Cell culture and reagents

The bladder cell lines T24 cells and 5637 cells were purchased from Stem Cell Bank (Chinese Academy of Sciences, Beijing, China) and cultured in McCoy’s 5 A medium (Thermo Fisher Scientific, Waltham, MA, USA, catalog no. 16600082) or RPMI 1640 medium (Thermo Fisher Scientific, Waltham, USA catalog no. 11875093) with 10% fetal bovine serum (FBS, AusGeneX, Gold Coast, Australia, catalog no. FBS500-S). Oxaliplatin (OXA, catalog no. S1224), SRT3025 (catalog no. S8481), and compound 3 K, the inhibitor of PKM2, (3 K, catalog no. S8616), were purchased from Selleck Chemicals (Houston, USA).

Synthesis of the 3025@ML

To synthesize SRT3025-loaded cell membrane hybrid liposomes (3025@ML), a mixture of 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Avantor Inc., Radnor, PA, USA, catalog no. S01003), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Avantor Inc., catalog no. S01004), and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DSPE-PEG2000, Avantor Inc., catalog no. F01008) in a 4:15:1 (n/n/n) ratio was used. SRT3025 was dissolved in chloroform and methanol mixture (9:1, v/v) and vortexed. The organic solvent was then evaporated under reduced pressure using a rotary evaporator at 37 °C and 30 rpm. Once lipid film was formed on the flask’s bottom, 1 mL of PBS was added to create a biphasic system, which was then further processed on the rotary evaporator at 45 °C and 60 rpm for 30 min. The resulting suspension was put in an ice bath and sonicated for 2 min to produce 3025@L.

Cell membrane vesicles were harvested and extracted from T24 cells, as reported by Zhang et al. ‘s method [18]. These vesicles were incubated with 3025@L at 37°C for 15 minutes and then ultrasonicated on ice for 3 minutes to completely fuse the liposome (Lipo) and tumor membrane (Mem), forming 3025@ML. Fluorescently labeled liposomes (Dio-Lipo) and tumor membranes (Dil-Mem) were prepared like 3025@ML, incorporating the dyes 3,3’-Dioctadecyloxacarbocyanine perchlorate (Dio) and 1,1’-Dioctadecyl − 3,3,3’,3’-tetramethylindocarbocyanine perchlorate (Dil) for experimental tracking. Figure 1 illustrates the study’s workflow and experimental elements.

Fig. 1

Experimental design flowchart

Characterization of the 3025@ML

To evaluate the stability of nanoparticles, the size distribution and zeta potential of the nanoparticles were determined by Zetatronix 919 (Opptronix, Shanghai, China). To verify the successful formation of the cell membrane hybrid liposomes (ML), Mem and Lipo were labeled with Dil and Dio dyes. Dio-Lipo and Dil-Mem were evaluated by co-localization analysis via an Axio Vert. A1 fluorescence microscope (Carl Zeiss, Oberkochen, Germany). In addition, Lipo, Mem, and ML were added to the SDS gel loading buffer for lysis and boiling. Subsequently, gels were stained with Coomassie brilliant blue to observe membrane protein expression. To examine the cytotoxicity of nanoparticles, 4 × 103 T24 and 5637 cells were seeded per well in a 96-well plate separately and cultured for 24 h. The cells were treated with 0, 1, 10, 100, and 1000 µg/mL of ML for 48 h. CCK8 assay was used to detect the cytotoxicity of ML.

Drug loading capacity and encapsulation efficiency

SRT3025 (1 mg/mL) was dissolved in methanol. The maximum absorption wavelength of SRT3025 was determined using ultraviolet-visible (UV-Vis) scanning spectroscopy. Various ratios of 3025@ML from 1:1 to 1:20 (w/w) (SRT3025:ML) were synthesized to determine the drug loading capacity. Drug Loading Capacity (DLC) and encapsulation efficiency (EE) were calculated using the following formulas:

$$DLC(\%) = \frac} + \text} \times 100\%$$

$$EE(\%) = \frac}} \times 100\%$$

Evaluating cellular uptake of 3025@ML

To evaluate the cellular uptake ability of 3025@ML, 2.5 × 105 T24 cells per well were allowed to grow in a 6-well plate for 48 h and incubated with PBS (as a control), 3025@ML and 3025@ML combination with filipin (inhibitors of caveolin-1) at 37 °C and 3025@ML at 4 °C. Following incubation with the respective treatments for 2 h, the cells were washed with cold PBS to remove excess particles. The cells were then trypsinized to detach from the culture plates, collected by centrifugation, and resuspended in cold PBS. Samples were immediately analyzed using flow cytometry to measure the fluorescence intensity of DiI, which corresponded to the amount of 3025@ML internalized by the cells.

Evaluating cell viability

T24 and 5637 cells were seeded at a density of 2.0 × 10³ cells per well in a 96-well plate. After 24 h of incubation to investigate the concentration of 3025@ML and 3 K that has an anti-tumor effect, the cells were treated with a medium containing either varying concentrations (0.1, 1, 10, 100 µM) of 3025@ML or 3 K for 48 h. To assess the impact of 3025@ML and 3 K on OXA, cells were plated following the previously described method and subjected to treatment with the control, 3025@ML, 3 K, OXA, 3025@ML + OXA, and 3 K + OXA. Cell viability was evaluated using the Cell Counting Kit-8 (CCK8, MedChemExpress, Monmouth Junction, USA, code HY-K0301). The intensity of the color change was quantified by measuring the absorbance at 450 nm using a Synergy LX multimode reader (BioTek, Winooski, USA).

$$\text (\%)=\frac}} \times 100 \%$$

Evaluating cell death and apoptosis

To evaluate cell death, T24 and 5637 cells were seeded at a density of 3.0 × 104 cells per well in a 24-well plate for 24 h. Subsequently, these cells were treated with control, OXA, 3025@ML + OXA, and 3025@ML alone for 48 h. Cell death was quantified by trypan blue staining. For apoptosis analysis, T24 cells were seeded at a density of 2.5 × 105 cells per well in a 6-well plate and treated with control, 3025@ML, OXA, or a combination of both drugs for 48 h. Apoptotic cells were quantified using the Annexin V-APC/PI apoptosis kit (Elabscience, Houston, USA, catalog no. E-CK-A217) and a NovoCyte flow cytometer system (Agilent, Santa Clara, USA).

Evaluating molecular mechanisms

Western blotting (WB) was performed to determine the level of pyruvate kinase M2 (PKM2), fatty acid synthase (FASN), and the regulation of apoptosis protein, such as BCL2-Associated X protein (Bax), B-cell lymphoma-2 (Bcl-2) and cleaved caspase-3. 2.5 × 105 T24 and 5637 cells were seeded separately per well in a 6-well plate and cultured for 24 h. These cells were treated with the 3025@ML, 3025@ML + OXA, OXA, 3 K, and 3 K + OXA for 48 h. After lysis with RIPA buffer, samples were centrifuged at 10,000× g for 30 min at 4 °C. The supernatant was collected, and the protein concentration was determined. Then, the proteins were denatured by boiling in an SDS-gel loading buffer for 10 min. Equal amounts of protein were subjected to 15% SDS-PAGE. Proteins were blotted onto PVDF Western blot membranes (Roche Diagnostics, Mannheim, Germany) and incubated with antibodies (Table 1). Inhibitors of caveolin-1 (filipin), alongside Dil-labeled 3025@ML, were investigated to evaluate the uptake mechanism of these hybrid liposomes in bladder cancer cells via flow cytometry.

Table 1 The following antibodies were used

The proteins were visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling, Danvers, USA, catalog no. 7074) by using a Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate (Waltham, USA, catalog no. 34096).

Gene expression evaluation

In order to investigate changes in gene expression profiles, T24 cells per well were seeded in 6 well plates for 48 h and treated by control and 3025@ML for another 24 h. RNA was extracted with TRIzol (Thermo Fisher Scientific, catalog no. 15596026), and mRNA expression was analyzed using Illumina next-generation sequencing. Differential expressions were assessed with the DESeq2 package in R. Additionally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to further explore the functional implications of the differentially expressed genes.

Animal model and in vivo studies

The animal experiments were conducted at the Southern University of Science and Technology under the approval of the Guangdong Medical Laboratory Animal Center Ethical Committee, with the assigned ethical approval number C202205-24 and D202410-5. BALB/c-nu male mice, 6–8 weeks old, were quarantined in an animal experiment center for 7 days to relieve mental stress. T24 cells were digested and harvested with PBS; the cell density was then adjusted to 0.5 × 105 cells/µL. The mice’s skin was carefully sterilized using 75% alcohol solution three times, and then the 80 µL cell suspension mixture was injected subcutaneously into the dorsal region of the mice. After that, all animals were quarantined for another 10–15 days until the tumors became visible to the naked eye. The control group was intraperitoneally injected with 25 µl DMSO once a day. The 3025@ML group was intraperitoneally injected with 25 µl 50 mg/kg 3025@ML once a day. Treatment continued for a total of 30 days. Tumor size was detected using a Vernier caliper, and tumor volume (mm3) was calculated as AL × AW2 × 0.52 [19], where AL and AW refer to the length and width of tumors, respectively. The mice were weighed every three days. To study the tissue distribution of the SRT3025 and OXA after 3025@ML + OXA administration, the subcutaneous tumor model was constructed in 5 nude mice in each group as previously described. The mice were intraperitoneally injected with 50 mg/kg 3025@ML every day and 10 mg/kg OXA once a week. The mice in the control group received the same dose of DMSO solution. After 15 days of administration, the mouse plasma and tissues were collected. Levels of the SRT3025 and OXA were measured by HPLC.

Statistical analysis

The data are presented as means ± SD (error bars) and were analyzed using GraphPad Prism software (Version 8.00). The Mann-Whitney rank sum test was used to evaluate the significant differences with a P value lower than 0.05 was considered significant.