Nanoparticles co-loaded with bacillus Calmette-Guérin pure protein derivative and doxorubicin for bladder tumor therapy

Abstract

Bladder perfusion chemotherapy remains the standard treatment for bladder cancer, yet its effectiveness is frequently limited by rapid drug clearance through urinary excretion and inadequate infiltration of immune cells into bladder tissue. To address these challenges, we developed an active-targeting nano-drug delivery system specifically designed for bladder tumors. This system utilizes a sialic acid-targeted poly (lactic-co-glycolic acid) (PLGA) platform to co-deliver doxorubicin (DOX) and the purified protein derivative (PPD) of bacillus Calmette-Guérin (BCG-PPD). By leveraging the selective binding of phenylboronic acid to sialic acid, the system enhances tumor-specific drug uptake, significantly amplifying DOX’s therapeutic efficacy and inducing immunogenic cell death. Furthermore, BCG-PPD exerts potent immunostimulatory effects, promoting dendritic cell (DC)-mediated tumor antigen processing and presentation, which in turn drives robust cytotoxic T lymphocyte (CTL) infiltration into the tumor microenvironment. The superior anti-tumor performance of this system was validated in an orthotopic bladder cancer mouse model. In conclusion, by synergistically combining targeted drug delivery with chemo-immunotherapy, our nanoparticle system presents a highly effective and promising new paradigm for bladder cancer therapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12896-026-01122-4.

Introduction

Bladder cancer (BCa) stands as one of the most prevalent malignancies globally, with non-muscle invasive bladder cancer (NMIBC) constituting 75% of all BCa instances [1, 2]. Despite standardized treatment and meticulous follow-up care, NMIBC exhibits a relapse rate exceeding 50%, and approximately 30% of patients experience disease progression [3–5]. Consequently, there is an urgent call for innovative treatment strategies that can effectively eliminate NMIBC.

Considering the unique structure of the bladder, transurethral intravesical administration is a common route for NMIBC clinical treatment [6]. Doxorubicin (DOX) is a standard agent in intravesical chemotherapy, primarily eradicating residual tumor cells through direct cytotoxicity. Notably, DOX can also induce immunogenic cell death (ICD) [7]. During ICD, dying tumor cells release damage-associated molecular patterns (DAMPs) that act as danger signals to recruit dendritic cells (DCs) to the tumor site. These DCs capture and process the tumor antigens, undergo maturation, and migrate to draining lymph nodes. Within the lymph nodes, mature DCs present tumor-derived peptides to naive T cells via MHC molecules, thereby activating and clonally expanding tumor-antigen-specific T lymphocytes to establish systemic antitumor immunity [8].

However, intravesical DOX monotherapy has not achieved satisfactory clinical outcomes due to multiple complex challenges. First, urine dilution and rapid clearance via physiological bladder emptying significantly reduce the effective intravesical concentration and shorten the retention time of DOX, thereby compromising its direct cytotoxic potency [9, 10]. Furthermore, although DOX-induced ICD exposes tumor antigens and provides the initial signal for immune recognition, the immunosuppressive tumor microenvironment often results in insufficient antigen presentation, making it difficult to activate effective anti-tumor immunity [11, 12]. Therefore, strategies aimed at enhancing drug uptake and converting tumor antigen exposure into potent and sustained immune activation are crucial for improving therapeutic efficacy.

Recent advancements in targeted drug delivery have dramatically refined the process of delivering medications [13]. Sialic acid (SA), a terminal sugar residue, is frequently overexpressed on tumor tissues, constituting a hallmark of aberrant sialylation in cancer compared to normal tissues [14, 15]. Consequently, SA is therefore considered a pivotal targeting point in intravesical therapy for bladder cancer. A promising ligand for this target is phenylboronic acid (PBA), which specifically binds to overexpressed SA on tumor cells to form a stable complex [16, 17]. Hence, PBA functions as a ligand, offering a novel strategy for targeted therapy of bladder cancer.

Bacillus Calmette–Guérin (BCG), a live attenuated strain of Mycobacterium bovis, is the cornerstone immunotherapy for NMIBC [18]. However, the therapeutic application of BCG is often limited by its adverse effects, which range from common local irritation to severe systemic complications [19]. To mitigate these safety concerns while retaining potent immunostimulatory activity, we focused on the BCG protein derivative (BCG-PPD), a preparation derived from BCG cultures that consists of purified proteins and metabolites. Given its strong immunogenicity, BCG-PPD may synergize with DOX-induced ICD by recruiting and maturing DCs, enhancing antigen presentation, and ultimately stimulating a potent tumor-specific T cell response.

In this study, we developed PBA-functionalized nanoparticles to enhance the tumor-specific delivery of DOX and BCG-PPD by selectively targeting SA overexpressed on bladder cancer cells. This targeting strategy significantly increases intracellular DOX accumulation, potentiating its direct cytotoxic effect and the induction of ICD with consequent tumor antigen exposure. Through combined ICD signals and BCG-PPD-derived chemokines, the treatment drives the recruitment of immature DCs to the tumor, facilitating antigen capture and DC maturation. The mature DCs subsequently migrate to lymph nodes, where they efficiently present the antigens to T cells, thereby orchestrating a potent tumor-specific T-cell response (Fig. 1). Together, this nanoparticle-based co-delivery system provides a novel and efficient platform for improving therapeutic outcomes in non-muscle-invasive bladder cancer (NMIBC) through enhanced drug uptake and synergistic immunopotentiation.

Fig. 1.

(A) Fabrication scheme of the multifunctional PLGA-PEG-PBA nanoparticles co-encapsulating DOX and BCG-PPD (Combo@NPs-T). (B) Schematic illustration of the therapeutic mechanism after intravesical administration. Phenylboronic acid (PBA) targets sialic acid (SA) on bladder cancer cells, enhancing specific cellular uptake and significantly increasing the intracellular accumulation of DOX. This potentiates the direct cytotoxic effect and induces immunogenic cell death (ICD), leading to tumor antigen release. Simultaneously, BCG-PPD acts synergistically by recruiting and maturing dendritic cells (DCs). The mature DCs subsequently migrate to lymph nodes, where they efficiently present the antigens to T cells, priming a robust adaptive immune response against the tumor.

Materials and methods

All materials, antibodies, and chemical reagents employed in this study are detailed in Table S1.

SA detection in human tissue and cell lines

Clinical Samples and Immunohistochemistry Pathologically confirmed bladder urothelial carcinoma tissue samples (n = 9) were obtained from the First Affiliated Hospital of Chongqing Medical University (Ethics Approval No. 2022-043). Paraffin-embedded tissue sections were prepared and processed for immunohistochemical staining. Quantitative analysis of staining intensity was performed using Image-Pro Plus software (v6.0) by measuring integrated optical density values.

Cell Culture and Immunofluorescence Staining Three bladder cancer cell lines (MB-49, T24, and UM-UC-3) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (ExcellBio) and 1% penicillin-streptomycin (Gibco), while the normal bladder epithelial cell line SV-HUC-1 was cultured in Ham’s F-12 K medium (Procell) with identical supplements.

For sialic acid detection, cells were seeded on 12-mm coverslips in 24-well plates and cultured for 24 h. After phosphate buffered saline‌ (PBS) washing, cells were fixed with 4% paraformaldehyde (15 min), blocked with Carbo-Free Blocking Solution (30 min), and incubated with Cy3-conjugated Sambucus Nigra Lectin (SNA; 2 h, dark). Following 4’,6-diamidino-2-phenylindole (DAPI) counterstaining (10 min), samples were imaged using a Leica fluorescence microscope (Germany). Cy3 fluorescence intensity was quantified using ImageJ software (v1.50i).

Cell viability detected by cell counting Kit-8 (CCK-8) assay

Cell Viability Assay Cells were seeded in 96-well plates at a density of 1,000 cells/well and allowed to adhere for 12 h prior to drug treatment. Following 24–48 h of drug exposure, 10 µL of CCK-8 reagent was added to each well and incubated at 37 °C for 1 h. Absorbance was measured using a Thermo Scientific microplate reader (USA).

Preparation and characterization of nanoparticles

Weigh PLGA-PEG-NH₂ into a flask and add chloroform to dissolve it until the solution becomes clear. Weigh carboxyphenylboronic acid (1.0 eq) into an EP tube, dissolve it with 0.5 ml of N, N - Dimethylformamide, and then add the solution to the flask. Add 1 - (3 - Dimethylaminopropyl) − 3 - ethylcarbodiimide hydrochloride (3.0 eq), N - Hydroxysuccinimide (3.0 eq), and triethylamine (3.0 eq), and stir the reaction mixture at 40℃ for 4 h. Wash the product with water three times. After drying, precipitate it in ice-cold diethyl ether, and then remove the diethyl ether using a vacuum pump. The product PLGA-PEG-PBA is obtained.

In the preparation of poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating DOX and BCG-PPD, we employed a multi-step emulsion technique [20, 21]. Initially, we prepared 1 ml of an aqueous DOX solution at a concentration of 2 mg/ml using ultrapure water and combined it with 2 ml of a dichloromethane solution containing PLGA-polyethylene glycol (PEG)-PBA at 25 mg/ml. We then introduced 100 µl of a BCG-PPD stock solution (2.14 mg/ml) into this mixture and subjected it to sonication for 3 min (5 s on, 5 s off, at 35% power). Next, we added 5 ml of a polyvinyl alcohol solution (2% w/v) to the emulsion and sonicated the blend for another 3 min. To solidify the nanoparticle shell, we incorporated 10 ml of an isopropanol solution (2% v/v) and removed the organic solvent by magnetic stirring for 4 h. Finally, the nanoparticle solution was centrifuged (13,000 rpm, 4 °C, for 10 min) to collect the nanoparticles, which were then stored at 4 °C. This process yielded the nanoparticles designated as DOX/BCG-PPD@PLGA-PEG-PBA (Combo@NPs-T). The DOX content within the nanoparticles and the drug release kinetics were quantified using high-performance liquid chromatography (Agilent 1260 Infinity II) and fluorescence spectrophotometry (SHIMADZU RF-6000). Additionally, the BCG-PPD content in the nanoparticles was measured using an Enhanced BCA Protein Assay Kit (Beyotime).

Cellular uptake and targeting verification of nanoparticles

MB49 cells were seeded into confocal dishes at a density of 1 × 10⁵ cells per dish in 1 mL of DMEM medium and incubated overnight. Subsequently, Combo@NPs-T and DOX/BCG-PPD@PLGA-PEG (referred to as Combo@NPs), containing DOX at a concentration of 10 µg/ml, were added and co-incubated for 1–2 h. The MB49 cells were then fixed with 4% paraformaldehyde (PFA) and the nuclei were stained with DAPI for 10 min. A confocal laser scanning microscope (CLSM; Nikon A1, Japan) was utilized to observe the cellular uptake of the nanoparticles, with flow cytometry employed for quantitative analysis.

To further confirm the targeting specificity, MB49 cells and SV-HUC-1 cells were cultured on confocal dishes at three time points: 0.5 h, 1 h, and 2 h. The MB49 cells were treated with Combo@NPs-T, Combo@NPs-T with free PBA, or Combo@NPs-T with chlorpromazine, while the SV-HUC-1 cells received Combo@NPs-T alone. Cellular uptake of the Combo@NPs-T was visualized under a CLSM.

To track the intracellular trafficking and lysosomal involvement in the uptake of nanoparticles, MB49 cells were co-incubated with Combo@NPs-T for varying durations (0.5, 1, and 2 h). Lysosomes were labeled with Lyso-Tracker Green and the nuclei were stained with Hoechst 33,342. Live-cell imaging was immediately carried out using a confocal laser scanning microscope.

To evaluate the targeting capabilities of the nanoparticles, they were incubated with tissue frozen sections at a concentration of 300 µg/ml of DOX. Furthermore, the fluorescence intensity of DOX in mouse tissues was assessed using fluorescence microscopy.

In vitro antitumor efficacy

MB49 cells were seeded in six-well plates at a density of 4 × 10⁵ cells per well and incubated overnight. The DMEM culture medium was then replaced with fresh medium containing nanoparticles. After 24 h of incubation, the cells were washed with PBS and digested with trypsin. To avoid interference from the spectral overlap between DOX autofluorescence and the PI channel, we stained MB49 cells with Annexin V-FITC and DAPI for flow cytometric analysis of apoptosis. Additionally, cells subjected to the same treatment were identified as live or dead through Calcein-AM and DAPI double staining and observed under a laser confocal microscope.

Evaluation of ICD induction by detection of DAMPs

MB49 cells were seeded in confocal dishes at a density of 1 × 10⁵ cells per dish and incubated overnight. The drugs and nanoparticles were then co-cultured with the MB49 cells for 24 h. Following this, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. To block non-specific antigens, goat serum was applied for 30 min. Subsequently, the MB49 cells were incubated overnight at 4 °C with either anti-Calreticulin (CRT) or anti-High Mobility Group Box 1 (HMGB1) antibody. Afterward, the cells were incubated with FITC-labeled Goat Anti-Rabbit IgG (H + L) for 2 h in the dark. The expression of CRT and HMGB1 was observed using a laser confocal microscope. Adenosine Triphosphate (ATP) levels were measured employing an Enhanced ATP Assay Kit.

Flow cytometric analysis of DC maturation

To evaluate nanoparticle-induced DC maturation, a Transwell co-culture system was employed. Briefly, MB49 bladder cancer cells were seeded into the upper chamber of a Transwell insert and allowed to adhere overnight. The following day, the MB49 cells were incubated with different nanoparticle formulations for 24 h. Then, the medium was replaced with fresh, nanoparticle-free medium. Subsequently, DC2.4 cells (Otwo Biotech Inc, Shenzhen, China) were seeded into the lower chamber of the Transwell plate. The two cell types were then co-cultured for 48 h. Following this, the DC2.4 cells were collected and stained with PE-conjugated anti-CD80 antibody and APC-conjugated anti-CD86 antibody for flow cytometer analysis.

In vivo anticancer therapy

All male C57BL/6 mice (6 weeks) were obtained from the Animal Experiment Center of Chongqing Medical University (Approval number: 2022-K52).

Subcutaneous Tumor Model: A total of 1 × 10⁶ MB49 cells were implanted into the right flank of male C57BL/6 mice. Once the MB49 tumors reached a volume of approximately 100 mm³, calculated using the formula V = 0.5 × L × W², the mice were randomly assigned to five distinct treatment groups. Each group received intratumoral injections of DOX at a dose of 3 mg/kg, administered every two days for a total of four injections. Mice were monitored every two days for body weight and tumor volume. Apoptosis in tumor tissues were detected using TUNEL staining three days after the final treatment. Tyramide signal amplification (TSA) was utilized for multiple immunofluorescence analysis. For safety evaluation, Hematoxylin and eosin (H&E) staining was also employed to evaluate the tissue architecture of key organs, namely the bladder, heart, liver, spleen, lungs, and kidneys.

Orthotopic Tumor Model: A total of 1 × 10⁶ MB49‑luc cells in 100 µL of PBS were intravesically inoculated into the bladder via urethral catheterization under anesthesia in male C57BL/6 mice. Tumor engraftment was confirmed five days post‑inoculation by bioluminescence imaging following intraperitoneal injection of D‑luciferin potassium salt (150 mg/kg). Mice with confirmed tumor growth were then randomly assigned to treatment groups. Mice received intravesical instillations of the nanoparticles at a DOX dose equivalent of 6 mg/kg. During each instillation, the urethra was clamped for 30 min to retain the drug within the bladder. Treatments were administered every three days for a total of four sessions. Following the final treatment, tumor burden was assessed by bioluminescence imaging as described above. The survival of mice in each group was monitored for a period of 90 days. For safety evaluation, routine blood tests and assessments of liver and kidney function were conducted.

In vivo flow cytometric analysis

Single-cell suspensions were prepared using tissue-specific protocols. For bladder tumor tissues, fresh specimens were minced into fine fragments and subsequently digested at 37 °C for 30 min in a dissociation solution containing 1 mg/mL collagenase IV, 1 mg/mL hyaluronidase, and 0.5 mg/mL DNase I. The resulting cell suspension was filtered through a 70 μm nylon mesh and subjected to red blood cell lysis. For spleen and lymph node tissues, mechanical dissociation was performed by gentle grinding, followed by filtration through a 70 μm mesh and red blood cell lysis. All procedures were carried out on ice or using pre-chilled reagents to maintain cell viability.

For immunophenotyping, cells were counted and adjusted to a concentration of 1 × 10⁶ cells per sample in cell staining buffer. Cells were first incubated with purified anti-mouse CD16/32 monoclonal antibody for 30 min at 4 °C to block Fc receptors. Subsequently, to analyze dendritic cells and T lymphocytes, cells were incubated for 40 min at 4 °C in the dark with distinct antibody cocktails. Dendritic cell staining was performed using a combination of anti-CD11c-PB450, anti-CD80-PE, and anti-CD86-APC antibodies, while T lymphocyte staining utilized anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE antibodies. Data were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analyzed using FlowJo software (version 10.8, BD Biosciences). Specific immune cell populations were identified and quantified based on established sequential gating strategies.

Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from mouse tumor tissues using the Eastep™ Super Total RNA Extraction Kit. Subsequently, cDNA was synthesized through reverse transcription with the aid of ABScript III RT Master Mix, which includes a gDNA Remover, specifically designed for qPCR. qRT-PCR was performed employing the 2X Universal SYBR Green Fast qPCR Mix. Each step was meticulously executed following the manufacturer’s protocol. The primer sequences are outlined in Table S2.

Data analysis

The data were analyzed employing GraphPad Prism version 9.0. Results are depicted as the mean ± standard deviation (SD). Statistical significance was determined using the unpaired Student’s t-test (two-tailed) and one-way analysis of variance, with the following notation for p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, indicating significance, while ‘ns’ denotes not significant.

Results and discussion

High expression of SA in bladder cancer and its correlation with clinical prognosis

To evaluate the therapeutic potential of SA as a target in NMIBC, we conducted validation experiments using clinical bladder cancer tissues and multiple bladder cancer cell lines. Sambucus nigra lectin (SNA), a highly specific SA-binding probe, was employed to detect SA expression. Notably, SA levels were markedly elevated in human BCa tissues compared to adjacent normal tissues (Fig. 2A-C). Consistent with this, three BCa cell lines (MB49, T24, and UM-UC-3) exhibited significantly higher SA expression in both membrane and cytoplasmic compartments relative to the normal bladder cell line SV-HUC-1 (Fig. 2D, E). Considering the crucial role of sialyltransferases in the biosynthesis of sialic acid and the modification of tumor cell surfaces, the elevated expression of these enzymes facilitates the transfer of additional sialic acid to the sugar chains on the surface of tumor cells [22, 23]. This results in a significant increase in the expression of sialic acid on the surfaces of tumor cells. We analyzed sialyltransferase expression profiles in the GEPIA database. In bladder urothelial carcinoma patients, high expression of ST6GALNAC3 (n = 1206, p < 0.01), ST8SIA6 (n = 1065, p < 0.05), and ST3GAL6 (n = 399, p < 0.01) correlated with reduced overall survival (Fig. 2F). These results collectively demonstrate SA overexpression in BCa and underscore its potential as a novel therapeutic target for NMIBC.

Fig. 2.

(A, B) SA expression in human tissues was detected by immunohistochemistry using Sambucus nigra lectin (SNA). Scale bars = 5 μm. (C) Integrated optical density (IOD) values from 9 cases of bladder urothelial carcinoma. ***p < 0.001. (D, E) Expression of SA in bladder tumor cell lines (MB49, T24, and UM-UC-3) and a normal bladder cell line (SV-HUC-1) was assessed using Cy3-labeled SNA. Scale bars = 300 μm; **p < 0.01, **p < 0.001. (F) Impact of sialyltransferase expression (ST6GALNAC3, n = 1206, p < 0.01; ST8SIA6, n = 1065, p < 0.05; ST3GAL6, n = 399, p < 0.01) on overall survival in bladder cancer patients

BCG-PPD-induced dendritic cell maturation

BCG, renowned for its potent immunomodulatory properties, serves dual clinical roles in tuberculosis prophylaxis and intravesical bladder cancer immunotherapy [24, 25]. BCG-PPD represents a purified biological preparation derived from mycobacterial culture filtrates, primarily containing antigenic proteins and their processed epitopes. Through mass spectrometry analysis, we identified BCG-PPD’s protein profile as predominantly comprising bioactive components inherent to BCG bacilli (Fig S1, Table S3).

To assess biocompatibility, MB49 cells were exposed to varying BCG-PPD concentrations, with viability assays demonstrating no significant cytotoxic effects (Fig S2). Crucially, we validated BCG-PPD’s preserved immunotherapeutic capacity through co-culture experiments with DC2.4 cells. Flow cytometric analysis revealed that BCG-PPD effectively promoted DC2.4 cells maturation, as evidenced by significant upregulation of co-stimulatory markers CD80 and CD86 (Fig S3). These findings collectively indicate that BCG-PPD maintains BCG’s immunostimulatory profile while exhibiting favorable safety characteristics, likely mediating anti-tumor effects through immune activation rather than direct cytotoxicity [26].

Preparation and characterization of nanoparticles

We synthesized nanoparticles using a multistep emulsion technique (Fig. 3A). The nuclear magnetic resonance (Fig S4) and Fourier transform infrared spectroscopy (Fig S5) of PLGA-PEG-PBA indicated that PBA has successfully coupled to PLGA-PEG-NH₂. Transmission electron microscopy revealed that the nanoparticles exhibited uniformly sized spherical structures (Fig. 3B). Table S4 provides a summary of the characterization of the DOX@NPs-T, Combo@NPs and Combo@NPs-T nanoparticles. The Combo@NPs-T exhibited a pink coloration, with a particle size measuring (263.47 ± 4.35 nm) and a zeta potential of (19.70 ± 0.86 mV). (Fig. 3C, D). Due to the modification by PBA, the Combo@NPs-T exhibited a negative charge, which is more conducive to being adsorbed by tumor cells [27, 28]. The particle size of Combo@NPs-T maintained stability in solutions at pH 5.5 and 7.4 over the course of the 35-day observation period (Fig. 3E).

Fig. 3.

(A) Schematic illustration of the synthesis of Combo@NPs-T nanoparticles. (B) Transmission electron microscopy (TEM) images of Combo@NPs-T nanoparticles at various magnifications. Scale bars: 2 μm, 500 nm, 200 nm, and 200 nm. (C) Particle size distribution and morphological characteristics of Combo@NPs-T nanoparticles. (D) Zeta potential of Combo@NPs-T nanoparticles. (E) Stability of Combo@NPs-T nanoparticles as monitored by particle size under different pH conditions (pH 5.5 and pH 7.4) during storage at 4 °C. (F) Cumulative release profile of DOX from Combo@NPs-T nanoparticles at pH 5.5 and pH 7.4

The drug loadings of DOX and BCG-PPD within the Combo@NPs-T nanodrugs were precisely quantified at 1.69 ± 0.02% and 0.36 ± 0.01%, respectively (Table S4). In clinical practice, the dwell time for intravesical drug perfusion is usually maintained for 0.5 to 2 h [3].The 2-hour release rate of DOX from Combo@NPs-T increased from 19.1% at pH 7.4 to 24.6% at pH 5.5 (Fig. 3F), a similar trend was also observed in DOX@NPs-T (Fig S6), which is likely attributable to the accelerated degradation of the PLGA matrix under acidic conditions [29]. The relatively low amount of free DOX released suggests that a substantial portion of the drug remains encapsulated within the intact nanoparticles. This favors nanoparticle-mediated drug delivery, as it ensures that the carriers themselves remain predominantly intact during the critical perfusion period, thereby promoting targeted accumulation and uptake by tumor tissue.

Cellular uptake and targeting verification of nanoparticles

To assess the targeting efficiency of Combo@NPs-T, we performed in vitro cellular uptake studies using confocal microscopy and flow cytometry. Compared to the control group, the Combo@NPs-T-treated cells exhibited a significantly higher DOX fluorescence intensity (Fig. S7A-C), indicating enhanced cellular internalization. To further elucidate the underlying uptake mechanism, we inhibited clathrin-mediated endocytosis with chlorpromazine, which led to a marked reduction in nanoparticle internalization (Fig. 4A) [30]. This result suggests that Combo@NPs-T primarily enters cells via clathrin-dependent endocytosis. Moreover, when free PBA was added as a competitive ligand, or when low sialic acid-expressing SV-HUC-1 cells were used, a significant decrease in cellular uptake was observed, confirming the critical role of sialic acid-mediated targeting. Lysosomal colocalization studies further demonstrated that the nanoparticles effectively escaped from lysosomes and, over time, released DOX into the nucleus (Fig. 4B). Collectively, these data indicate that Combo@NPs-T can specifically bind to sialic acid receptors overexpressed on cancer cells and efficiently deliver DOX into the nucleus via receptor-mediated endocytosis and lysosomal escape, thereby significantly improving drug targeting and cellular uptake.

Fig. 4.

(A) Uptake of Combo@NPs-T by MB49 cells under different conditions. Cells were incubated for 0.5, 1, and 2 h in the following groups: MB49 + Combo@NPs-T (Control), MB49 + Combo@NPs-T + free PBA (PBA), MB49 + Combo@NPs-T + chlorpromazine (Chlorpromazine), and normal SV-HUC-1 cells + Combo@NPs-T (SV-HUC-1). Scale bars = 50 μm. (B) Intracellular trafficking and lysosomal escape of nanoparticles monitored using LysoTracker-labeled lysosomes. (C) Viability of MB49 cells after treatment with Blank@NPs-T nanoparticles. (D, E) Apoptosis of MB49 cells induced by PBS, Blank NPs, DOX NPs, Combo NPs, and Combo NPs-T (DOX concentration: 0.6 µM), as analyzed by flow cytometry. (F) Live/dead staining of MB49 cells treated with various nanoparticles. Live cells were stained with Calcein-AM, and dead cell nuclei were stained with DAPI (scale bars = 200 μm). (ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Safety evaluation of the PLGA-PEG nanoparticle delivery system

As a nano-carrier, PLGA is considered to exhibit minimal systemic toxicity in biomedical applications [31, 32]. We selected PLGA as the nanoparticle carrier for the delivery of BCG-PPD and DOX drugs. By attaching the hydrophilic molecule PEG to PLGA, we can enhance the hydrophilicity of the PLGA nanoparticles, thereby prolonging their residence time in vivo [33, 34]. Additionally, both PLGA and PEG have received approval from the U.S. Food and Drug Administration for use as drug delivery carriers and drug conjugates [35]. We selected PLGA (lactide: glycolide ratio of 50:50) due to its rapid degradation profile, which can improve the biological safety of the nanoparticles [36]. According to the CCK8 assay (Fig. 4C), Blank@NPs-T exhibited minimal cytotoxicity after 48 h of co-culture, suggesting that nanoparticle-based drug delivery systems are safe. The in vivo safety was further validated through subsequent animal experiments.

Anti-tumor effect of nanoparticles in vitro

Doxorubicin is a widely used drug in cancer chemotherapy that induces cell death through multiple intracellular interactions, producing reactive oxygen species (ROS) and DNA adducts, thereby inducing apoptosis [37, 38]. To screen for therapeutic doses, the CCK8 assay was used to evaluate the cytotoxic effect of different concentrations of DOX on MB49 cells (Fig S8). Results indicated that the drug’s half maximal inhibitory concentration (IC50) for the MB-49 cell line was 0.35 µg/ml (0.6 µM) after a 24-hour exposure period (Fig S9). Then, the cytotoxic impact of nanoparticles on tumor cells was examined by apoptosis flow cytometry and live/dead cell staining methodologies. Treatment with Combo@NPs-T induced the highest apoptotic rate at 48.79%, followed by Combo@NPs (23.35%), DOX@NPs (15.51%), Blank@NPs (6.22%), and PBS control (2.67%), demonstrating its significantly enhanced pro-apoptotic potency (Fig. 4D, E). In live/dead cell staining, the Combo@NPs-T group exhibited a reduced number of living cells (Calcein AM) compared to the other groups, and concurrently, a higher count of dead cells (DAPI) (Fig. 4F). These results indicate that Combo@NPs-T could effectively induce apoptosis in tumor cells and exhibited a potent anti-tumor effect.

The release of DAMPs and DCs maturation in vitro

ICD, a special form of apoptosis, relies upon the release of DAMPs, such as Calreticulin (CRT), High Mobility Group Box 1 (HMGB1) and ATP. Doxorubicin has been proven to be one of the chemotherapy drugs capable of inducing ICD [39]. During the process of ICD, CRT rapidly translocates from the endoplasmic reticulum lumen to the cell surface, HMGB1 is passively released from the nucleus into the extracellular space, and ATP is abundantly secreted from organelles such as mitochondria and the endoplasmic reticulum [40–42]. To assess the ICD-inducing potential, we conducted an in vitro immunofluorescence analysis of CRT and HMGB1 in MB49 cells, and detection of the ATP content in the supernatant. In the Combo@NPs-T group (Fig. 5A and B), a significant increase in green fluorescence on the cell membrane (indicating CRT surface exposure) was observed concurrently with a marked reduction of nuclear fluorescence (reflecting HMGB1 release). Furthermore, the Combo@NPs-T group demonstrated a significantly higher level of ATP in the supernatant compared to all other groups (Fig. 5C). These results collectively indicate the successful induction of the ICD effect.

Fig. 5.

(A) Confocal microscopy images showing the expression and localization of CRT and HMGB1 in MB49 cells. (B) Quantitative analysis of the average fluorescence intensity for CRT and HMGB1 expression in MB49 cells. (C) ATP content in the supernatant across different treatment groups. (D) Schematic of the in vitro co-culture of MB49 cells and DC2.4 cells. (E) Expression levels of the maturation markers CD80 and CD86 on DC2.4 cells after co-culture with MB49 cells and different nanoparticles. (F) Percentage of matured DC2.4 cells under each experimental condition. (ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Acting as key danger signals, these DAMPs enhance antigen uptake by DCs and facilitate their functional maturation [43]. Building on our prior finding that BCG-PPD independently promotes DC maturation (Fig. S3), we next investigated whether DOX-induced ICD and BCG-PPD could act synergistically. For this purpose, DC2.4 cells were co-cultured with MB49 cells that had been pre-treated with the different nanoparticles (Fig. 5D). Flow cytometry results showed that the DOX@NPs group exhibited a significantly higher percentage of mature DCs compared to the control group, indicating that DOX-induced ICD effectively promoted DC maturation (Fig. 5E, F). Furthermore, the proportion of mature DCs in the Combo@NPs group was significantly higher than that in the DOX@NPs group, suggesting an additional enhancement effect contributed by BCG-PPD (Fig. 5E, F). Notably, the Combo@NPs-T group demonstrated the highest level of DC maturation among all groups, highlighting that this targeted combination strategy maximizes the induction of DC maturation (Fig. 5E, F).

Anti-tumor efficacy of nanoparticles in a subcutaneous tumor model

In vitro experiments, we fully validated the targeting ability of the Combo@NPs-T, its cytotoxic effect on tumor cells, and its promotion of DCs maturation. To preliminarily clarify the antitumor effect of Combo@NPs-T, subcutaneous tumor model was first conducted. It is worth noting that the combined therapeutic strategy of DOX and BCG bacteria is based on previous research, and this integrated approach may explain the superior efficacy and enhanced safety compared to simple mixtures of free drugs [44]. Given this established synergistic foundation, the present study aimed to validate the therapeutic efficacy of the DOX and BCG-PPD combination specifically when co-delivered via our novel targeted nanoplatform.

When the subcutaneous tumor volume reaches approximately 100mm³, intratumoral administration is initiated with three doses given every other day, and tumor growth was observed in each group within the 21 days following the start of treatment (Fig. 6A). The sizes of the tumor in various groups are shown in the Fig. 6B. Due to intratumoral administration, there was no significant difference in tumor size and tumor mass between the Combo@NPs-T group and the Combo@NPs group, but there was a significant difference compared to other groups (Fig. 6C, D). These results indicate that the combination of DOX and BCG-PPD in our delivery system demonstrates a potent anti-tumor effect in vivo. Throughout the treatment phase, no considerable weight reduction was observed in the mice across all groups (Fig. 6E). The H&E staining results showed no significant damage to the main organ tissue structure (Fig S10). These results indicated that the nanoparticles have a certain level of safety.

Fig. 6.

(A) Schematic diagram of the treatment regimen in the subcutaneous tumor model. (B) Representative photographs of excised tumors from each experimental group. (C) Tumor growth curves of each group over time. (D) Quantitative comparison of final tumor weights among the groups. (E) Changes in body weight of mice across different treatment groups throughout the study. (F) TUNEL fluorescence staining of tumor tissue sections from different treatment groups, indicating apoptotic cells (scale bars = 100 μm). (G) Immunofluorescence staining of tumor tissues for CD80/CD86 (dendritic cell/macrophage activation markers) and CD3/CD8 (T‑cell markers) (scale bars = 100 μm). (H–L) Relative mRNA expression levels of TNF‑α, IL‑12, IFN‑γ, CXCL9, and CXCL10 in tumor tissues, as determined by quantitative reverse transcription polymerase chain reaction (qRT‑PCR). (ns: not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

To verify the tumor apoptosis induced by nanomedicines, immunofluorescence staining of TUNEL was conducted (Fig. 6F). The Combo@NPs and Combo@NPs-T groups exhibited a greater extent of tumor cell apoptosis within the tumor compared to the other groups. During the apoptosis process, tumor antigens and immunostimulatory substances are released. Immature DCs are capable of capturing and processing these antigens. BCG-PPD drives the full maturation of these DCs and promotes their migration to the draining lymph nodes, where they present antigens to activate tumor-specific T cells. Subsequently, the activated tumor-antigen-specific CTLs infiltrate the tumor tissue, exerting a potent cytotoxic effect on the tumor [45, 46]. To better understand the maturation of DCs and the infiltration of CD8⁺ T cells within tumor tissues, we performed immunofluorescence staining experiments. In comparison to DOX and DOX@NPs, our results revealed a higher abundance of matured DCs and CD8⁺ T cells in the Combo@NPs and Combo@NPs-T groups (Fig. 6G). These indicates that the synergistic effect of DOX and BCG-PPD markedly bolstered the anti-tumor immune response, demonstrating the effectiveness of combination therapy.

In our study, the central antitumor effect is designed to be driven primarily by T cells specific for tumor-associated antigens, not by T cells reactive to BCG-PPD components. BCG-PPD is utilized chiefly for its potent immunoadjuvant activity. We acknowledge that BCG-PPD-specific T cells could potentially be induced. Although such T cells would not directly recognize tumor cells due to antigen mismatch, they might contribute indirectly by secreting Th1-type cytokines [47, 48]. This cytokine milieu could help sustain a pro-inflammatory tumor microenvironment and non-specifically amplify the effector functions of tumor-specific T cells.

To investigate this cytokine-mediated component of the immune response, we analyzed key factors linked to effective T-cell immunity. Guided by TCGA data, we focused on TNF-α, IL-12, IFN-γ, CXCL9, and CXCL10, as their expression in human bladder cancer tissues positively correlates with immune cell infiltration (Fig S11). These molecules constitute a functional network critical for cell-mediated immunity, with IL-12 acting as a pivotal inducer of Th1 responses, IFN-γ and TNF-α as major effector cytokines, and CXCL9/CXCL10 as IFN-γ-inducible chemokines responsible for recruiting cytotoxic T lymphocytes (CTLs) [49, 50]. qRT-PCR analysis confirmed that both combination therapy groups (Combo@NPs and Combo@NPs-T) significantly upregulated all five mediators compared to controls (Fig. 6H-L), indicating the successful creation of a Th1-polarized, immune-active microenvironment. Notably, while cytokine levels were numerically higher with targeted delivery (Combo@NPs-T), only CXCL9 showed a statistically significant increase over the non-targeted combo group.

In the subcutaneous tumor model, we validated the benefits of the nanoparticles co-delivery of DOX and BCG-PPD. The therapeutic advantage of Combo@NPs-T over Combo@NPs was not pronounced in this model. This observation may be attributable to the specific experimental conditions, particularly the subcutaneous tumor model and the direct intratumoral route of administration. To further substantiate the superiority of targeted drug delivery, a clinically pertinent model is warranted.

Anti-tumor effect of nanoparticles in a orthotopic tumor model

To further clarify the preferential targeting and anti-tumor efficacy of the nanoparticles, we employed an orthotopic bladder cancer model alongside bladder perfusion administration (Fig. 7A). The bladder orthotopic model was established by intravesical injection of MB49-luc. Five days after inoculation, the bioluminescence signals were detected by in vivo imaging (Fig. 7B), and abnormal hyperechoic signal was also observed in the bladder (Fig S12), definitively confirming the successful establishment of the orthotopic model. Following treatment, the Combo@NPs-T group exhibited a significantly reduced bioluminescence intensity in the bladder compared to the Combo@NPs and Control groups (Fig. 7C). This therapeutic efficacy was further corroborated by corresponding decreases in bladder size, bladder weight, and residual tumor burden (Fig. 7D-F). Consistently, the Combo@NPs-T group also showed a significant extension in overall survival (Fig. S13). Collectively, these data demonstrate that the targeted combination nanodrug, Combo@NPs-T, exerts a superior antitumor effect, likely through enhanced drug delivery and synergistic cytotoxicity.

Fig. 7.

(A) Schematic of the experimental protocol for nanoparticle treatment in an orthotopic bladder tumor model. (B, C) Tumor burden monitored by in vivo imaging at 5 and 15 days post-inoculation. (D) Representative photographs of bladders harvested from each experimental group after treatment. (E) Quantification of bladder mass across treatment groups. (F) Hematoxylin and eosin (H&E) staining of bladder tissue sections (scale bars = 1 mm). (G, H) Fluorescence images of frozen sections from normal bladder tissue and tumor tissue following incubation with Combo@NPs‑T or Combo@NPs. (*p < 0.05, **p < 0.01, ***p < 0.001)

To further validate the enhanced drug delivery enabled by PBA–sialic acid targeting in vivo, we executed frozen section assessments for validation. Notably, despite the potential concern regarding PBA stability in ROS‑enriched tumor microenvironments, the PBA‑functionalized nanoparticles (Combo@NPs‑T) showed markedly higher accumulation in tumor tissues than in normal bladder tissues (Fig. 7G, H), underscoring that the targeting function of PBA remains effective. This finding suggests that the PBA moiety in our system functions as a relatively stable, covalently anchored targeting ligand. Consequently, PBA-mediated targeting effectively elevated intratumoral drug concentrations, thereby potentiating the antitumor efficacy in vivo.

To further assess the activation of antitumor immunity, we performed flow cytometry to assess DCs within the bladder (Fig. 8A, B), lymph nodes (Fig. 8C, D) and spleen (Fig. 8E, F) individually. The findings indicated that the proportion of mature DCs in the Combo@NPs-T group was significantly higher compared to the other groups, exhibiting a statistically significant difference. We further examined the T lymphocytes in the spleen (Fig. 8G, H) and bladder (Fig. 8I, J), and the results indicated that the proportion of CD8⁺ T lymphocytes in the Combo@NPs-T group was higher than in the other groups, with a statistically significant difference. Consequently, Combo@NPs-T has the potential to eliminate tumor cells and effectively enhance DCs maturation, as well as promote CD8⁺ T lymphocytes infiltration at the tumor site, thereby exerting potent anti-tumor immune responses.

Fig. 8.

(A, B) Proportion and statistical analysis of mature DCs in the mouse bladder. (C, D) Proportion of mature DCs in draining lymph nodes, as assessed by flow cytometry. (E, F) Proportion and statistical analysis of mature DCs in the mouse spleen. (G, H) Proportion and statistical analysis of CD8⁺ T cells in the mouse spleen. (I, J) Proportion and statistical analysis of CD8⁺ T cells in the mouse bladder. (*p < 0.05, **p < 0.01, ***p < 0.001)

At the end of the experiment, we conducted biochemical and hematological analyses on venous blood samples from each group. Due to the large bladder tumor in situ, causing urinary tract obstruction, the blood urea nitrogen (BUN) in the PBS group was significantly higher compared with the Combo@NPs-T group, and there was no statistical difference in the other indicators (Fig S14).

This work has certain limitations that highlight important areas for future research. First, while the present work focuses on elucidating the essential pathway of tumor-antigen-directed immunity, the specific contribution of BCG-PPD-specific T cells remains to be defined. Second, the generation and phenotype of tumor-specific memory T cells were not directly evaluated. Finally, the precise mechanisms of T-cell activation and the subsequent tumor killing remain to be established. Addressing these specific gaps will be crucial for enhancing our mechanistic understanding and therapeutic application of this combination strategy.

Conclusion

In conclusion, this research has triumphantly devised a novel combination therapy encapsulated in nanoparticles, labeled DOX/BCG-PPD@PLGA-PEG-PBA, which actively targets SA to evoke ICD, promote the maturation of DCs, and intensify the infiltration of CTLs, ultimately bolstering cancer immunotherapy. Our research, possessing high clinical significance and translational potential, presents a novel approach to perfusion therapy for NMIBC.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Jindong Zhang: Conceptualization, Data curation, Formal analysis, Writing – original draft. Shuai Su: Data curation, Formal analysis, Investigation. Maoyu Liu: Formal analysis, Methodology, Writing – original draft. Yu Luo: Investigation, Methodology. Chengcheng Wei: Resources, Software. Yang Cao: Resources, Supervision. Honglin Cheng: Project administration, Supervision. Shenyin Zhu: Conceptualization, Writing–review & editing. Delin Wang: Conceptualization, Project administration, Writing – review & editing.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials.

Declarations

Ethical approval

This study received approval from the Medical Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (Approval Number: 2022-043), and written informed consent was obtained from all participants. All procedures adhered to the ethical standards set by the Institutional Ethics Committee on human experimentation and were in compliance with the Helsinki Declaration. Additionally, the Animal Ethics Committee of Chongqing Medical University (Approval number: 2022-K52) granted approval for the animal study.

Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) used WeTab AI Pro in order to improve language and readability. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the publication.

Competing interests

The authors declare no competing interests.