Targeted Nanoparticles for PROTAC-Mediated JAK1/JAK2 Degradation for Bladder Cancer Treatment

Abstract

Purpose

Although inhibition of the JAK–STAT signaling pathway has shown promise in cancer therapy, the therapeutic potential of PROTAC-mediated JAK1/JAK2 degradation in bladder cancer remains to be fully explored. To improve the delivery and therapeutic efficacy of PROTAC-mediated JAK1/JAK2 degradation in bladder cancer, this study developed PPcJ, a cRGD-functionalized PLGA–PEG nanoparticle encapsulating the PROTAC-based JAK1/JAK2 degrader JAPT8.

Methods

PPcJ nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, and high-performance liquid chromatography. Flow cytometry and confocal microscopy were used to evaluate cytotoxicity, anti-tumor effect, and intracellular localization, while Western blotting assessed the expression of JAK/STAT pathway and apoptosis-related proteins. In vivo tumor-associated fluorescence, antitumor efficacy, survival, histopathology, and serum biochemical parameters were assessed in tumor-bearing mice.

Results

PPcJ exhibited a spherical nanoscale morphology. PPcJ achieved near-plateau cellular uptake at low concentrations and partially evaded lysosomal degradation, which significantly induced apoptosis in bladder cancer cells and suppressed key components of the JAK/STAT signaling pathway. In vivo, cRGD modification enabled higher tumor-specific accumulation and prolonged retention. PPcJ inhibited tumor growth by 86.2% and prolonged mean survival from 22.4 ± 4.2 to 41.0 ± 2.2 days, without significant changes in body weight, major-organ histology, or serum AST, ALT, urea, and creatinine levels under the tested conditions.

Conclusion

These findings provide a preclinical proof-of-concept that the PPcJ nano-delivery system improves the delivery and antitumor performance of JAPT8. This strategy warrants further evaluation in systemic therapeutic approaches for bladder cancer.

Graphical Abstract

Introduction

Bladder cancer is a highly prevalent urological malignancy associated with rising global incidence and mortality.¹ While conventional treatments such as cisplatin-based chemotherapy and Bacillus Calmette-Guérin (BCG) immunotherapy remain foundational,² their clinical utility is frequently compromised by severe local inflammation, off-target organ toxicity, and limited therapeutic efficacy.^3–5 Therefore, developing novel targeted strategies is urgently required.

Recently, JAK inhibitors have emerged as promising therapeutic agents due to their profound impact on tumor-associated signaling networks. Preclinical and clinical evidence indicates that combining JAK inhibitors with immune checkpoint blockade substantially enhances therapeutic efficacy across various malignancies, such as melanoma, pancreatic cancer, and Hodgkin lymphoma.⁶ As the JAK/STAT pathway is a pivotal axis regulating immune activation and cytokine production, its suppression is inextricably linked to tumor cell apoptosis.⁷ In the context of bladder cancer, the aberrant activation of the JAK/STAT signaling axis—particularly the hyperactivation of STAT3—plays a critical role in driving urothelial carcinoma proliferation, metastasis, and resistance to apoptosis.^8,9 Previous preclinical attempts to target this pathway in bladder cancer models have demonstrated that pharmacological inhibition of JAK can effectively suppress tumor progression and sensitize malignant cells to conventional therapies.¹⁰

Currently, first-generation JAK inhibitors, such as ruxolitinib and tofacitinib, are FDA-approved primarily for hematological malignancies and autoimmune diseases, and their clinical application is now rapidly expanding into solid tumors like bladder cancer. However, these ATP-competitive inhibitors typically require high doses for efficacy. This approach inevitably induces off-target risks and various adverse effects, significantly hampering their broader clinical utility.¹¹ Therefore, there is an urgent unmet need to develop next-generation therapeutic strategies, such as proteolysis-targeting chimeras (PROTACs), to achieve highly specific JAK degradation and minimize systemic toxicity. Motivated by the need for safer and more effective JAK-targeted therapies, our group previously developed a PROTAC-based JAK1/JAK2 degrader, JAPT8. Unlike conventional occupancy-based inhibitors, JAPT8 binds JAK1/JAK2 with an E3 ubiquitin ligase, triggering the ubiquitination and subsequent proteasomal degradation of the target proteins. Crucially, the catalytic and recyclable nature of PROTACs allows JAPT8 to retain its structural integrity after degrading the target, enabling potent and sustained suppression of the JAK-STAT pathway at lower overall drug exposures, which potentially reduce off-target effects and adverse reactions.¹²

Nevertheless, the bivalent architecture of PROTACs, consisting of two ligands connected by a linker, often results in a high molecular weight of approximately 900–1100 Da. Together with their complex structural properties, this may lead to limited aqueous solubility, poor membrane permeability, and unfavorable pharmacokinetic profiles, thereby restricting their therapeutic application.^13,14 To address these delivery limitations and improve the therapeutic performance of JAPT8, we developed cRGD-functionalized PLGA–PEG nanoparticles for the targeted delivery of JAPT8 (PPcJ). The PLGA–PEG was designed to improve the delivery of JAPT8, whereas cRGD functionalization was introduced to facilitate its interaction with integrin-expressing tumor cells.¹⁵ We characterized the physicochemical properties and encapsulation efficiency of PPcJ and evaluated its near-plateau cellular uptake, cytotoxicity, apoptosis-inducing activity, and regulation of JAK/STAT signaling in MB49 bladder cancer cells. Its biodistribution, antitumor efficacy, survival benefit, and preliminary systemic safety were further investigated in a murine bladder cancer model. The study was designed to determine whether nanoparticle-mediated delivery could improve the therapeutic potential of PROTAC-based JAK1/JAK2 degradation in bladder cancer.

Materials and Methods

Material

Nanomaterials and Chemical Reagent

PLGA-PEG-cRGD and PLGA-PEG were purchased from Evonik (Essen, Germany). Sodium cholate hydrate was purchased from Alfa Aesar (Heysham, UK). Dichloromethane and dimethyl sulfoxide (DMSO) were obtained from Sangon Biotech (Shanghai, China). Coumarin 6 was purchased from J&K (Beijing, China).

Cell Culture Reagents and Biological Material

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, TrypLE, and phosphate-buffered saline (PBS) were obtained from Gibco (Grand Island, CA, USA). The 4% paraformaldehyde fixative solution, BCA Protein Assay Kit, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime Biotech (Shanghai, China). Hoechst and DAPI were from Thermo Fisher Technology (Waltham, MA, USA). Cell lysis buffer was provided by Epizyme (Shanghai, China). For Western blot analyses, the following primary antibodies were used: β-tubulin, Caspase-3, Bcl-2, STAT1, Phospho-STAT1, STAT3, Phospho-STAT3, JAK1, JAK2, Phospho-JAK1, and Phospho-JAK2, which were purchased from Proteintech (Chicago, IL, USA) and Cell Signaling Technology (Boston, MA, USA). The secondary antibodies included anti-rabbit IgG (Cell Signaling Technology, 7074S; 1:1000) and anti-mouse IgG (Cell Signaling Technology, 7076S; 1:1000).

Cell Culture and Animal Experiment

Murine bladder carcinoma (MB49) cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO₂ (Thermo, Waltham, MA, USA). MB49 murine bladder cancer cells were purchased from QuiCell (Catalog No. QuiCell-M398, China; RRID: CVCL_7076).

Six-week-old male C57BL/6 mice (for in vivo tumor therapy and survival evaluations) and BALB/c nude mice (for in vivo fluorescence imaging) were obtained from Shanghai JieSiJie Laboratory Animal (Shanghai, China). Animals were housed under specific pathogen-free conditions with controlled environmental parameters: 12 h light/dark cycle, ambient temperature 24 ± 1°C, and ad libitum access to standardized food and water. All animal experiments were approved by the animal ethics committee of Fudan University (Ethical approval number: 2021- HSYY-JS −188) and strictly complied with both institutional and national ethical guidelines for the care and use of laboratory animals. The sample size (n=5 mice per group) for in vivo efficacy studies was determined based on established pre-clinical tumor models in the nanomedicine field, effectively balancing the ethical requirement for animal reduction (the 3Rs principle) with the necessity of statistics.

Preparation and Characterization of PPcJ Nanoparticles

Preparation of PPcJ Nanoparticles: PLGA-PEG-cRGD was dissolved in dichloromethane (20 mg/mL), while JAPT8 was separately dissolved in DMSO (10 mg/mL). Subsequently, 20 mg of PLGA-PEG-cRGD and 1.5 mg of JAPT8 (an optimal drug-to-polymer feeding ratio determined through preliminary formulation screening to maximize encapsulation efficiency while preventing drug precipitation) were co-dissolved in 5 mL of 1.5% (w/v) sodium cholate hydrate aqueous solution. The mixture was sonicated for 1 min (15 s pulse duration, 5 s interval between pulses) on ice by SCIENTZ-950E sonicator. Subsequently, PPcJ nanoparticles were prepared by adding 10mL of water and stirring at room temperature. The mixture was then transferred to a glass vial and subjected to continuous magnetic stirring at 700 rpm overnight at a constant temperature of 25 °C. Finally, the nanoparticles were collected by centrifugation at 60,000 rpm for 45 min to remove the residual DMSO and unencapsulated JAPT8.

Preparation of PLGA-PEG Nanoparticles: PLGA-PEG was dissolved in dichloromethane to achieve a final concentration of 20 mg/mL. Subsequently, 20 mg of the PLGA-PEG was introduced into 5 mL of 1.5% (w/v) sodium cholate hydrate aqueous solution. The mixture was then processed identically to the PPcJ nanoparticle synthesis protocol described above. As a non-targeted control, PLGA-PEG nanoparticles loaded with JAPT8, lacking the cRGD conjugation, were prepared following the identical procedure and designated as PPJ.

For fluorescent-labeled nanoparticle preparation, PPcJ/Coumarin 6 (PPcJ/C6) and PPcJ/DiR formulations were synthesized by partial substitution of JAPT8 with either Coumarin 6 or DiR, respectively, while maintaining the identical protocol of PPcJ described above. Specifically, for DiR encapsulation, a dye stock solution was prepared in anhydrous ethanol (5 mg/mL) and incorporated during the synthesis to achieve a final working concentration of 20 μg/mL, while maintaining the identical formulation protocol of PPcJ described above. For Coumarin 6 encapsulation, C6 was dissolved into the acetone (1mg/mL) and incorporated into the organic phase during nanoparticle preparation to achieve a final C6 loading of 0.3% (w/w, relative to the total polymer mass). The total polymer mass, aqueous phase volume, sodium cholate concentration, sonication conditions, stirring conditions, and purification procedure were kept identical to those used for PPcJ preparation. The final concentration of fluorescently labeled nanoparticles was calculated based on the total nanoparticle mass.

The Polymer dispersity index (PDI), zeta potential, and size of PPcJ/PLGA-PEG nanoparticles were measured by Nano Zetasizer (Microtrac, NewYork, PA, USA). The nanoparticle morphology was observed using a Transmission Electron Microscope (TEM) (Hitachi, Tokyo, Japan) at an acceleration voltage of 80 kV.

Fourier infrared spectroscopy (FTIR) was measured by the potassium bromide tablet method. The nanoparticles were dried, ground, and then mixed with potassium bromide. The scanning conditions were set as follows: spectral range was 525–4000 cm⁻¹, scanning times were 32 times, and resolution was 8 cm⁻¹.

To determine the encapsulation efficiency (EE), the unencapsulated JAPT8 was separated from the nanoparticles by centrifugation at 60,000 rpm for 45 min at 4°C. The concentration of free JAPT8 in the supernatant was quantified by high-performance liquid chromatography (HPLC). The EE were calculated using the following equations: EE (%) = [(Total amount of JAPT8 added − Amount of free JAPT8)/Total amount of JAPT8 added] × 100%. For the HPLC analysis, the mobile phase was acetonitrile and water (initial ratio was 40:60, then increased to 80:20 in 11 minutes), the flow rate was 1.0 mL/min, and the detection wavelength was 192.8 nm. The HPLC analysis was carried out on a Waters HPLC Alliance e2695 separating module (Waters, Milford, MA, USA) using the column (5 μm, 4.6 × 250 mm) (Agilent, Santa Clara, CA, USA).

Cell Viability Assay and Uptake Assay of PPcJ

The cell viability profiles of PLGA-PEG, PPcJ nanoparticles, and free JAPT8 in MB49 cells were assessed using a CCK-8 proliferation assay. Cells were cultured in 96-well plates overnight. Then, the cells were treated with corresponding drugs at concentrations ranging from 0.05 to 50 μg/mL (concentration of JAPT8). For the PLGA-PEG control group, cells were treated with empty nanoparticles at a mass concentration exactly equivalent to the amount of polymer carrier present in the corresponding PPcJ doses. The incubation was continued for another 72 h. A continuous 72-hour exposure was utilized to determine the maximum pharmacological potency, providing sufficient time for the PROTAC-mediated ubiquitin-proteasome degradation and subsequent metabolic arrest to manifest.

Then we added CCK-8 solution (10 μL) to each well. The microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to read absorbance at 450 nm. The IC50 values were calculated utilizing a nonlinear regression analysis (log(inhibitor) vs response, four parameters) via GraphPad Prism software.

To determine the incubation time of PPcJ, we added PPcJ/C6 to DMEM medium and incubated for 2h-8h. The obtained cells were washed and detected by flow cytometry using an Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA). This time window was optimized based on preliminary time-course evaluations to capture the active endocytic phase while avoiding non-specific accumulation or exocytosis.

To optimize nanoparticle incubation concentration, MB49 cells were exposed to PPcJ/C6 gradients (1–10 μg/mL) in serum-free DMEM for 4 h. Cells were trypsinized, washed, and analyzed using an Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Waltham, MA, USA).

To observe cells more vividly, MB49 cells were cultured on a Millicell EZ SLIDE 8-well glass (Merck, Kenilworth, NJ, USA) overnight. Then the medium containing PPcJ/C6 was added and incubated for 1h-5h. After washing, fixing, and staining with Hoechst, cellular internalization was visualized using a confocal microscope (Leica, Wetzlar, Germany).

For lysosomal localization analysis, MB49 cells cultured in 6-well plates were incubated with 5 μg/mL PPcJ/C6 for 4 h. Following PBS washes, live cells were stained with LysoTracker Red (Beyotime, Shanghai, China) and Hoechst. Then the cells were visualized using a fluorescence microscope (Yuehe, Shanghai, China).

In vitro Apoptosis Assay

Apoptosis was analyzed using an Annexin V-FITC Apoptosis detection kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. Briefly, cells were co-incubated with PBS/PLGA-PEG/JAPT8/PPcJ for 4h. Following this initial exposure, the drug-containing medium was removed. The cells were washed with PBS and then incubated in fresh, drug-free complete medium for an additional 24 h or 72 h. The culture medium was implemented timely replenishment during the 72-h incubation period. This protocol was designed to mimic in vivo pharmacokinetic clearance, allowing for the evaluation of both early apoptosis initiation (24 h) and late-stage cell death (72 h) driven exclusively by the internalized nanocarriers. Subsequently, cells were harvested, washed, and resuspended in the binding buffer, then stained with Annexin V-FITC and propidium iodide for 5–10 min at room temperature. The apoptotic analysis was determined by FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA).

Western Blotting

Cellular and tissue protein lysates were prepared using ice-cold RIPA buffer and detected using the BCA protein assay kit (Beyotime, Shanghai, China). Equal protein quantities were loaded in each lane, separated by SDS-polyacrylamide gel electrophoresis, and subsequently transferred onto PVDF membranes (0.22 μm, Millipore). Membranes were blocked for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies. Following three 5-min TBST washes, membranes were incubated with secondary antibodies for 1 h at 25°C. Proteins were detected using BeyoECL Plus substrate (Beyotime, P0018S) with an Integrated chemiluminescence analyzer (Epizyme, Shanghai, China). Throughout this study, β-tubulin was utilized as the consistent and universal internal loading control for the normalization of all investigated proteins.

In vivo Imaging of PPcJ/DiR

Male BALB/c nude mice (6–8 weeks) received subcutaneous inoculation of 1×10⁶ MB49 cells suspended in 100 μL PBS in the right flank. Tumor growth was monitored daily until reaching 75–100 mm³ volume (calculated as π/6×length×width²). The mice were intravenously injected with 100 μL of the PPcJ/DiR or PLGA-PEG/DiR nanoparticle suspension (equivalent to a DiR concentration of 20 μg/mL). In vivo fluorescence imaging of mice was performed at different time points using an ABL X5 imaging system (Tanon, Shanghai, China), and the mice were sacrificed 24 h after the administration of PPcJ/DiR or PLGA-PEG/DiR. The tumors and main organs were collected and imaged on the imaging system. Fluorescence images were acquired at 0, 2, 6, 12, 16, and 24 h post-injection. To accurately evaluate the accumulation and retention dynamics, the fluorescence intensity of the tumor regions was quantitatively analyzed by selecting appropriate regions of interest (ROIs) across a full cohort of mice (n = 3 per group).

The DiR-loaded nanoparticles were prepared using a single emulsion-solvent evaporation method. Briefly, 0.5 mL of polymer solution (PLGA-PEG or PLGA-PEG-cRGD in dichloromethane, 20 mg/mL) and 10 μL of DiR solution (in absolute ethanol, 5 mg/mL) were mixed as the organic phase. The mixture was added into 2.5 mL of 1% (w/v) sodium cholate aqueous solution, followed by probe sonication in an ice bath. The resulting emulsion was stirred overnight at room temperature to completely evaporate the organic solvents. After purification, the DiR-loaded nanoparticles were resuspended in physiological saline. For in vivo fluorescence imaging, the tumor-bearing mice were intravenously injected with 100 μL of the nanoparticle suspension, containing approximately 2 μg of DiR per mouse.

In vivo Antitumor and Biosafety Studies

When the subcutaneous tumors reached volume of approximately 100mm³, the tumor-bearing male mice were randomly divided into five treatment groups (n=5 per group). Furthermore, predefined endpoints were strictly implemented: animals would be humanely euthanized if the tumor volume exceeded 2000mm³, if body weight loss exceeded 20% or if severe tumor ulceration were observed. The investigators responsible for tumor-volume measurement, fluorescence ROI analysis, and histological evaluation were blinded to treatment allocation until the analyses were completed.

The tumor-bearing male mice were intravenously injected with PPcJ, non-targeted PLGA-PEG-JAPT8 (hereafter referred to as PPJ), JAPT8, PLGA-PEG, or PBS when the tumor size reached 100 mm³ every 48 hours for four times. The administered dosage for JAPT8-containing formulations (PPcJ, PPJ, and free JAPT8) was 5 mg/kg (equivalent to JAPT8), which was calculated and determined based on the effective concentrations observed in the in vitro studies. PLGA-PEG was administered at an equivalent mass concentration to the nanocarrier in the PPcJ group. The body weight and tumor volume were recorded every 2–3 days during the injections, and the tumor volume was calculated using the following formula: V = width² × length × π/6. Twenty-four hours post-final administration, mice were euthanized by cervical dislocation under isoflurane anesthesia. Tumors and major organs (heart, liver, spleen, lungs, kidneys) were excised, fixed in 10% neutral buffered formalin, and embedded in paraffin. Sections were stained with hematoxylin-eosin (HE), dUTP nickel end labeling (TUNEL), and Ki-67. After the color reaction, the images of staining were captured using a microscope (Nikon, H550S, Tokyo, Japan). Serum was collected to measure the levels of aspartate aminotransferase, alanine transaminase, urea, and serum creatinine.

Statistical Analysis

All quantitative data are expressed as mean ± standard deviation (SD) unless otherwise specified. Statistical analyses were performed using GraphPad Prism 9.0. Concentrations of free JAPT8 and PPcJ are expressed as JAPT8-equivalent concentrations. The blank PLGA–PEG control was matched according to the corresponding polymer content in the PPcJ formulation. For the comparison of means between multiple groups at a single time point, a one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was applied. For longitudinal data, such as tumor volume growth curves, a two-way repeated measures ANOVA followed by Tukey’s post-hoc test was utilized. Survival analysis was performed using the Kaplan-Meier method, and statistical differences between survival curves were determined using the Log-rank (Mantel-Cox) test. The IC₅₀ values of free JAPT8 and PPcJ were determined by four-parameter logistic nonlinear regression using the “log(inhibitor) versus response—Variable slope” model. A P value of <0.05 was considered statistically significant, and was marked as “*”; A P value of <0.01 was marked as “**”, a P value of <0.001 was marked as “***”, and a P value of <0.0001 was marked as “****”.

Results

PPcJ Nanoparticles Exhibit Nanoscale Morphology and Efficient JAPT8 Encapsulation

PPcJ and PLGA-PEG nanoparticles were prepared as described in Preparation and Characterization of PPcJ Nanoparticles, and the structure of JAPT8 is presented in Figure S1. Dynamic light scattering analysis and TEM images (Figure 1A and B) revealed that the PLGA-PEG nanoparticles possessed a spherical morphology with a mean diameter of 216.03 ± 7.49 nm. Following the encapsulation of JAPT8 (Figure 1C and D), the particle size of PPcJ increased to 240.53 ± 9.04 nm. Both PLGA-PEG and PPcJ nanoparticles exhibited a polydispersity index (PDI) below 0.3, reflecting a narrow and homogeneous size distribution in suspension. Furthermore, the zeta potential shifted from −12.19 mV for PLGA-PEG to −8.95 mV for PPcJ (Figure S2), consistent with the surface modification and drug loading. FTIR analysis (Figure 1E) identified characteristic peaks at 3319.5, 2160.3, and 1634.9 cm⁻¹. These were accurately attributed to the N-H stretching vibrations of the amide group in the cRGD peptide, the C≡N stretching originating from the encapsulated JAPT8, and the characteristic Amide I band (C=O stretching) of the cRGD peptide, respectively. Finally, encapsulation efficiency was quantified via HPLC at 192.8 nm, confirming a high JAPT8 content of 86.35 ± 0.64% (Figures 1F and S3). Collectively, these characterization data verify the successful construction of the PPcJ nanoplatform.

Figure 1.

Physicochemical characterization and JAPT8 encapsulation of PPcJ nanoparticles. (A) Particle size distribution and PDI of PLGA-PEG by Nano Zetasizer. (B) Representative TEM images of PLGA-PEG. (Scale bars: 1 μm for the left image, 500 nm for the right insert). (C) Particle size distribution and PDI of PPcJ by Nano Zetasizer. (D) Representative TEM images of PPcJ. (Scale bars: 1 μm for the left image, 500 nm for the right insert). (E) Absorbance values of PPcJ in the presence of JAPT8 under the 525–4000 cm⁻¹ IR light. The upward-pointing arrows indicate the specific wavenumbers (3319.5, 2160.3, and 1634.9 cm⁻¹) of the characteristic absorption peaks. (F) The encapsulation efficiency of JAPT8 was determined by HPLC at 192.8 nm. These data confirm the successful synthesis of PPcJ nanoparticles, featuring nanoscale morphology and highly efficient drug loading capacities.

PPcJ Enhances Cellular Uptake and Exerts Potent Cytotoxicity in Bladder Cancer Cells

To evaluate the in vitro cytotoxicity, bladder cancer cells were incubated with varying concentrations of PLGA-PEG, JAPT8, and PPcJ nanoparticles. As depicted in Figure 2A, PPcJ markedly inhibited the viability of MB49 cells in a concentration-dependent manner. The IC₅₀ of PPcJ was 2.20 μg/mL (95% CI: 1.59–3.03). In contrast, free JAPT8 (IC₅₀:10.10 μg/mL; 95% CI: 8.01–12.75) showed relatively limited effects within the tested concentration range. These findings indicate that PPcJ exhibits a superior inhibitory effect on bladder cancer cells than PLGA-PEG and JAPT8. As a targeted nanodelivery system, PPcJ is rationally designed to enhance specific drug accumulation within the tumor tissue compared to free JAPT8, thereby maximizing local therapeutic efficacy.

Figure 2.

In vitro cellular uptake, cytotoxicity, and lysosomal colocalization of PPcJ. (A) Cell viability of MB49 cells with various treatments by CCK-8. Concentrations of free JAPT8 and PPcJ are expressed as JAPT8-equivalent concentrations. The blank PLGA–PEG control was matched according to the corresponding polymer content in the PPcJ formulation. Cellular uptake of PPcJ/C6 in MB49 cells detected by flow cytometry in different incubation times (B) and concentrations (C). (D) Representative immunofluorescence images of PPcJ/C6 in MB49 cells incubating for 1h-5h (blue, Hoechst; green, PPcJ/C6). Scale bar: 100 µm. (E) Representative fluorescence images of PPcJ/C6 and lysosome in MB49 cells (blue, Hoechst; green, PPcJ/C6; red, lysosome). Scale bar: 100 µm. These findings demonstrate that PPcJ achieves rapid, targeted cellular uptake and partially lysosomal escape, thereby exerting potent cytotoxicity against bladder cancer cells.

To elucidate the cellular internalization profile, flow cytometry was utilized to assess the time- and concentration-dependent uptake of PPcJ. As shown in Figures 2B and S4, fluorescence intensity was measured at 0, 2, 3, 4, 5, and 8 h after treatment. Cellular uptake increased progressively during the early incubation period and reached its highest numerical level at 4 h (Figure S4). Furthermore, the fluorescence intensity at 5 h was not significantly different from that at 4 h, indicating that uptake had reached a near-plateau during this period (Figure 2D). Additionally, PPcJ cellular uptake did not increase significantly above 5 μg/mL, even at a higher dose of 10 μg/mL (Figures 2C and S5). These results suggest that the cellular uptake condition of PPcJ is 4 h incubation at 5 μg/mL.

To visualize the subcellular distribution of PPcJ nanoparticles within bladder cancer cells, confocal microscopy was used to examine the colocalization between the green fluorescence of PPcJ/C6 and the red fluorescence of LysoTracker Red. Notably, only partial colocalization was observed between the nanoparticles and lysosomes (Figure 2E). Quantitative analysis of Pearson’s Correlation Coefficient is 0.42 ± 0.14, confirming that a significant portion of PPcJ effectively evaded lysosomal entrapment. Therefore, this facilitated lysosomal escape provides a crucial pathway for the subsequent release and delivery of JAPT8 into the cytoplasm.

PPcJ Induces Apoptosis by Suppressing the JAK/STAT Signaling Pathway

The anti-tumor effect of PPcJ was evaluated in vitro. As shown in Figure 3A and quantified in Figure S6, the PLGA-PEG blank nanocarriers exhibited excellent biosafety, with a minimal total apoptosis rate at both 24 h (1.23%) and 72 h (2.15%), which was statistically comparable to the PBS control group. At 24 h, the targeted delivery platform began to demonstrate a therapeutic advantage, as the PPcJ group induced a higher total apoptosis rate (11.94%) compared to the free JAPT8 group (5.83%). By 72 h, this cytotoxic effect was markedly amplified. While free JAPT8 significantly increased the total apoptotic population, the PPcJ formulation induced a much more profound overall apoptotic effect. These quantitative results indicate that utilizing the PLGA-PEG nanocarrier with targeted modifications significantly enhances the cellular internalization and in vitro therapeutic efficacy of JAPT8. To elucidate the underlying molecular mechanisms, the expression of apoptosis-related proteins was examined via Western blotting (Figures 3B, S7A and B). The Caspase-3 and Bcl-2 expression revealed no significant differences between the PBS and blank PLGA–PEG groups. Compared to the PBS group, both free JAPT8 and PPcJ significantly upregulated the expression of the apoptosis executioner Caspase-3 and downregulated the anti-apoptotic protein Bcl-2. Conversely, empty PLGA-PEG nanoparticles exhibited no evident pro-apoptotic effects.

Figure 3.

PPcJ induces apoptosis in bladder cancer cells by suppressing the JAK-STAT signaling pathway. (A) Flow cytometry scatterplot for apoptosis MB49 cells treated with PBS, PLGA-PEG, JAPT8, and PPcJ. Western blot analysis was used to detect the expression of proteins related to apoptosis (B) and the JAK/STAT signaling pathway (C), and β-tubulin was used as an internal reference protein. Together, these results mechanistically validate that the PPcJ triggers apoptosis by effectively silencing the JAK/STAT signaling cascade.

To verify the signaling pathway through which PPcJ functions, the expression of key components of the JAK-STAT pathway was assessed. As illustrated in Figures 3C and S8A–F, JAPT8 and PPcJ nanoparticles downregulated the expression of the upstream initiators JAK1 and JAK2, and consequently significantly downregulated their phosphorylated active forms (p-JAK1 and p-JAK2). This upstream dual-inhibition effectively blocked the downstream signaling cascade, leading to a marked reduction in the expression of the downstream effectors STAT1 and STAT3, as well as their phosphorylated (p-STAT1 and p-STAT3) active forms. Importantly, this inhibitory effect was most pronounced in the PPcJ group. Collectively, PPcJ nanoparticles effectively induce apoptosis in bladder cancer cells by suppressing the JAK-STAT signaling pathway.

cRGD-Modified PPcJ Exhibits Tumor-Targeting Capability and Superior in vivo Biosafety

To evaluate whether cRGD enhances the tumor targeting of the nanoplatform, we established a subcutaneous bladder cancer mouse model. DiR-labeled non-targeted (PLGA-PEG-DiR) and targeted (PLGA-PEG-cRGD-DiR) nanoparticles were synthesized and intravenously administered; quantitative tumor-region-of-interest (ROI) analysis was performed using all mice in the imaging cohort (n = 3 per group). In vivo imaging (Figures 4A and S9) showed that PLGA‑PEG‑DiR exhibited appreciable accumulation at the tumor site up to 12 h post-injection, after which the signal faded. In contrast, the cRGD-modified nanoparticles displayed distinct fluorescence in the tumor region as early as 6 h post-injection. ROI analysis showed higher mean tumor-associated fluorescence for PLGA–PEG–cRGD–DiR at 12, 16, and 24 h (Figure S9). Ex vivo imaging of tumors and major organs further confirmed that cRGD promoted tumor-associated DiR fluorescence and retention of the nanoplatform within tumor tissues. This indicates that the inherent tumor-targeting of cRGD synergizes with the enhanced permeability and retention (EPR) effect of the nanocarrier, effectively promoting the specific accumulation and prolonged retention of PPcJ in tumor tissues.

Figure 4.

Tumor-associated accumulation and long-term retention of PPCJ in tumor tissues without systemic toxicity. (A) In vivo fluorescence imaging of mice bearing MB49 tumors after intravenous injection of PPcJ/DiR or PLGA-PEG/DiR, and ex vivo image in major organs and the tumor. The dotted white circle indicates the tumor region. (B) H&E staining of major organs extracted from mice. Scale bar: 100 µm. These imaging and histological results demonstrate that the cRGD modification improves PPcJ/DiR with active tumor-targeting capability while maintaining excellent systemic biocompatibility.

Importantly, the biosafety of PPcJ was also evaluated. The HE staining images (Figure 4B) revealed no obvious damage in the heart, liver, spleen, lungs, or kidneys. Furthermore, no significant changes were observed in organ function indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine (CR) (Figure S10A-D). Collectively, these histological and serological results demonstrate that the PPcJ nanodelivery system possesses excellent biocompatibility and does not induce observable systemic toxicity or off-target organ damage. Collectively, this strategy enhances local drug concentration while reducing systemic toxicity.

PPcJ Suppresses in vivo Tumor Growth and Prolongs Survival

Based on the above results, the antitumor efficacy of PPcJ in vivo was further investigated. As shown in Figure 5A, tumors in the PLGA-PEG group showed no significant difference compared with the PBS group. In contrast, tumors in the JAPT8 group were relatively smaller than those in the PBS group, indicating that JAPT8 exhibited a certain therapeutic effect. The PPcJ group showed the most potent tumor growth inhibition. Consequently, PPcJ treatment significantly extended animal survival; whereas mice in the PBS group survived up to day 27, the PPcJ group maintained a 100% survival rate through day 36, with the maximum observed survival extending to 42 days (Figure 5B). The mean survival time of mice in the PBS group was 22.4 ± 4.2 days, whereas those receiving the targeted PPcJ treatment exhibited a significantly prolonged mean survival time of 41.0 ± 2.2 days (Figures 5B and S11). Furthermore, WB results (Figure 5C) revealed that PPcJ upregulated the pro-apoptotic protein Caspase-3 and downregulated the anti-apoptotic protein Bcl-2, consistent with the induction of apoptosis. Meanwhile, consistent with our in vitro findings, PPcJ treatment remarkably induced the degradation of JAK1 and JAK2 in the tumor microenvironment, leading to a significant reduction in their phosphorylated active forms (p-JAK1 and p-JAK2). Consequently, the downstream signaling was also powerfully inhibited in vivo, as evidenced by the decreased expression of STAT1, STAT3, and their corresponding phosphorylated forms (p-STAT1 and p-STAT3). These in vivo results comprehensively demonstrate that PPcJ effectively suppresses tumor growth by blocking the JAK/STAT signaling pathway.

Figure 5.

PPcJ inhibits tumor growth and prolongs survival in bladder cancer model by inhibiting the JAK/STAT signaling pathway. (A) Morphology of tumors extracted from mice with different treatments at the study endpoint. (B) Kaplan–Meier survival analysis of tumor-bearing mice. (C) Western blot analysis of apoptosis-related proteins and JAK/STAT signaling pathway-related proteins in tumor tissues; β-tubulin was used as the loading control. (D) Tumor volume kinetics after different treatments. (E) Body weight monitoring during treatment as an indicator of systemic safety. (F) Representative images of hematoxylin and eosin (H&E) staining, TUNEL staining, and immunohistochemical analysis of tumor tissues from mice receiving different treatments. Data were obtained from five mice per group (n = 5). Scale bar: 100 μm. These in vivo findings support that PPcJ suppresses tumor growth, delays tumor-associated mortality, and enhances the therapeutic activity of JAPT8. ***P < 0.001.

Throughout the treatment period, no significant fluctuations in body weight were observed across any groups (Figure 5E), indicating the excellent biocompatibility and systemic safety of the nanoplatform. Taken together, these findings indicate that PPcJ demonstrates favorable outcomes in terms of animal survival, tumor suppression, and biocompatibility.

Tumor volume increased significantly in the PBS group, whereas PPcJ significantly inhibited tumor proliferation (Figure 5D). Owing to the robust Enhanced Permeability and Retention (EPR) effect in the subcutaneous tumor model, both the non-targeted PPJ and targeted PPcJ nanoplatforms profoundly suppressed tumor growth compared to the free JAPT8 group. Notably, the targeted PPcJ group consistently exhibited the lowest mean tumor volume, achieving a tumor inhibition rate of 86.2% relative to the PBS control at the end of the observation period (Figure 5D).

These therapeutic effects were further corroborated by histological and immunohistochemical analyses. H&E staining revealed that tumor cells in the PBS group displayed dense arrangement with large, deeply stained nuclei (Figure 5F). In contrast, the PPcJ group induced phenomena such as nuclear pyknosis and karyolysis, along with a sharp decrease in cell number, indicative of tumor tissue apoptosis and necrosis. Moreover, immunohistochemical staining for Ki-67, a well-established marker of cellular proliferation, demonstrated a profound reduction in proliferative activity following PPcJ treatment (Figure 5F). Finally, TUNEL staining exhibited widespread, intense green fluorescence in the PPcJ group compared to the negligible signal in the PBS control, confirming extensive in vivo apoptosis. Taken together, these results confirm that PPcJ exerts effective antitumor activity in vivo.

Discussion

Bladder cancer is one of the most common malignancies of the urinary system.^16,17 Despite ongoing improvements in surgical and chemotherapeutic approaches, patient prognosis remains poor owing to the aggressive nature of the disease, including its high potential for dissemination and metastasis, especially in advanced and metastatic settings.^18,19

Aberrant activation of the JAK/STAT signaling pathway plays a pivotal role in tumor apoptosis regulation.²⁰ This dysregulation promotes the proliferation and infiltration of immunosuppressive cells while upregulating immune checkpoint molecule expression on tumor cells, thereby impeding the apoptotic process. Recent research has revealed that JAK inhibitors potentiate antitumor immune responses in specific cancer.^6,21 Early-phase clinical trials have further demonstrated synergistic activity between JAK inhibitors and immune checkpoint inhibitors. For instance, the combination of ruxolitinib and nivolumab in Hodgkin lymphoma resulted in an overall response rate of 53%, with 6 of 19 patients attaining complete metabolic remission.²² While first‑generation JAK inhibitors exhibit broad‑spectrum activity against multiple JAK subtypes, they interfere not only with tumor‑related aberrant pathways but also with normal JAK/STAT signaling in healthy cells, thus lacking target specificity.⁷ Consequently, the design of next‑generation selective JAK inhibitors has emerged as a key direction in drug development.

Previously, our group synthesized JAK1/JAK2 degraders (JAPT8) based on proteolysis-targeting chimeras (PROTACs), which utilize E3 ligases to promote the ubiquitination and degradation of JAK1 and JAK2.¹² Based on this foundation, we developed a targeted nanoparticle, termed PPcJ, for bladder cancer therapy. Nanotechnology offers a novel chemical-biological strategy for treating bladder cancer, capitalizing on its ability to improve drug solubility, enhance bioavailability, and minimize systemic toxicity.^23,24 PPcJ encapsulated JAPT8 and was surface-functionalized with the targeting peptide cRGD. The design aims to utilize the active targeting mediated by cRGD to enhance the specific accumulation of JAPT8 in tumor tissues, enabling precise therapeutic delivery.

Furthermore, while our pre-conjugation strategy ensured highly uniform cRGD functionalization on the PPcJ nanoplatform, the precise percentage of surface functionalization is a critical parameter in nanomedicine. Existing literature suggests that an optimal surface ligand density (typically ranging from 5% to 20%) is required to maximize receptor-mediated endocytosis.^25,26 Excessively high functionalization percentages can inadvertently compromise the anti-fouling properties of the PEG corona, accelerate systemic clearance, or trigger competitive receptor binding, often leading to restricted tumor penetration and the so-called hook effect.^27,28 Our in vivo biodistribution and antitumor efficacy results indicate that the current cRGD functionalization achieved a favorable tumor targeting.

The JAK-STAT signaling pathway plays a central role in regulating essential cellular functions, including proliferation, migration, differentiation, and apoptosis.^29,30 In vitro studies showed that PPcJ induced apoptosis in bladder cancer cells within 72 h after a 4 h exposure. Western blot analysis revealed that PPcJ upregulates the pro-apoptotic protein Caspase-3 while downregulating the anti-apoptotic protein Bcl-2, thereby reducing tumor cell viability, and this finding was further supported by TUNEL staining. In addition, PPcJ achieves rapid cellular drug uptake when incubated with bladder cancer cells at a relatively low concentration. This approach not only avoids the adverse effects linked to high drug concentrations but also enables effective evasion of lysosomal degradation, thereby enhancing therapeutic efficacy against bladder cancer via lysosomal escape.

Upon stimulation by various cytokines, the JAK-STAT pathway contributes to complex immunoregulatory processes.^31,32 For example, cancer cell recognition is predominantly initiated by interferons and STAT1/STAT2 signaling, whereas immune evasion is strongly linked to IL-6–STAT3 pathway activation.^33,34 Targeting JAK and inhibiting its phosphorylation effectively disrupts cytokine-driven aberrant immune and inflammatory responses, underscoring the rationale for employing JAK inhibitors in cancers.³⁵ Our study showed that PPcJ downregulated the expression of JAK1, JAK2, STAT1, STAT3, and their phosphorylated forms p-STAT1 and p-STAT3, suggesting that it promotes apoptosis in bladder cancer cells via suppression of the JAK-STAT pathway.

Crucially, the remarkable in vivo success of the PPcJ nanoplatform is intrinsically linked to its high physiological stability. The hydrophilic PEG corona provides robust steric hindrance, protecting the PLGA core from protein adsorption and aggregation in the complex circulatory system.¹⁵ This colloidal stability not only minimizes premature drug leakage but also evades rapid clearance by the reticuloendothelial system.³⁶ Consequently, this prolonged blood circulation window maximizes the opportunities for the nanoparticles to extravasate into the tumor microenvironment via the EPR effect and subsequently achieve active cRGD-mediated internalization.^37,38 Indeed, our in vivo results strongly supported the potential of PPcJ, demonstrating significantly extended survival (mean survival time of 41.0 ± 2.2 days vs 22.4 ± 4.2 days), effective tumor suppression (86.2% tumor inhibition rate), and excellent biocompatibility. This efficacy under a four doses regimen underscores the sustained target degradation capability of the PROTAC mechanism in vivo, where optimized continuous dosing could yield more profound survival benefits. Fluorescence imaging confirmed the strong tumor-targeting capability of this nano-delivery system, while immunohistochemical analysis further verified its capacity to effectively inhibit tumor cell proliferation. Collectively, these findings highlight the promising potential of PPcJ for precision bladder cancer therapy.

Ultimately, the PLGA-PEG nanocarrier is completely hydrolyzed into biologically benign lactic acid and glycolic acid. These natural metabolites enter the Krebs cycle and are eliminated as carbon dioxide and water. This metabolic clearance efficiently prevents long-term material accumulation, explaining the systemic biosafety observed during our in vivo treatments.³⁹

To facilitate the future translation of the PPcJ nanoplatform into clinical practice, it is essential to consider its intended treatment settings and routes of administration. For advanced or metastatic bladder cancer, intravenous administration represents the primary clinical route, where the nanoparticles leverage the EPR effect and cRGD-mediated active targeting to reach metastatic lesions. Compared to current standard therapies, such as conventional systemic chemotherapy or non-targeted small-molecule inhibitors, PPcJ offers distinct clinical advantages. It successfully resolves the poor aqueous solubility of the hydrophobic PROTAC (JAPT8), enhances tumor-specific accumulation to strictly limit off-target toxicities, and enables sustained targeted protein degradation. However, for localized non-muscle-invasive bladder cancer, direct intravesical instillation represents a highly attractive alternative strategy. Intravesical instillation maximizes local drug concentrations while minimizing systemic toxicity. Notably, the cRGD peptides on PPcJ can anchor to integrin-overexpressing cells, potentially preventing the rapid drug washout typical of conventional therapies. However, its inability to treat deep muscle-invasive or metastatic lesions. Ultimately, the feasibility of intravesical PPcJ for early-stage NMIBC requires future validation in orthotopic models. Furthermore, to bridge the translational gap between current pre-clinical models and true clinical applicability, our future investigations will strictly incorporate patient-derived tumor organoids and a broader panel of human bladder cancer cell lines. This organoid-based approach will be pivotal for rigorously validating the therapeutic efficacy of PPcJ within a complex tumor microenvironment.

Conclusion

To overcome the limitations of inadequate targeting and poor bioavailability of JAK inhibitors in bladder cancer treatment, we developed PPcJ, a cRGD-functionalized PLGA-PEG nanodelivery system. In vitro and in vivo experiments demonstrated that PPcJ facilitated efficient cellular internalization and tumor-associated accumulation compared with the controls. Mechanistically, PPcJ markedly downregulated key components of the JAK/STAT pathway, promoted apoptosis, suppressed tumor growth, and prolonged survival in tumor-bearing mice. Studies further validated its favorable tumor targeting, potent antitumor activity, improved survival, and biocompatibility without evident systemic toxicity. Collectively, this study provides a pre-clinical proof-of-concept that the PPcJ nanodelivery system can enhance targeted PROTAC therapy. Further comprehensive investigations, including validation in orthotopic tumor models and long-term systemic toxicity evaluations, are warranted to robustly explore its true feasibility for future clinical translation.

Funding Statement

This work was supported by Natural Science Foundation of Shanghai Municipal Science and Technology Commission [Grant No. 19ZR1408000].

Ethical Approval

All animal care, housing, and experimental protocols were performed in accordance with both institutional and national ethical guidelines for the care and use of laboratory animals. The study protocols were formally reviewed and approved by the animal ethics committee of Fudan University (Ethical approval number: 2021- HSYY-JS −188).

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. Specific contributions were as follows: Weihong Ding, Zongguang Tai, and Guowei Xia: Conceptualization; Xinyue Zhang and Hongdan Wu: Methodology; Congrui Zhou and Ke Xu: Validation; Weihong Ding and Congrui Zhou: Formal analysis; Xinyue Zhang and Zongguang Tai: Investigation; Guowei Xia and Zongguang Tai: Resources; Weihong Ding and Xinyue Zhang: Writing—original draft; Zongguang Tai and Guowei Xia: Writing—review and editing; Guowei Xia and Weihong Ding: Funding acquisition.

Disclosure

The authors report no conflicts of interest in this work.