Materials
Au nanoparticles (Au NPs) were constructed via a previously reported method [23], briefly, 10 mL of HAuCl4·3H2O (16961-25-4, 99%, Sigma-Aldrich) aqueous solution (10 mM) was added to 10 mL of water in a 25 mL beaker with a strong magnetic stirring. Then, 7.5 mg/ml AA powder was rapidly added to the water solution and allowed to react for 5 min. Hexadecyltrimethylammonium bromide (CTAB, 57-09-0, 99%) was purchased from Sigma-Aldrich (Shanghai, China). Silver nitrate (AgNO3, 7761-88-8, 99%) was purchased from Aladdin (Shanghai, China). Ascorbic acid (AA, 50-81-7, 99%) was purchased from Sigma-Aldrich (Shanghai, China). Hydrogen hexachloroplatinate hexahydrate (H2PtCl6, 26023-84-7, 99.9%) was purchased from Sigma-Aldrich (Shanghai, China). 200–300 U urease (9002-13-5, 98%) was purchased from Macklin (Shanghai, China). Sulfo-Cy5 NHS ester (sulfo-NHS-Cy5, 2230212-27-6, 95%) was purchased from Aladdin (Shanghai, China). HS-PEG2000-NH2HCL (Q-0146715, 95%) was purchased from JenKem Technology (Beijing, China). Acetyloximic acid (AHA, A106239, 98%) was purchased from Aladdin (Shanghai, China). H2O2 quantification assay kit was purchased from Sangon Biotech (BC3595, Shanghai, China). Urease activity detection kit was purchased from Solarbio (BC4115, Beijing, China). DMEM (high glucose), penicillin–streptomycin, trypsin-EDTA, fetal bovine serum (FBS), and phosphate-buffered saline (PBS, pH 7.4) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The Cell Count Kit-8 (CCK8 kit) was purchased from LABLEAD (CK001, Beijing, China). Methylcellulose (9004-67-5), Annexin V-FITC Apoptosis Detection Kit (C1062), Reactive Oxygen Species Assay Kit (S0033) and Hoechst 33,342 (C1026, 1000×) were purchased from Beyotime Biotechnology Co., Ltd. Matrigel was purchased from Corning (354248, New York, America).
The size, shape and HRTEM of nanoparticles were observed using TEM (JEM-F200, JEOL, Japan). ICP-MS was tested using Agilent 7800 (America). The UV–Vis–NIR absorption spectra of the nanoparticles were tested using a spectrophotometer (Thermo Fisher). The size distribution and Zeta potential of nanoparticles were evaluated by a Zetasizer nano ZS particle analyzer (Malvern Instruments Co., Ltd.). The movement of Au-Pt@ur NPs was recorded by Particle Metrix (ZetaView). Fluorescence analysis of mucus penetration and cytotoxicity were evaluated by a Multi-Mode Microplate Reader (Synergy Mx, Bio-Tek Instruments Inc., Winooski, US). Fluorescence images were detected using CLSM (confocal laser scanning microscope, Leica DMI8). Flow cytometer analysis were tested by BD Biosciences (New Jersey, US). The bioluminescence of BCa was monitored by IVIS Lumina XRMS Series III (PerkinElmer Inc., Waltham, US) HE stained tissues were imaged by an inverted microscope (TS 100, Niikon Ti, Japan). The thermal maps were provided by a thermal imager (FLIR).
Synthesis of Au-Pt nanoparticles
The synthesis of Pt-modified Au NPs were silver ions-assisted [41–42]. 200 µl Au NPs (~ 50 pM), 500ul 200 mM CTAB, 10 ul 2 mM AgNO3 were added together and diluted with sterile double distilled Water (dd water) to a 5 ml reaction system. The reaction mixture was heated to 50 °C in a dd water bath and stirred for 200 rpm 10 min before adding 40 ul 100 mM AA. Continuously stirring for 1 h then add 60 ul 10 mM H2PtCl6 and react for another 1 h. Afterward, microparticles were removed by centrifugation at a rotating speed of 3000 rpm for 10 min and wash twice with dd water to obtain the preliminary product Au-Pt NPs. Au-Pt NPs exhibit strong solubility in dd water and can be stored under 4 °C.
Synthesis of Au-Pt@ur nanoparticles
2 ml of pre-synthesized Au-Pt NPs was mixed with 6.25 × 10− 5 mM HS-PEG2000-NH2HCL and stirred at low speed (100 rpm) for 12 h, then 4 × 10− 3 µM urease was added and reacted for another 12 h. To remove excess impurities and unreacted reagents, the Au-Pt@ur NPs were centrifuged using an ultrafiltration centrifuge tube (MW: 30KD, Millipore) under 3000 rpm 15 min. This centrifugation step was repeated twice to ensure thorough purification and gain the final product Au-Pt@ur NPs (~ 4.625 pM). 16.6 mM sulfo-Cy5 NHS ester can be added for 30 min to get Au-Pt@ur-cy5 NPs, and addition of 0.5 mM AHA can effectively inhibit the activity of urease to obtain the Au-Pt-cy5 NPs, the appeal ultrafiltration method also available here. All the resulting products should be stored at 4 °C until use.
Pt activity assay
The catalytic activity of Pt immobilized on Au-Pt@ur NPs was assessed indirectly by monitoring the consumption of H2O2. Au-Pt@ur NPs was reacted with 3.2 mM H2O2 [8]. For 20 min and the concentration of hydrogen peroxide left from the reaction was detected using a H2O2 quantification assay kit.
Urease activity assay
The enzymatic activity of urease immobilized on Au-Pt@ur NPs was evaluated with urease activity detection kit from Solarbio. The production of 1 µg of NH3−N per mg of protein per min is defined as an enzyme activity unit. The enzymatic activity of urease we purchased and adopted is found to be 200–300 U/mg.
Optical video recording
Particle Metrix was utilized to observe and record the movement of Au-Pt@ur NPs. 80 µL Au-Pt@ur NPs solution (4.625 pM) was added into 990 µL of urea solution in different concentration (0, 100, 300, 500 mM), with or without 3.2 mM H2O2. The mixture thoroughly homogenized to ensure uniform distribution of the Au-Pt@ur NPs within the solution. The videos of the movement of nanoparticles were recorded for up to 20 s via at the frame rate of about 25 fps.
Mean-square-displacement analysis
The movement videos of Au-Pt@ur NPs in different concentration of solution were analyzed using a self-developed program based on Python to extract their movement trajectories. Thereafter, MSD was calculated using the following formula: MSD(Δt) = [(xi(t + Δt) – xi(t))2] (I = 2, for two dimensional analysis). Subsequently, the following formula was applied to obtain the diffusion coefficient (D), MSD (Δt) = 4DtΔt, which works for small particles with low rotational diffusion for small time intervals. More than 30 particles were analyzed to obtain statistics in each experimental group.
Mucus-penetration assay
The mucus layer was gently scraped off and placed in a centrifuge tube at 1000 g, centrifuged for 30 min, centrifuged twice, then the supernatant was extracted and sterilized under UV for 30 min. Sterilized mucus layer were seeded onto a polyester membrane filter of a Transwell chamber (0.4 μm pore size) with the thickness of about 20 μm, and place in incubator overnight. After that, 80 ul 4.625pM Au-Pt@ur-cy5 NPs + 20 µl solvent were mixed well in 1 ml no FBS DMEM and added 100 ul onto the topside of each mucus layer, respectively, with 1 mL of PBS filling the lower chamber. During 0–5 h, we collected 100 µL of PBS buffer containing the leakage from the upper chamber for fluorescence analysis via a Multi-Mode Microplate Reader per hour.
Cell culture
bCa cells (T24 and 253 J) and normal urothelium cells (SVHUC-1) were cultured in DMEM. HUVECs were cultured in F12K. All cell lines were supplemented with 10% fetal bovine serum and 100 U/mL 1% penicillin/streptomycin and maintained at 37 °C. T24, 253 J, SVHUC-1 and HUVECs cells were obtained from the National Collection of Authenticated Cell Cultures, China.
Penetration efficiency in 2D cell culture models and MCSs
T24 or 253 J cells were suspended in DMEM (containing 0.12% w/v methylcellulose), seeded into 8 laser confocal Petri dishes (105 cells per microplate) and incubated for 12 h. Then, medium of each dish was discarded and washed 3 times with PBS before administration. Each group was administered according to 80 ul nanoparticles + 20 ul solvent, mixed with serum-free DMEM presented as 1 ml system, namely, Au-Pt-cy5 + PBS, Au-Pt-cy5 + U, Au-Pt-cy5 + H, Au-Pt-cy5 + H + U, Au-Pt@ur-cy5 + PBS, Au-Pt@ur-cy5 + U, Au-Pt@ur-cy5 + H, Au-Pt@ur-cy5 + H + U. As for the preparation of MCSs, 5 × 106/ml T24 cells or 8 × 106/ml 253 J cells were suspended in DMEM (containing 0.12% w/v methylcellulose) and mixed evenly. Then, 20 µl of the cell suspension was dropped on the lid of the cell culture plate to form uniform droplets and 10 ml PBS was added to the plate for keeping the droplets moist. After being placed in a 37 °C incubator for 60 h, dense spheroids were transferred to a low adhesion 24-well plate and equally divided into 8 groups with the same appeal to administration. After 12 h, fluorescence imaging was performed under CLSM after three gentle rinses with PBS and ImageJ software was used for analysis.
Penetration efficiency in murine orthotopic bCa models
All animal procedures were approved by the Laboratory Animal Management Committee at Zhejiang Provincial People’s Hospital (No. 20240614132209126857). All animal procedures were performed according to the guidelines of the Administration Committee of Experimental Animals in Zhejiang Province and the Ethics Committee of Zhejiang Provincial People’s Hospital. Six- to eight-week-old nu/nu female mice were anesthetized by inhalation of 1% isoflurane in an oxygen gas mixture and kept on a heated platform during catheterization procedures. Lubricated angiocatheters were inserted into the urethra. After full insertion, the bladder was flushed with 80 µl of sterile PBS and pretreated with 80 µl of poly-L-lysine for 15 min. A single-cell suspension of 5 × 105 GFP-transfected T24 cells in 100 µl of PBS was inoculated into the bladder and preserved for 1 h. During the entire procedure, the mice were kept under anesthesia for 2 h before the catheter was gently removed from the urethra. The mice were monitored every day for any signs of pain and distress. After 1 week, the nu/nu female mice bearing bCa were anesthetized by inhalation of 1–2% isoflurane in an oxygen gas mixture and kept on a heated platform during catheterization procedures. Lubricated angiocatheters were inserted into the urethra. After full insertion, the bladder was flushed with 80 µL of sterile PBS. 80 ul Au-Pt@ur NPs were mixed with 20 ul different solutions to form 4 groups (PBS, H2O2, urea, H2O2 + urea), and were intravesically instilled and preserved for 2 h, respectively. The bladder was washed twice with PBS. The mice were sacrificed immediately. The tissues (including the bladder, heart, liver, spleen, lung and kidney) were harvested for further histopathological examination by HE staining. The bladders were frozen and sectioned (20 μm thick) in a cryostat and the sections were examined by using CLSM.
Cytotoxicity assay
T24, 253 J, and SVHUC-1 were seeded in 96-well plates with 8,000 cells per well for tumor cells and 12,000 per well for SVHUC-1. 200 ul of DMEM (with 10% serum) was added and placed in an incubator for 24 h. Configure different concentrations of nanomaterials separately, we selected the interval of 0.5-4 times after testing, for example, 80 ul of Au-Pt@ur NPs and 20 ul of urea + H2O2 were prepared into 1 ml with serum-free DMEM and added 100 ul to each well. After administration for 12 h, the cell viability was measured with CCK-8 cell viability assay kit. And the cell viability was measured in the same way after administration and radiated for 600 s under 1.5 W·cm− 2.
Flow cytometry analysis
Generally, T24 cells were seeded into 24-well plates at a density of 1 × 105 cells per well and incubated for 12 h. 80 ul of Au-Pt@ur NPs and 20 ul of solvent (H2O2, urea, H2O2 + urea) were administrated for another 12 h, and we irradiated the cells with a laser power density of 1.5 W·cm− 2. Afterward, stained with Annexin V-FITC/PI at room temperature for 15 min washed twice with PBS buffer (pH 7.4) and digested with trypsin. Detached cells were collected using centrifugation at 300 g for 3 min to remove trypsin and washed with PBS buffer (pH = 7.4). The cells were resuspended in PBS buffer (pH 7.4) and transferred to a flow cytometer (BD Biosciences, New Jersey, US). The average fluorescence intensity was determined by counting every 5000 cells. The experiment was independently repeated three times, and the data were analyzed using FlowJo software. The results are presented as the mean ± the standard deviation (S.D.). Detection of intracellular ROS generation is in the same way, just replaced the fluorescent-labeled materials with DCFH-DA and incubated at 37 °C for 20 min.
In vitro angiogenesis characterization
To detect the formation of tubes, 200 µL of HUVECs were seeded into 48-well plate at a concentration of 3 × 104 cells/mL with material of each group. The plate was Preplanking with 4°C Matrigel. Then the plate was incubated in 36℃ for 6 h, HUVECs were photographed by microscope to count the number of tubes.
Photothermal therapy of Au-Pt@ur NPs treated bCa models
The nu/nu female mice bearing bCa originating from luciferase-transfected T24 cells were obtained by the same method as appeals. They were anesthetized by inhalation of 1–2% isoflurane in an oxygen gas mixture and kept on a heated platform during catheterization procedures. Lubricated angiocatheters were inserted into the urethra. After full insertion, the bladder was flushed with 80 µl of sterile PBS. 80 ul Au-Pt@ur NPs were mixed with 20 ul different solutions to form 4 groups (PBS, H2O2, urea, H2O2 + urea), and were intravesically instilled and preserved for 2 h, respectively. Saline was also intravesically instilled as in the control group. After each administration, irradiated the lower abdomen with a laser power density of 1.5 W·cm− 2 with irradiation time of 600 s. Intravesical instillation and photothermal therapy were performed every five days for a total of five times. The mice were intraperitoneally injected with 100 mg/kg D-luciferin to monitor the in vivo bioluminescence of tumors using the IVIS Spectrum system with an excitation wavelength of 640 nm and an emission wavelength of 660 nm every 5 d. The thermal maps were provided by a thermal imager (FLIR). For the case whose orthotopic bCa got chemo-resected within five rounds of intravesical therapy, the duration of observation on its tumor growth and the change body weight was extended to another 3 weeks. Finally, all murine were sacrificed, and tissues (including bladder, heart, liver, spleen, lung, and kidney) were harvested for further histopathological examination by HE staining. The tissues were imaged using an inverted microscope. 5 groups (Control, Au-Pt@ur PBS, Au-Pt@ur + U, Au-Pt@ur + H, Au-Pt@ur + H + U) intravesically instilled and preserved for 2 h, respectively. All murine were sacrificed, and bladders were harvested for further histopathological examination by DHE staining and imaged by microscope.
Statistical analysis
The testing values of repeated measurements were averaged to obtain the data of general analysis experiments, and relevant data are expressed as mean ± SD. The statistical analysis was performed using GraphPad Prism software (ver 9.5.0). Statistical significances were examined by t test with *P < 0.05.
