) and received intratumoral injections every other day for 12 days. Each treatment was administered in a volume of 100 μL. The Que-loaded formulations contained 170 μg of Que per injection, corresponding to approximately 10 mg/kg.^58,59 Body weight and tumor volume were monitored every other day. At the experimental endpoint, tumors were excised, weighed and processed for H&E and TUNEL staining. CD8⁺ T-cell infiltration was evaluated by immunofluorescence staining of tumor sections. Major organs were also collected for histological examination.

For immune profiling, mature DCs were analysed in tumor-draining lymph nodes, T-cell subsets in the spleen and macrophage populations and regulatory T cells in tumor tissues by flow cytometry. The immune-cell populations and phenotypic markers used for flow-cytometric analysis are described in the Supporting Information.

All animal experiments complied with the National Research Council’s Guide for the Care and Use of Laboratory Animals and the AVMA Guidelines for the Euthanasia of Animals, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Binzhou Medical University (now Shandong Medical and Pharmaceutical University; Protocol No. BZMU-2023-419). BALB/c mice were anesthetized using a small-animal anesthesia machine (RWD-R540). The oxygen flow rate was adjusted to 400 mL/min, and the isoflurane vaporizer was set to 3% for induction. After loss of the righting reflex and stabilization of breathing, the isoflurane concentration was reduced to 1%, and the absence of a withdrawal response to toe pinch was confirmed before the procedure. During the operation, respiratory rate and mucosal color were monitored, and anesthetic depth was checked every 10–15 min. At the study endpoint, mice were deeply anesthetized with isoflurane, and euthanasia was subsequently performed by cervical dislocation. Death was confirmed by cessation of breathing, absence of heartbeat, pupil dilation, and complete muscle relaxation. Carcasses were placed in designated biohazard bags, immediately frozen and subsequently sent to the institutional animal facility for harmless disposal. The reporting of in vivo experiments was performed in accordance with the ARRIVE guidelines.

Measurement of Tumor-Associated Cytokines and Serum SP

At the experimental endpoint, tumor tissues and serum samples were collected from mice in the PBS, LCP/Q, R-LCP, R-LCP/Q and AMG9810 groups. Tumor-associated TNF-α and IL-6 levels and SP concentrations were quantified using commercial ELISA kits according to the manufacturers’ instructions.

Behavioural Assessment and TRPV1-Related Analgesic Evaluation in vivo

Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed on days 0, 2, 4, 6, 8, 10 and 12 throughout the experimental period. Behavioural assessments were performed by an investigator blinded to treatment allocation. Detailed behavioural testing procedures are provided in the Supporting Information.

To assess the contribution of TRPV1 signalling to cancer-associated hyperalgesia, a designated group of tumor-bearing mice received a single intrathecal administration of AMG9810 (100 nM, 10 μL) when the tumor volume reached approximately 100 mm³.

At the experimental endpoint, L4–L6 dorsal root ganglia (DRGs) were collected, fixed, cryosectioned, and stained with an anti-TRPV1 primary antibody and a fluorescent secondary antibody. TRPV1 expression was quantified according to mean fluorescence intensity. Detailed immunofluorescence procedures are provided in the Supporting Information.

Short-Term Systemic Safety and Haemocompatibility Evaluation

Short-term systemic safety was evaluated by haematological, coagulation, and serum biochemical analyses. Serum free haemoglobin (FHb) was measured at 6 h after the first administration and at the experimental endpoint to assess in vivo haemocompatibility. At the endpoint, haematological and coagulation parameters, including RBC, WBC, PLT and PT, were determined. Serum biochemical parameters, including ALT, AST, TBIL, BUN, creatinine, CK, LDH and calcium, were also measured.

Haemocompatibility was further evaluated using an in vitro haemolysis assay, with PBS and distilled water serving as the negative and positive controls, respectively.

Statistical Analysis

Quantitative data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 9.5. Comparisons between two groups were conducted using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparisons test. Time-course data, including tumor growth and behavioural measurements, were analysed using two-way ANOVA with multiple comparisons adjusted for multiplicity. In vitro experiments were independently repeated three times. Exact sample sizes are indicated in the corresponding figure legends. A two-sided p value < 0.05 was considered statistically significant.

Results and Discussion

Synthesis and Characterization of R-LCP/Q Nanoparticles

R-LCP/Q nanoparticles were successfully constructed as a lipid-coated calcium phosphate nanoplatform for the co-delivery of Que and ginsenoside Rg3.⁶⁰ In this formulation, Rg3 was incorporated into the PEGylated lipid layer as a bioactive structural component in place of conventional cholesterol, while the CaPO₄ cores provided the inorganic component of the hybrid nanostructure. A schematic representation of nanoparticle preparation and the proposed therapeutic mechanism is shown in Scheme 1.

Scheme 1.

Schematic illustration of R-LCP/Q nanoparticle preparation and the proposed mechanisms underlying tumor suppression, antitumor immune activation and cancer pain relief.

Dynamic light scattering showed narrow hydrodynamic size distributions, with mean diameters of 106.9 ± 1.08 nm for LCP/Q and 124.7 ± 3.24 nm for R-LCP/Q (Figure 1A). TEM imaging further revealed relatively uniform spherical morphologies for both LCP/Q and R-LCP/Q (Figure 1B and C). Both formulations displayed negative surface potentials (Figure 1D). R-LCP/Q showed only minor changes in hydrodynamic size during one week of incubation in PBS and PBS containing 10% FBS, indicating acceptable colloidal stability under the tested conditions (Figure S1). Elemental mapping demonstrated the spatial distribution of C, N, O and Ca within R-LCP/Q, while XPS supported the corresponding surface elemental composition; collectively, these findings were consistent with the hybrid inorganic-lipid composition of the nanoplatform (Figure 1E and F).

Figure 1.

Physicochemical characterization of LCP/Q and R-LCP/Q nanoparticles. (A) Hydrodynamic size distributions of LCP/Q and R-LCP/Q measured by dynamic light scattering. (B and C) Representative TEM images of LCP/Q and R-LCP/Q, respectively. Scale bars = 200 nm. (D) Zeta potentials of LCP/Q and R-LCP/Q. (E) Elemental mapping images of R-LCP/Q. Scale bar = 100 nm. (F) Survey XPS spectrum of R-LCP/Q. (G) UV–Vis absorption spectra of free Que, R-LCP and R-LCP/Q. Where applicable, quantitative data are presented as mean ± SD (n = 3).

The characteristic UV-Vis absorption peak of Que at 372 nm was retained in the R-LCP/Q formulation, supporting Que incorporation (Figure 1G). The encapsulation efficiency and drug-loading content of Que were 83.25% and 32.15%, respectively. Collectively, these physicochemical analyses support the successful preparation of R-LCP/Q nanoparticles with relatively uniform morphology, acceptable colloidal stability, and efficient Que loading.

In vitro Acidity-Responsive Dissociation and Drug Release

To evaluate the acidity-responsive behaviour of R-LCP/Q, the release profiles of Ca²⁺, Que, and Rg3 were examined at pH 7.4, 6.5 and 5.5. R-LCP/Q exhibited progressively enhanced cargo release with decreasing pH, with the most pronounced release observed at pH 5.5 (Figure S2A–C). At pH 5.5, the cumulative release of Ca²⁺, Que, and Rg3 reached 69.76%, 71.34%, and 85%, respectively, within 12 h, whereas release remained lower at pH 7.4 and showed an intermediate response at pH 6.5. These findings demonstrate a clear acidity-dependent release profile of the inorganic and therapeutic components of R-LCP/Q.

Consistent with the release behaviour, TEM imaging showed marked structural disruption of R-LCP/Q after incubation at pH 5.5 for 24 h, whereas the nanoparticles largely retained their morphology at pH 7.4 (Figure S3). These morphological changes are consistent with acid-induced destabilization of the CaPO₄-based nanostructure and the resulting acceleration of Ca²⁺ and drug release. Collectively, the results indicate limited release under physiological conditions, an intermediate response under mildly acidic conditions, and substantially enhanced degradation and cargo liberation under stronger acidity. This behaviour is consistent with a model in which mildly acidic extracellular tumor conditions may initiate partial release, whereas more acidic intracellular compartments following cellular uptake may further promote nanoparticle degradation and Ca²⁺/drug liberation.

Cellular Uptake, Tumor-Associated Distribution and in vitro Antitumor Activity

To investigate whether Rg3 functionalization enhanced cellular uptake, RhB-labelled LCP/Q and R-LCP/Q were incubated with 4T1 cells. R-LCP/Q produced a markedly stronger intracellular fluorescence signal than LCP/Q after 4 h (Figure 2A and B). In the glucose-competition experiment, D-glucose pretreatment markedly reduced the uptake of RhB-labelled R-LCP/Q under the tested conditions. In a separate colocalization assay, RhB-labelled R-LCP/Q showed substantial spatial overlap with GLUT1-associated fluorescence in 4T1 cells (Figure 2C and D). Taken together, the colocalization and glucose-competition findings support the involvement of a glucose-sensitive, GLUT-1-associated process in the enhanced cellular uptake of R-LCP/Q.^61,62 However, these findings do not establish receptor specificity or prove that GLUT-1 is the exclusive route of R-LCP/Q internalization.

Figure 2.

Cellular uptake and in vitro antitumor activity of R-LCP/Q. (A) Representative fluorescence images of 4T1 cells incubated with RhB-labelled LCP/Q, R-LCP/Q or R-LCP/Q under D-glucose competition for 4 h. Scale bar = 100 μm. (B) Quantitative analysis of intracellular RhB fluorescence intensity following the indicated treatments. (C) Representative fluorescence images showing the spatial distribution of GLUT-1-associated fluorescence and RhB-labelled R-LCP/Q in 4T1 cells. Scale bar = 50 μm. (D) Line-profile analysis of the corresponding R-LCP/Q-associated RhB and GLUT-1-associated fluorescence signals. (E) Concentration-dependent viability of 4T1 cells following 24 h treatment with LCP/Q, R-LCP or R-LCP/Q. (F) Viability of 4T1 cells following 24 h treatment with PBS, free Que, free Rg3, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q at the selected treatment concentrations. (G) Representative calcein-AM/PI live/dead fluorescence images of 4T1 cells after the indicated treatments. Scale bar = 50 μm. (H) Annexin V-FITC/PI flow-cytometric analysis of apoptosis in 4T1 cells treated with PBS, LCP/Q, R-LCP, R-LCP/Q or BAPTA + R-LCP/Q for 24 h. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

To further evaluate tumor-associated distribution in vivo, IR-780-labelled LCP/Q and R-LCP/Q were intravenously administered to 4T1 tumor-bearing mice. Serial fluorescence imaging showed greater tumor-associated fluorescence accumulation of R-LCP/Q than LCP/Q (Figure S4A and B). Ex vivo imaging of major organs from the R-LCP/Q group further characterized its tissue distribution at the terminal time point (Figure S4C and D). Together, these findings indicate that Rg3 functionalization is associated with enhanced cellular uptake and tumor-associated accumulation of the nanoplatform.

We next evaluated the in vitro antitumor activity and cytocompatibility of R-LCP/Q. R-LCP/Q showed limited cytotoxicity towards the tested non-malignant and immune-related cell models, including HEK-293T, MCF-10A, RAW264.7 cells and DCs under the examined conditions (Figure S5). By contrast, R-LCP/Q produced concentration-dependent inhibition of 4T1 cell viability (Figure 2E). Considering tumor-cell inhibition together with cytocompatibility, 8 μg/mL R-LCP/Q was selected for subsequent mechanistic experiments.

At the selected treatment condition, free Que, free Rg3, LCP/Q, R-LCP, and R-LCP/Q were further compared (Figure 2F). R-LCP/Q produced the greatest reduction in 4T1 cell viability among the tested formulations, supporting the therapeutic advantage of integrating Que, Rg3, and the CaPO₄-based carrier within a single formulation.

Consistent with the CCK-8 results, live/dead staining showed the greatest proportion of dead cells after R-LCP/Q treatment (Figures 2G and S6). Annexin V-FITC/PI flow cytometry showed apoptotic rates of 10.22%, 27.2%, 28.6%, and 33.8% in the PBS, LCP/Q, R-LCP, and R-LCP/Q groups, respectively. Notably, BAPTA pretreatment reduced R-LCP/Q-associated apoptosis to 10.99% (Figures 2H and S7), supporting a substantial contribution of intracellular Ca²⁺ accumulation to R-LCP/Q-induced tumor-cell death.

R-LCP/Q Induces Intracellular Ca²⁺ Accumulation

Following cellular internalization, degradation of the CaPO₄ component is expected to release Ca²⁺ under acidic intracellular conditions.⁶³ Intracellular Ca²⁺ levels were therefore assessed using Fluo-4 AM. R-LCP and R-LCP/Q treatment produced markedly stronger Fluo-4 fluorescence than PBS and LCP/Q, indicating increased intracellular Ca²⁺ accumulation (Figures 3A and S8).

Figure 3.

Intracellular Ca²⁺ accumulation, mitochondrial depolarization, and ROS generation in 4T1 cells. (A) Representative Fluo-4 AM fluorescence images showing intracellular Ca²⁺ levels in 4T1 cells following the indicated treatments. (B) Representative JC-1 fluorescence images showing changes in ΔΨm; red fluorescence represents JC-1 aggregates associated with preserved ΔΨm, whereas green fluorescence represents JC-1 monomers associated with mitochondrial depolarization. (C) Representative DCFH-DA fluorescence images showing intracellular ROS generation following the indicated treatments. Scale bars = 50 μm. Representative images are from three independent experiments.

Importantly, BAPTA pretreatment markedly attenuated the fluorescence increase induced by R-LCP/Q. Together with the pH-responsive Ca²⁺ release results, these findings are consistent with intracellular Ca²⁺ elevation associated with acid-responsive CaPO₄ degradation and provide a mechanistic basis for evaluating the downstream mitochondrial effects of R-LCP/Q.

Ca²⁺-Associated Mitochondrial Dysfunction

Given the intracellular Ca²⁺ accumulation observed above, mitochondrial membrane potential (ΔΨm) was assessed using JC-1 staining.⁶⁴ PBS-treated cells predominantly exhibited red JC-1 aggregate fluorescence, consistent with preserved mitochondrial membrane potential, whereas R-LCP/Q-treated cells showed a pronounced shift towards green monomer fluorescence (Figure 3B), indicating mitochondrial depolarization.⁶⁵

Quantitative analysis showed a marked shift from JC-1 aggregate fluorescence towards JC-1 monomer fluorescence after R-LCP/Q treatment, whereas BAPTA pretreatment substantially attenuated this shift (Figure S9). Together with the intracellular Ca²⁺ findings, these results support a functional link between R-LCP/Q-induced Ca²⁺ accumulation and mitochondrial membrane depolarization.

Intracellular ROS Generation

Given the mitochondrial depolarization observed above, intracellular ROS generation was assessed using DCFH-DA. R-LCP/Q treatment produced the strongest DCF fluorescence among the tested groups, indicating increased intracellular ROS generation (Figures 3C and S10).

Importantly, BAPTA pretreatment markedly attenuated the R-LCP/Q-induced ROS signal. Together with the intracellular Ca²⁺ and mitochondrial membrane-potential findings, these results support a mechanistic sequence in which R-LCP/Q-induced intracellular Ca²⁺ accumulation contributes to mitochondrial depolarization and subsequent oxidative stress. However, because a dedicated ROS-scavenger rescue experiment was not performed, these data do not establish ROS as an essential downstream mediator of R-LCP/Q-induced cytotoxicity.

R-LCP/Q Promotes ICD-Associated DAMPs Release and DCs Maturation

Both ROS and ginsenoside Rg3 have been implicated in the induction of ICD.⁶⁶ We therefore examined established ICD-associated danger signals, including CRT exposure and extracellular ATP and HMGB1 release, which are important hallmarks of ICD.^67,68 R-LCP/Q treatment induced the most pronounced increase in cell-surface CRT exposure among the tested groups (Figure 4A and B). Consistently, extracellular ATP and HMGB1 levels in the culture supernatants were markedly increased following R-LCP/Q treatment compared with PBS, LCP/Q and R-LCP (Figure 4C and D).⁶⁹ Extracellular ATP can function as a “find-me” signal during ICD-associated immune activation.69 Together, these findings are consistent with established features of ICD.^70,71

Figure 4.

In vitro assessment of ICD-associated DAMPs and DCs maturation. (A) Representative fluorescence images of cell-surface CRT exposure in 4T1 cells following the indicated treatments. Scale bar = 50 μm. (B) Quantitative analysis of cell-surface CRT mean fluorescence intensity (MFI). (C) Quantification of extracellular ATP in 4T1 cell culture supernatants by chemiluminescence assay. (D) Quantification of extracellular HMGB1 in 4T1 cell culture supernatants by ELISA. (E) Representative flow-cytometric analysis of DCs maturation following exposure to conditioned media from treated 4T1 cells. Mature DCs were defined as CD11c⁺CD80⁺CD86⁺ cells. (F) Quantification of mature DCs proportions. Data are presented as mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

We next evaluated whether tumor-cell-conditioned media were associated with DC maturation. Conditioned medium derived from R-LCP/Q-treated 4T1 cells increased the proportion of mature DCs to 42.5 ± 1.7%, compared with 10.9 ± 1.8%, 16.9 ± 1.5% and 25.9 ± 3.3% in the PBS, LCP/Q and R-LCP groups, respectively (Figure 4E and F). These findings support enhanced ICD-associated immunogenic signalling and subsequent DCs maturation following R-LCP/Q treatment.

Because conditioned-medium experiments cannot completely distinguish ICD-dependent DCs maturation from possible direct or residual effects of nanoplatform components, these findings are interpreted as supportive rather than definitive evidence linking R-LCP/Q-induced ICD-associated signals to DCs activation.

Antitumor Efficacy of R-LCP/Q in vivo

The antitumor efficacy of R-LCP/Q was subsequently evaluated in the 4T1 tumor-associated pain model. When the tumor volume reached approximately 100 mm³, mice were randomly allocated to the PBS, LCP/Q, R-LCP or R-LCP/Q treatment groups (n = 5 per group) and tumor growth was monitored throughout the 12-day treatment period (Figure 5A).

Figure 5.

In vivo antitumor efficacy of R-LCP/Q in 4T1 tumor-bearing mice. (A) Schematic illustration of the in vivo experimental schedule. (B) Tumor growth curves during the treatment period. (C) Representative photographs of excised tumors at the experimental endpoint. (D) Average tumor weights following the indicated treatments. (E) Representative H&E-stained tumor sections. (F) Representative TUNEL-stained tumor sections. Data are presented as mean ± SD (n = 5 per group). Scale bars in (E and F) = 125 μm. **p < 0.01 and ***p < 0.001.

R-LCP/Q produced the greatest inhibition of tumor growth among the tested formulations (Figure 5B and C), whereas LCP/Q and R-LCP produced more moderate effects. Consistent with the tumor-volume data, the mean tumor weight in the R-LCP/Q group was reduced to 35.71% of that in the PBS group at the experimental endpoint (Figure 5D), further supporting the superior antitumor efficacy of R-LCP/Q.

Histological analysis showed extensive tumor-cell damage, nuclear shrinkage, cytoplasmic vacuolization, and reduced cellularity following R-LCP/Q treatment (Figure 5E). TUNEL staining further demonstrated the strongest apoptotic signal in the R-LCP/Q group among the tested formulations (Figure 5F).

Collectively, these findings demonstrate that R-LCP/Q provides greater in vivo antitumor efficacy than the corresponding single-component nanoparticle formulations under the present experimental conditions, consistent with its enhanced antitumor activity observed in vitro.

R-LCP/Q Alleviates Cancer-Associated Hyperalgesia

Previous studies have implicated TRPV1-related signalling in the analgesic effects of Que.²⁸ We therefore evaluated whether R-LCP/Q could alleviate cancer-associated mechanical and thermal hypersensitivity in the 4T1 tumor-associated pain model. MWT and TWL were assessed on days 0, 2, 4, 6, 8, 10, and 12 according to the experimental schedule shown in Figure 5A.

Following treatment initiation, MWT and TWL progressively declined in the PBS group, indicating increasing mechanical and thermal hypersensitivity during tumor progression. In contrast, LCP/Q and R-LCP/Q treatment attenuated these behavioural changes, with R-LCP/Q producing the most pronounced alleviation of mechanical and thermal hypersensitivity (Figure 6A and B). R-LCP produced a more moderate improvement, suggesting that tumor suppression and associated changes in the local tumor environment may themselves contribute to pain relief.

Figure 6.

Behavioural assessment, TRPV1–SP-associated changes, and histological evaluation of pulmonary metastasis. (A) MWT and (B) TWL measured during the treatment period. (C) Representative immunofluorescence images of TRPV1 expression in L4–L6 DRGs at the experimental endpoint. (D) SP concentrations measured by ELISA. (E and F) Tumor-associated TNF-α (E) and IL-6 (F) levels measured by ELISA. (G) Representative H&E-stained lung sections for histological assessment of pulmonary metastatic foci following the indicated treatments. Data are presented as mean ± SD (n = 5 per group). Scale bars in the corresponding histological and immunofluorescence images = 125 μm. ns ≥ 0.05, *p < 0.05, **p < 0.01 and ***p < 0.001.

To examine the contribution of TRPV1 signalling, a separate group of tumor-bearing mice received the selective TRPV1 antagonist AMG9810. Pharmacological TRPV1 blockade also increased MWT and TWL in the absence of R-LCP/Q treatment, supporting an important contribution of TRPV1 signalling to nociceptive hypersensitivity in this model. The pharmacological blockade of TRPV1 with AMG9810, even in the absence of R-LCP/Q therapy, was associated with elevated MWT and TWL, reinforcing the mechanistic contribution of TRPV1 signaling to pain sensitization in this model. In addition, a key control comparison of tumor volumes between the AMG9810 and PBS groups demonstrated that the observed analgesic effects were not consequent to tumor burden reduction (Figure S12).

Association of Analgesia with Modulation of the TRPV1–SP Axis

To further investigate the neurobiological changes associated with pain relief, TRPV1 expression in L4–L6 DRGs and circulating SP levels were examined after treatment. R-LCP/Q treatment was associated with markedly reduced TRPV1 immunofluorescence in DRGs compared with PBS and R-LCP treatment (Figures 6C and S11). Low TRPV1 immunofluorescence was also observed in the AMG9810 group. Serum SP concentrations were similarly reduced following R-LCP/Q and AMG9810 treatment (Figure 6D).

In parallel, tumor-associated TNF-α and IL-6 levels were reduced following R-LCP/Q treatment (Figure 6E and F), suggesting attenuation of the local inflammatory milieu that may additionally contribute to peripheral nociceptive sensitization. Collectively, these findings indicate that the analgesic response to R-LCP/Q is associated with modulation of TRPV1–SP signalling together with reduced tumor-associated inflammation.

Nevertheless, the present design does not establish that Que directly suppresses TRPV1 independently of tumor control. Because direct nociceptive modulation and analgesia secondary to tumor inhibition were not experimentally separated, their relative contributions cannot be fully distinguished in the present study.

R-LCP/Q Promotes Antitumor Immune Activation in vivo

To determine whether the ICD-associated responses observed in vitro were accompanied by antitumor immune activation in vivo, representative immune-cell populations were analysed in tumor-draining lymph nodes, spleens, and tumor tissues following treatment. R-LCP/Q markedly increased the proportion of CD11c⁺CD80⁺CD86⁺ mature DCs in tumor-draining lymph nodes to 27.6%, compared with 16.2%, 19.2% and 20.8% in the PBS, LCP/Q and R-LCP groups, respectively (Figures 7A and S13A). These findings indicate enhanced DCs maturation and lymph-node immune priming following R-LCP/Q treatment.

Figure 7.

In vivo antitumor immune activation following R-LCP/Q treatment. (A) Flow-cytometric analysis of mature dendritic cells (CD11c⁺CD80⁺CD86⁺) in tumor-draining lymph nodes. (B) Flow-cytometric analysis of splenic CD4⁺ and CD8⁺ T-cell populations. (C and D) Flow-cytometric analysis of M1-like macrophages (CD11b⁺F4/80⁺CD86⁺) (C) and M2-like macrophages (CD11b⁺F4/80⁺CD206⁺) (D) in tumor tissues. (E) Flow-cytometric analysis of tumor-infiltrating regulatory T cells (Tregs; CD4⁺CD25⁺FoxP3⁺). (F) Quantification of the intratumoral M1/M2 macrophage ratio following the indicated treatments. (G) Immunofluorescence analysis of CD8⁺ T-cell infiltration in tumor tissues. Data are presented as mean ± SD (n = 5 per group). Scale bar in (G) = 125 μm. ***p < 0.001.

Consistent with enhanced DC maturation, the proportion of splenic CD8⁺ T cells increased to 30.2% in the R-LCP/Q group, compared with 12.5%, 17.8%, and 24.2% in the PBS, LCP/Q, and R-LCP groups, respectively (Figures 7B and S13B). Immunofluorescence analysis of tumor sections further showed increased CD8⁺ T-cell infiltration following R-LCP/Q treatment (Figures 7G and S13C). Together, these findings support both systemic and intratumoral antitumor immune activation.

Ginsenoside Rg3 has previously been reported to modulate tumor-associated immune responses and macrophage phenotypes.^40,72 We therefore further examined macrophage and regulatory T-cell populations within tumor tissues. R-LCP/Q treatment shifted the macrophage phenotype distribution towards an M1-like profile and away from an M2-like profile (Figures 7C, D and S13D, E). Specifically, the M2-like population decreased from 38.7% to 25.2%, whereas the M1-like population increased from 7.46% to 20.8%. Accordingly, the M1/M2 ratio reached 0.786 ± 0.039 in the R-LCP/Q group, significantly higher than in the other treatment groups (Figure 7F). The frequency of tumor-infiltrating CD4⁺CD25⁺FoxP3⁺ Tregs was also reduced from 16.1% to 9.68% following R-LCP/Q treatment (Figures 7E and S13F).

Collectively, these immune-cell changes are consistent with attenuation of an immunosuppressive tumor immune phenotype and enhanced antitumor immune activation following R-LCP/Q therapy.

Histological Assessment of Pulmonary Metastasis

Given the reported involvement of SP signalling in tumor progression and metastasis,^23,73 pulmonary tissues were histologically examined at the experimental endpoint. Numerous metastatic foci were apparent in lung sections from PBS-treated mice, whereas fewer metastatic foci were observed in the examined sections following LCP/Q or R-LCP treatment (Figure 6G). In the examined sections from the R-LCP/Q and AMG9810 groups, no obvious metastatic foci were identified.

These histological observations suggest that R-LCP/Q treatment may reduce pulmonary metastatic burden in this model. However, because the current assessment was based on endpoint histological examination rather than systematic quantitative analysis of whole-lung metastatic burden, this potential antimetastatic effect should be interpreted as preliminary.

Short-Term Systemic Safety and Haemocompatibility

Short-term systemic safety was evaluated by histopathological examination, body-weight monitoring, and haematological and biochemical analyses. No overt histopathological abnormalities were observed in the major organs, including the heart, liver, spleen, lungs, and kidneys, after R-LCP/Q treatment compared with the PBS control (Figure S14). Body weights remained relatively stable throughout the treatment period in all groups (Figure S15).

Haematological and biochemical analyses further showed that R-LCP/Q treatment did not result in significant changes in ALT, AST, TBIL, LDH, BUN, creatinine, CK, RBC, PLT, PT or serum calcium compared with the PBS group (Figure S16). The WBC count was modestly but significantly increased in the R-LCP/Q group. Overall, the measured parameters did not reveal overt short-term treatment-associated abnormalities in hepatic or renal function, coagulation, or most haematological indices, apart from the modest increase in WBC count.

Haemocompatibility was further evaluated by serum FHb measurements in vivo and an erythrocyte haemolysis assay in vitro. Serum FHb concentrations measured 6 h after the first administration and at the experimental endpoint did not differ significantly between the PBS and R-LCP/Q groups (Figure S17A and B). In the in vitro haemolysis assay, supernatants from erythrocytes exposed to LCP/Q or R-LCP/Q remained comparable to the PBS negative control, whereas distilled water produced complete haemolysis (Figure S17C). Quantitative analysis showed haemolysis rates below 5% for both nanoparticle formulations (Figure S17D). Collectively, these findings support favourable short-term tolerability and haemocompatibility of R-LCP/Q under the tested conditions.

Limitations of the Study

Several limitations should be considered when interpreting the present findings. First, the intraplantar 4T1 tumor model provides a useful model of tumor-associated peripheral hyperalgesia but does not reproduce the full anatomical and biological complexity of cancer pain in patients, particularly skeletal metastasis-associated pain. Validation in additional clinically relevant models will therefore be necessary.

Second, although Rg3 functionalization enhanced cellular uptake and tumor-associated accumulation, the GLUT-1 colocalization and D-glucose competition experiments support, but do not definitively establish, a specific GLUT-1-mediated uptake mechanism. Receptor-specific pharmacological blockade or genetic interrogation of GLUT-1 would be required to determine the precise contribution of GLUT-1 to R-LCP/Q internalization.

Third, the greater antitumor efficacy of R-LCP/Q compared with the individual nanoparticle formulations should not be interpreted as pharmacological synergy because a formal combination-index or equivalent synergy analysis was not performed. Similarly, although pharmacological TRPV1 blockade with AMG9810 supports the involvement of TRPV1 signalling in nociceptive hypersensitivity, the present design does not fully distinguish direct nociceptive modulation by R-LCP/Q from secondary pain relief resulting from tumor suppression.

Fourth, the in vivo behavioural and immunological experiments used a relatively small exploratory sample size (n = 5 per group), and immune profiling was performed primarily at the experimental endpoint. Larger independently replicated studies with longitudinal immune characterization will be required to confirm the robustness and temporal dynamics of these responses.

Fifth, intratumoral administration was used for therapeutic evaluation in the present proof-of-concept study. Although intravenous fluorescence imaging demonstrated tumor-associated accumulation of R-LCP/Q, formal pharmacokinetic analysis, quantitative systemic biodistribution, and therapeutic and analgesic efficacy following systemic administration remain to be established.

Finally, the present safety evaluation was limited to short-term histological, biochemical, haematological, and haemocompatibility endpoints. Longer-term toxicity, repeated-dose immunogenicity, and systemic inflammatory responses require further investigation. In addition, pulmonary metastasis was evaluated primarily by endpoint histology rather than systematic quantitative analysis of whole-lung metastatic burden, and the apparent antimetastatic effect should therefore be considered preliminary.

Conclusion

In conclusion, we developed a multifunctional R-LCP/Q nanoplatform that integrates tumor suppression, antitumor immune modulation, and cancer pain relief. Rg3 functionalization enhanced cellular uptake and tumor-associated accumulation of the nanoplatform. Acid-responsive degradation of the CaPO₄ component promoted intracellular Ca²⁺ accumulation, mitochondrial membrane depolarization, ROS generation, and tumor-cell death, with BAPTA rescue experiments supporting a substantial Ca²⁺-dependent contribution to these effects. R-LCP/Q also induced ICD-associated hallmarks and was accompanied by enhanced DCs maturation, increased CD8⁺ T-cell responses, and attenuation of immunosuppressive immune-cell phenotypes.

In parallel, R-LCP/Q alleviated mechanical and thermal hyperalgesia in association with modulation of TRPV1–SP signalling, while the analgesic response to the TRPV1 antagonist AMG9810 further supported the involvement of TRPV1 signalling in nociceptive hypersensitivity. Histological examination additionally suggested a preliminary reduction in pulmonary metastatic burden following R-LCP/Q treatment. The formulation showed favourable short-term tolerability and haemocompatibility under the tested conditions.

Collectively, these findings provide preclinical proof of concept for a multifunctional nanotherapeutic strategy that integrates tumor control, antitumor immune activation, and cancer-associated pain relief, while further mechanistic validation, systemic therapeutic evaluation, and longer-term safety studies remain warranted.

Funding Statement

This work was supported by the Education Department of Shandong Province (No. 2022KJ091), the State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology (No. CRE-24C003), the National Natural Science Foundation of China (Grant Nos. 51903015 and 81772281), the Scientific Research Foundation of Binzhou Medical University (No. 50012304274), the Shandong Province Taishan Scholar Project (No. ts201712067), and the Scientific Research Startup Fund of Yantai Affiliated Hospital of Binzhou Medical University (Grant No. BY2019KYQD50).

Data Sharing Statement

The raw and processed data supporting the findings of this study are available from the corresponding author, Guofeng Zhang, upon reasonable request.

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 declare that they have no competing interests in this work.