Using Lipid Nanoparticles to Deliver P21 mRNA Directly into the Bladder as a Tumor Suppressor Treatment for Bladder Cancer

The coding sequence of human p21 (NM_000389↗) was cloned into the pcDNA3.1 vector (General Biology). The EGFP coding sequence was synthesized and cloned into pSG5L (General Biology). A C‐terminal HA tag was added using the ClonExpress II One Step Cloning Kit (Vazyme, C112‐01). The sequences of all constructs were confirmed by Sanger sequencing.

Linearized DNA templates were generated by PCR using a forward primer containing the T7 promoter sequence and a reverse primer containing a 100‐nucleotide Poly (T) tract to encode the Poly (A) tail. The purified PCR products served as templates for IVT. Transcription was performed using the T7 RNA Polymerase Mix (APExBIO, K1083) supplemented with ATP (APExBIO, K1043), CTP (APExBIO, K1045), GTP (APExBIO, K1044), and N1‐methylpseudo‐UTP (APExBIO, B8049). EzCap (APExBIO, B8177) was included to facilitate co‐transcriptional 5′‐capping. Following incubation at 37°C for 3 h, the DNA templates were degraded by DNase I digestion (Thermo Fisher Scientific, EN0521) at 37°C for 15 min. The synthesized mRNA was purified using the RNA Clean & Concentrator kit (APExBIO, K1069), and its concentration was determined prior to downstream applications.

The human bladder carcinoma cell line T24 was obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The human bladder carcinoma cell line J82 and the human embryonic kidney cell line HEK293T were purchased from the American Type Culture Collection (ATCC). J82 and T24 cells were cultured in McCoy's 5A Medium (BasalMedia, L630KJ) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10 099 141). HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (BasalMedia, L110KJ) supplemented with 10% FBS. All cells were maintained at 37°C in a humidified incubator containing 5% CO₂. Cell lines were kept under sterile conditions and routinely tested for mycoplasma contamination.

HEK293T cells were co‐transfected with the pLenti‐Luc plasmid and packaging plasmids using polyethyleneimine (Polysciences, USA). Viral supernatants were harvested 48 h post‐transfection and used to infect newly plated T24 cells in the presence of polybrene to enhance transduction efficiency. Forty‐eight hours post‐infection, cells were subjected to selection with 2 μg/mL puromycin (APExBIO, B7587). Stable cell lines were established after 3–5 days of selection.

Cells were seeded in 6‐well plates and transfected upon reaching 60%–70% confluence. For each well, 2 μg of mRNA and 4 μL of Lipofectamine 2000 (Thermo Fisher Scientific, 11 668 019) were diluted separately in 100 μL of Opti‐MEM medium (Thermo Fisher Scientific, 11 058 021) and incubated for 5 min at room temperature. The mRNA and the Lipofectamine mixture were combined, incubated for an additional 10 min, and added dropwise to the culture medium. All in vitro experiments were independently repeated at least three times.

Cells or tissues were lysed in radio immunoprecipitation assay (RIPA) buffer (NCM Biotech, WB3100) supplemented with protease inhibitor cocktail (Roche, 04693159001) and 1 mM phenylmethylsulfonyl fluoride (PMSF) (Amresco, 0754). Lysates were clarified by centrifugation at 13 000 rpm for 20 min at 4°C. Protein concentrations were determined using a bicinchoninic acid (BCA) assay Kit (Sangon Biotech, C503021). Samples were mixed with SDS loading buffer (Epizyme, LT101S), denatured at 95°C for 10 min, separated by SDS‐PAGE, and transferred onto nitrocellulose membranes (Millipore, HATF00010).

Membranes were blocked with 5% non‐fat milk in Tris‐buffered saline containing 0.1% Tween 20 (TBST) and incubated overnight at 4°C with primary antibodies. Primary antibodies included: Anti‐p21 (CST, 2947, 1:1000), anti‐p21 (Abcam, ab188224, 1:1000), anti‐phospho‐Rb (Ser807/811) (CST, 8516, 1:1000), anti‐Cyclin E (CST, 20808, 1:1000), anti‐Cyclin B (CST, 12231, 1:1000), anti‐PARP1 (CST, 9542 T, 1:1000), anti‐γ‐H2A.X (CST, 9718S, 1:1000), anti‐PCNA (HuaBio, ET1605‐38, 1:2000), anti‐Vimentin (Sigma, V6389, 1:1000), anti‐VE‐cadherin (Wanleibio, WL02033, 1:1000), anti‐GAPDH (Proteintech, 81 640–5‐RR, 1:5000), anti‐β‐actin (Proteintech, 81 115–1‐RR, 1:5000), and anti‐EGFP (Proteintech, 66 002–1‐Ig, 1:10000).

After washing, membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies for 1 h at room temperature. Secondary antibodies included HRP‐Goat Anti‐Mouse IgG (H + L) (APExBIO, K1221, 1:10000), HRP‐Rabbit Anti‐Goat IgG (H + L) (APExBIO, K1224, 1:10000), and HRP‐Goat Anti‐Rabbit IgG (H + L) (APExBIO, K1223, 1:10000). Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Tanon, 180–501) and imaged with the ChampChemi system.

Cells were cultured on sterile glass coverslips in 6‐well plates. After transfection, cells were washed, fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X‐100. Non‐specific binding was blocked with a solution containing 2% goat serum and 2% bovine serum albumin (BSA) for 1 h. Cells were then incubated with anti‐HA primary antibody (CST, 3724S, 1:300) for 1 h, followed by fluorescent secondary antibodies for 30 min. Secondary antibodies used were goat anti‐mouse IgG (H + L) Alexa Fluor 488 (Invitrogen, A32723, 1:400) and goat anti‐rabbit IgG (H + L) Alexa Fluor 568 (Invitrogen, A32731, 1:400). Nuclei were counterstained with DAPI. Coverslips were mounted using ProLong Diamond Antifade Mountant (Invitrogen, P36961↗), and images were acquired using a Nikon ECLIPSE E800 fluorescence microscope. Image analysis was performed using ImageJ software.

Cells were harvested and counted using an automated cell counter (JIMBIO iCytal). For cell viability assays, cells were trypsinized 6 h post‐mRNA transfection and seeded into 96‐well plates at a density of 1 × 10⁴ cells/well. Cell viability was assessed at 24 h post‐transfection using the CCK‐8 Kit (APExBIO, K1018) according to the manufacturer's instructions. Absorbance at 450 nm was measured using a microplate reader (Tecan Infinite 200 PRO).

At 6 h after transfection of EGFP or p21 mRNA into T24 cells, a wound‐healing assay was performed. Briefly, a scratch was created in a confluent cell monolayer using a sterile pipette tip to generate a cell‐free gap. Cell migration into the gap was monitored and imaged at 0, 24, and 48 h. Images were captured using a Nikon ECLIPSE E800 microscope and analyzed with ImageJ software.

T24 cells at 60%–70% confluence in 6‐well plates were transfected with EGFP or p21 mRNA for 6 h, then harvested and re‐seeded into new 6‐well plates at a density of 500 cells/well. Cells were cultured in complete medium for 14 days, with medium replacement every 2–3 days. Colonies were subsequently washed with PBS, fixed with 4% paraformaldehyde for 15 min, and stained with 0.1% crystal violet for 20 min at room temperature. After gentle washing and air‐drying, colonies were photographed, and the number of colonies was quantified using ImageJ.

Cells were seeded in 6‐well plates and transfected with mRNA at 60%–70% confluence. Twenty hours post‐transfection, cells were harvested, and apoptosis was assessed using the Annexin V‐FITC Apoptosis Detection Kit (LiankeBio, AP101) according to the manufacturer's protocol. Stained samples were analyzed on a CytoFLEX S flow cytometer (Beckman), and data were analyzed using FlowJo software.

A bladder cancer tissue microarray (ZL‐BlaU961) containing 59 clinical cases was used to evaluate p21 protein expression in human bladder tissues. Formalin‐fixed, paraffin‐embedded sections were baked at 59°C for 60 min, deparaffinized in xylene, rehydrated through graded ethanol, and subjected to antigen retrieval in EDTA buffer (Weiao, WH1034) using heat‐induced epitope retrieval under high‐temperature and high‐pressure conditions. Endogenous peroxidase activity was blocked with 3% H₂O₂, followed by blocking with 5% BSA (Weiao, WH2051). Sections were incubated with an anti‐p21 primary antibody (Cell Signaling Technology, 2947, 1:50) at 4°C overnight, followed by HRP‐based detection using a universal immunohistochemistry kit (DAKO, K5007). Immunoreactivity was visualized with DAB (Weiao, WB0167) and counterstained with hematoxylin (Weiao, WH1145). Sections were then dehydrated, cleared, and mounted.

p21 staining was quantified by calculating H‐scores from five non‐overlapping fields per sample using the IHC Profiler plugin in ImageJ according to the formula: H‐score = 0 × negative + 1 × low positive + 2 × positive + 3 × high positive. Cases were grouped according to pathological stage (Tis, T1, T2, T3, and T4) for comparison. Statistical analyses were performed using GraphPad Prism 10.

Tumor models were established using female BALB/c nude mice (5–6 weeks old). All animal experiments were conducted in compliance with the Animal Ethics and Welfare Guidelines of the Chinese Ministry of Public Health and were approved by the Animal Care and Experiment Committee of Shanghai Jiao Tong University School of Medicine (Accreditation No. JUMC2023‐047‐A).

Mice were anesthetized with isoflurane, and a catheter was inserted into the bladder through the urethra. The bladder was drained, washed with PBS, and instilled with 50 μL of 0.5 M silver nitrate (AgNO₃) for 15 s to disrupt the urothelial barrier. After three PBS washes, 100 μL of T24‐Luc cell suspension (2 × 10⁶ cells) was instilled. A non‐invasive purse‐string suture was applied to the urethra for 2 h to improve tumor cell retention.

Tumor burden was monitored using the IVIS Spectrum Imaging System (PerkinElmer). Tumor‐bearing mice were intraperitoneally injected with D‐luciferin (150 mg/kg). Mice were anesthetized via isoflurane inhalation, and imaging was performed 5 min post‐injection to capture the peak bioluminescent signal.

Lipid nanoparticles (LNP) were formulated using a lipid mixture of SM‐102 (APExBIO, C1042), 1,2‐Distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) (Avanti Polar Lipids, 850 365), cholesterol (APExBIO, B1702), and PEG2000‐DMG (Avanti Polar Lipids, 880 151) at a molar ratio of 50:10:38.5:1.5, dissolved in ethanol. mRNA was diluted in 50 mM citrate buffer (pH 3.2). The lipid and mRNA solutions were mixed at a volumetric ratio of 1:3 using a Feather Microfluidic Chip (APExBIO, RM1002) on a NanoDispatcher L device (APExBIO, RM1001). The resulting formulations were diluted twofold with 50 mM citrate buffer (pH 3.2) and dialyzed against PBS (pH 7.4) for > 15 h using Slide‐A‐Lyzer dialysis cassettes (Thermo Fisher Scientific, 66 830). Formulations were concentrated using Amicon Ultra centrifugal filters (MilliporeSigma, UFC90008↗) and sterilized by filtration through a 0.22‐μm membrane. Particle size and polydispersity index (PDI) were measured using a Zetasizer (Malvern Panalytical). mRNA encapsulation efficiency and concentration were quantified using the Quant‐iT RiboGreen RNA Assay (Thermo Fisher Scientific, R11490↗).

For biodistribution studies, mice received an intravesical instillation of 10 μg Luc‐LNP and were subjected to in vivo bioluminescence imaging at 6, 24, 48, and 72 h after administration. At the indicated time points, mice were euthanized, and major organs, including the bladder, heart, lung, spleen, kidney, and liver, were collected for ex vivo bioluminescence imaging. Total flux in each organ was quantified using Living Image software.

For therapeutic efficacy studies, tumor‐bearing mice were randomized into three groups (n = 7) on day 3 post‐implantation. Mice received intravesical instillations of 100 μL PBS, EGFP‐LNP (10 μg in 100 μL PBS), or p21‐LNP (10 μg in 100 μL PBS) every 3 days for a total of 8 doses. Tumor growth was monitored by IVIS imaging after intraperitoneal D‐luciferin injection (150 mg/kg), and total flux (photons/s) in the bladder region was quantified.

Bladder tissues and major organs, including the heart, liver and kidney were harvested, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with Hematoxylin and Eosin (H&E) for pathological evaluation.

Data are presented as mean ± standard deviation (SD) or standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software. Comparisons among multiple groups were performed using one‐way ANOVA followed by Tukey's post hoc test. A p‐value < 0.05 was considered statistically significant.

Given the recurrent inactivation of CDKN1A/p21 and the widespread disruption of the p53/cell‐cycle axis in bladder cancer, we evaluated the relevance of p21 loss using public datasets, clinical specimens, and bladder cancer cell lines. Pan‐cancer analysis based on TCGA datasets showed that bladder cancer exhibited one of the highest frequencies of CDKN1A alterations among the tumor types analyzed, affecting approximately 9%–10% of cases (Figure 1A). In addition, transcriptomic analysis showed that CDKN1A expression was significantly reduced in bladder cancer tissues compared with normal bladder tissues (Figure 1B), indicating recurrent downregulation of p21 during bladder tumorigenesis.

To further assess the clinical relevance of p21 loss at the protein level, we performed immunohistochemical staining of p21 in a bladder cancer tissue microarray and quantified p21 H‐scores according to pathological stage. Representative images showed a progressive reduction in p21 staining from Tis (carcinoma in situ) to more advanced lesions (Figure 1C). Quantitative analysis demonstrated that T1 (lamina propria invasion), T2 (muscularis propria invasion), T3 (perivesical soft tissue invasion), and T4 (invasion into adjacent organs or pelvic/abdominal wall) tumors all exhibited significantly lower p21 H‐scores than Tis lesions, whereas no significant differences were observed among the T1–T4 groups despite an overall decreasing trend with advancing stage (Figure 1D). These findings suggest that p21 downregulation is associated with bladder cancer progression.

To restore p21 function, we generated p21 mRNA using a T7 promoter‐based in vitro transcription (IVT) strategy, as illustrated in Figure S1A. We next examined basal p21 expression in bladder cancer cell lines and found that both T24 and J82 cells expressed very low basal p21 protein levels. In contrast, transfection of p21 mRNA led to robust p21 protein expression in both cell lines, as demonstrated by western blotting (Figure 1E). Immunofluorescence analysis further showed that the translated p21 protein was predominantly localized in the nucleus, whereas the EGFP control displayed diffuse cytoplasmic distribution (Figure 1F). Together, these genomic, histopathological, and cellular data support the selection of CDKN1A/p21 as a tumor suppressor replacement target in bladder cancer and confirm that IVT‐derived p21 mRNA can be efficiently translated to produce properly localized p21 protein in bladder cancer cells.

To assess the functional impact of p21 restoration, T24 bladder cancer cells were transfected with p21 mRNA or EGFP mRNA. While all groups exhibited comparable cell density at baseline, p21 mRNA‐transfected cells displayed pronounced morphological changes at 24 h, including cell rounding, detachment, and reduced density, whereas NC and EGFP cells reached near confluence (Figure 2A). Quantitative cell counting showed inhibition of T24 cell proliferation following p21 mRNA treatment (Figure 2B).

To further assess cell viability, cells were re‐counted 6 h post‐transfection—when mRNA expression had reached peak levels—and reseeded for Cell Counting Kit‐8 (CCK‐8) assay. At 24 h, EGFP mRNA had no significant effect on viability, whereas p21 mRNA markedly reduced cell viability relative to controls (Figure 2C).

Cell migratory capacity was assessed using a wound‐healing assay. Scratches were introduced 6 h after transfection, and wound closure was monitored at 0, 24, and 48 h. Compared with the NC and EGFP groups, p21 mRNA‐transfected cells displayed markedly delayed wound closure over time (Figure 2D,E). By 48 h, NC and EGFP cells achieved > 80% wound recovery, whereas p21 mRNA‐treated cells exhibited only ~20% closure. Because wound‐healing measurements can be influenced by both cell migration and cell proliferation, and because p21 restoration exerted a strong anti‐proliferative effect in parallel assays, the delayed wound closure observed here is interpreted primarily as impaired closure capacity rather than as definitive evidence of a direct anti‐migratory effect. Consistent with this growth‐suppressive phenotype, colony formation assays showed a substantial reduction in clonogenic capacity following p21 mRNA transfection (Figure 2F,G). Together, these data indicate that p21 mRNA markedly suppresses the proliferative potential of T24 bladder cancer cells and delays wound closure in vitro.

To further characterize the effect of p21 restoration at the molecular level, we assessed biomarkers associated with proliferation and invasion. Western blot analysis showed that proliferating cell nuclear antigen (PCNA), a canonical proliferation marker, was markedly downregulated after p21 mRNA transfection (Figure 2H), in agreement with the observed inhibition of cell growth and clonogenicity. By contrast, the migration/invasion‐related markers Vimentin and VE‐cadherin did not show obvious changes (Figure 2H). These findings suggest that p21 restoration primarily exerts an anti‐proliferative effect, while its impact on migration or invasion‐associated programs appears limited under the conditions tested.

To elucidate the molecular mechanisms underlying p21‐mediated phenotypes, we performed immunoblot analysis. Consistent with p21's role as a potent CDK inhibitor, p21 overexpression led to a significant reduction in phosphorylated retinoblastoma protein (Rb) (Ser807/811) (Figure 3A). This hypo‐phosphorylation of Rb prevents the release of E2F transcription factors, thereby repressing E2F target genes, including Cyclin E [19]. Accordingly, we observed a sharp decline in Cyclin E protein level (Figure 3A). Cyclin B levels were also significantly downregulated, suggesting that the inhibitory effect of p21 extends beyond the G1/S transition and also influences G2/M‐associated cell‐cycle control.

Notably, p21 mRNA‐transfected cells exhibited elevated γ‐H2A.X levels, indicating increased DNA double‐strand break‐associated signaling, consistent with replication stress induced by abrupt cell‐cycle arrest (Figure 3A). In agreement with these observations, apoptosis was evidenced by increased PARP1 cleavage (Figure 3A) and a higher proportion of Annexin V/PI‐positive cells detected by flow cytometry (Figure 3B). Collectively, these results indicate that p21 mRNA‐mediated restoration of p21 effectively enforces cell‐cycle arrest and triggers apoptotic cell death in bladder cancer cells.

To achieve efficient in vivo mRNA delivery, we encapsulated mRNA into lipid nanoparticles (LNP) using a microfluidic mixing platform to ensure rapid and homogeneous self‐assembly (Figure 4A). Structural characterization by transmission electron microscopy (TEM) showed that the resulting p21‐LNP exhibited a uniform and well‐dispersed spherical morphology, with particle diameters slightly below 100 nm (Figure 4B).

Consistent with the TEM observations, dynamic light scattering (DLS) analysis further confirmed that luciferase‐ (Luc‐), EGFP‐, and p21‐LNP all displayed narrow size distributions with high batch uniformity (Figure 4C–E). In addition, the polydispersity index (PDI) remained below 0.15 across all formulations (Figure 4F), indicating a highly homogeneous nanoparticle population. Together, these results demonstrate that the microfluidic formulation strategy yields stable mRNA‐LNP preparations with favorable physicochemical properties for subsequent in vivo administration.

To assess whether intravesical instillation could serve as an effective route for local delivery of mRNA‐LNP, we first used Luc‐LNP as a reporter system. Following intravesical administration of 10 μg Luc‐LNP into the mouse bladders, bioluminescence was detected in the bladder region (Figure 5A). Time‐course analysis showed that the luciferase signal peaked at approximately 6 h after instillation and remained detectable for up to 72 h, demonstrating sustained local expression after bladder administration (Figure 5A,B).

To further evaluate the in vivo distribution of the formulation, mice were sacrificed at 6, 24, 48, and 72 h after intravesical administration of Luc‐LNP, and six organs, including the bladder, heart, lung, spleen, kidney, and liver, were collected for ex vivo bioluminescence analysis. The strongest and most sustained signal was detected in the bladder, where luciferase activity remained measurable up to 72 h (Figure 5C,D). At 6 h and 24 h, relatively stronger transient signals were also observed in the spleen and liver, whereas these signals became undetectable after 48 h. In contrast, the heart, lung, and kidney showed minimal or negligible signal throughout the observation period (Figure 5C,D). These results indicate that intravesical delivery of Luc‐LNP enables prolonged local expression in the bladder with limited and transient distribution to distant organs.

To further assess acute systemic safety, H&E staining was performed on major organs after intravesical administration of PBS or Luc‐LNP. No obvious histopathological abnormalities, degenerative changes, or inflammatory lesions were observed in the heart, liver, kidney, or bladder at either 6 h or 72 h after treatment (Figure 5E), supporting the favorable short‐term tolerability of intravesical LNP delivery.

To evaluate therapeutic efficacy in vivo, we established an orthotopic bladder cancer model using luciferase‐labeled T24‐Luc cells (Figure 6A). Bladder mucosa was transiently disrupted with 0.5 M silver nitrate to facilitate tumor cell adherence, followed by intravesical instillation of T24‐Luc cells. Bioluminescence imaging on days 5, 11, and 18 revealed progressively increasing signals confined to the bladder, indicating stable tumor engraftment and continuous growth (Figures 6B and S2A).

We next used this orthotopic model to assess the therapeutic efficacy of intravesical p21‐LNP treatment. Tumor‐bearing mice were randomized to receive intravesical instillation of 100 μL per mouse of PBS, EGFP‐LNP (10 μg), or p21‐LNP (10 μg) every 3 days for a total of 8 doses, beginning on day 3 after tumor implantation (Figure 6C). Tumor progression was monitored by bioluminescence imaging. While tumor burden remained comparable across all groups on day 4, a distinct divergence in growth kinetics emerged by day 10 (Figure 6D,E). Compared with the PBS and EGFP‐LNP groups, the p21‐LNP‐treated group exhibited significantly reduced bioluminescent tumor signals, and this suppressive effect was maintained until the study endpoint (Figure 6D,E).

Importantly, no significant body weight loss was observed during the treatment period (Figure 6F), suggesting good tolerability of repeated intravesical administration. Histological analysis further supported the imaging results. Bladders from the PBS and EGFP‐LNP groups showed marked urothelial thickening, invasive tumor growth, and nuclear atypia, whereas bladders from the p21‐LNP‐treated group largely retained normal urothelial architecture and showed no obvious tumor‐associated thickening or invasion (Figure 6G). At 48 h after the final intravesical administration on day 24, bladder tissues were collected for western blot analysis, which further confirmed that p21 protein was significantly elevated in the p21‐LNP‐treated group (Figure 6H), demonstrating successful local restoration of p21 expression in vivo.

Together, these findings show that intravesical delivery of p21 mRNA‐LNP effectively restores p21 expression and suppresses orthotopic bladder tumor progression.

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