Inducing mitochondriopathy-like damages by transformable nucleopeptide nanoparticles for targeted therapy of bladder cancer

ABSTRACT Mitochondriopathy inspired adenosine triphosphate (ATP) depletions have been recognized as a powerful way for controlling tumor growth. Nevertheless, selective sequestration or exhaustion of ATP under complex biological environments remains a prodigious challenge. Harnessing the advantages of in vivo self-assembled nanomaterials, we designed an Intracellular ATP Sequestration (IAS) system to specifically construct nanofibrous nanostructures on the surface of tumor nuclei with exposed ATP binding sites, leading to highly efficient suppression of bladder cancer by induction of mitochondriopathy-like damages. Briefly, the reported transformable nucleopeptide (NLS-FF-T) self-assembled into nuclear-targeted nanoparticles with ATP binding sites encapsulated inside under aqueous conditions. By interaction with KPNA2, the NLS-FF-T transformed into a nanofibrous-based ATP trapper on the surface of tumor nuclei, which prevented the production of intracellular energy. As a result, multiple bladder tumor cell lines (T24, EJ and RT-112) revealed that the half-maximal inhibitory concentration (IC50) of NLS-FF-T was reduced by approximately 4-fold when compared to NLS-T. Following intravenous administration, NLS-FF-T was found to be dose-dependently accumulated at the tumor site of T24 xenograft mice. More significantly, this IAS system exhibited an extremely antitumor efficacy according to the deterioration of T24 tumors and simultaneously prolonged the overall survival of T24 orthotopic xenograft mice. Together, our findings clearly demonstrated the therapeutic advantages of intracellular ATP sequestration-induced mitochondriopathy-like damages, which provides a potential treatment strategy for malignancies.


Determination of critical aggregation concentration (CAC)
To determine the Critical Aggregation Concentration (CAC) of NLS-FF-T, pyrene was utilized as a fluorescent probe.Initially, 50 μL of pyrene solution in acetone (480 μM) was added to a 5 mL centrifuge tube and the acetone was allowed to evaporate completely.A range of NLS-FF-T solutions with different concentrations were added to the tube while the concentration of pyrene was kept constant at 6 μM.The excitation spectra were recorded at an emission wavelength of 393 nm and the intensity ratio (I338/I335) was analyzed as a function of NLS-FF-T concentration.The CAC was determined by identifying the intersection point at low concentration on the plot.

Animals and cells
All animal procedures were carried out in compliance with ethical regulations and were approved by the Institutional Animal Care and Use Committee of the National Center for Nanoscience and Technology (NCNST21-2208-0609). Female BALB/c nude mice (about 6-8 weeks old) were procured from Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China) and housed in ventilated cages (N=5) with a 12-h light-dark cycle (8:00-20:00 light/20:00-8:00 dark), constant room temperature (22°C) and relative humidity (30-70%), with ad libitum access to food and water.The T24 cell lines were obtained from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China) and were cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose containing 10% fetal bovine serum and 1% penicillin sulfate and streptomycin at 37°C with 5% CO 2 .

Circular dichroism (CD) spectra of IAS system
The secondary structure of the NLS-FF-T was monitored by a circular dichroism (CD) spectrum (JASCO Corporation, JC-1500).NLS-FF-T (0.5 mg/mL) was dissolved in PB solutions and kept at 37°C under constant shaking.The CD signals were recorded at 24 h.CD Pro was utilized to analyze the β-sheet percentage in the secondary structure of NLS-FF-T.
The percentage results of NLS-FF-T with or without KPNA2 were calculated by the three standard algorithms (CONTINLL, SELCON3, CDSSTR).

Fourier transform infrared (FTIR) spectroscopy measurement of IAS system
Molecular arrangement of NLS-FF-T was analyzed by FTIR.The solution of NLS-FF-T (0.5 mg/mL) was incubated with or without KPNA2 for 8 h, and dialyze (MWCO: 3500 Da) against PB for 48 h.The resulting solution was lyophilized for FTIR characterization.

Evaluation ATP sequestration ability of IAS system
To evaluate the ATP sequestration ability of IAS system, ATP hydrolysis rate in alkaline phosphatase (ALP) following treatment with NLS-FF-T was conducted.Initially, ATP (200 μM) was mixed with NLS-FF-T nanofibers or nanoparticles and dissolved in DEA buffer (10 mM, pH 9.8).The mixture was then incubated for varying amounts of time followed by inactivation at 75°C for 5 min.The remaining ATP was determined using an ATP assay kit, which utilized the generated chemiluminescence to calculate ATP hydrolysis rate.All hydrolysis reactions were conducted at 37°C with continuous agitation.

Cytotoxicity assay of IAS system
The Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate the cytotoxicity of NLS-FF, FF-T, NLS-T and NLS-FF-T against T24 cells.Initially, 100 μL of T24 cell suspension was seeded into a 96-well plate at a density of 5 × 10 3 cells/mL and cultured overnight in a humidified environment containing 5% CO 2 at 37°C .Subsequently, DMEM culture medium containing NLS-FF, FF-T, NLS-T and NLS-FF-T at a predetermined range of concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200, 400, 800 and 1600 μM) was incubated with T24 cells at 37°C for 24 h.Next, the T24 cells in the 96-well plate were washed with a PBS solution and then treated with 100 µL of freshly prepared 10% CCK-8 solution in cell culture medium.The absorbance of CCK-8 was measured using a microplate reader.

Cellular imaging study of IAS system
To begin, a 1 mL cell suspension of T24 cells was seeded in a confocal imaging chamber at a density of 5 × 10 5 cells/mL and cultured for one night in a humidified environment containing 5% CO 2 at 37°C .Following this, the T24 cells were incubated with NLS-FF, FF-T, NLS-T and NLS-FF-T at a concentration of 50 μM in serum-free cell culture medium at 37°C for 2 h.Next, the T24 cells were washed with PBS buffer three times and fixed with 4% paraformaldehyde for 30 min.Finally, the T24 cells were imaged using a Confocal Laser Scanning Microscope (CLSM) under 40 × objective (Ex/Em: 640 nm/725 nm).

Protein extraction and western blotting
The T24 cells were incubated with PBS, NLS-FF, FF-T, NLS-T and NLS-FF-T at a concentration of 50 μM at 37°C.Afterward, the cells were homogenized in RIPA extraction buffer (LABLEAD Inc., China, R1091) and centrifuged at 14,000 g for 15 minutes at 4°C.The protein concentrations in the supernatant were measured using the BCA Protein Assay Kit according to the manufacturer's instructions.The samples were then separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes.The PVDF membranes were blocked with NcmBlot blocking buffer (NCM Biotech) for 10 minutes, followed by incubation with primary antibodies and corresponding secondary antibodies overnight.Finally, detection and imaging were performed using a chemiluminescence imaging system with HRP substrate.

Intracellular ATP levels assay
A 2 mL suspension of T24 cells was seeded at a density of 5 × 10 5 cells per well in a 6 well-plate and cultured overnight in a humidified environment containing 5% CO 2 at 37°C .The T24 cells were then incubated with NLS-FF, FF-T, NLS-T and NLS-FF-T at a concentration of 50 μM in serum-free cell culture medium at 37°C for 12 h.After lysing by homogenization, the T24 cell samples were collected by centrifugation at 10000 g for 10 min at 4°C .Finally, the ATP levels were measured using ATP Content Assay Kit in accordance with the manufacturer's instructions.

Intracellular morphology observation of IAS system
T24 cells were collected by centrifugation at 3000 rpm for 5 min after administration of NLS-FF-T (50 μM).The collected cells were fixed with 2.5% glutaraldehyde at 4°C overnight and then treated with 1% osmium dichromate for 2 h after three rinses with PBS.
Dehydration process was carried out with ascending concentrations of alcohol solutions (50, 70, 80, 90, 100, 100, and 100%) for 10 min each.The cells were then exposed to a mixture of alcohol/acetone (1:1) for 10 min, subsequently, a mixture of acetone/EPON-812 (1:1) and then a mixture of acetone/EPON-812 (1:2) for 1 h each.Pure EPON-812 was used for further cell sample infiltration overnight at 4°C.The curative binding of EPON-812 with cells was carried out at 37, 45, and 60°C for 24 h respectively.Finally, the intracellular morphology of NLS-FF-T was observed by Transmission Electron Microscopy (TEM) after staining with 2% uranyl acetate and 3% lead citrate.

Immunohistochemistry (IHC) assay
To investigate the expression of KPNA2 in bladder tumor tissues from 30 patients combined with normal bladder tissues from 20 patients, immunohistochemistry (IHC) was conducted by Wuhan Servicebio Technology Co., Ltd.Fresh bladder cancer specimens and normal bladder urothelium specimens were collected and fixed for analysis after appropriate informed consent was obtained.The experiments performed using human specimens were reviewed and approved by the Committees for Ethical Review of the Fourth Hospital of Harbin Medical University (2022-SCILLSC-27).

Microscale Thermophoresis (MST) ligand binding measurements.
To investigate the binding affinity of FF-T and NLS-FF-T with KPNA2, Protein Labeling Kit RED-NHS was initially used to label KPNA2 following the manufacturer's instructions.The labeled KPNA2 was then incubated with concentration gradients of FF-T and NLS-FF-T.Finally, the prepared samples were loaded into silica capillaries and analyzed with Monolish NT.115.

Ex vivo dose-dependent fluorescence imaging of IAS system
To construct T24 xenograft mice, a density of 5 × 10 6 T24 cells resuspended in Matrigel

Intratumoral morphology observation of IAS system
After intravenous administration of NLS-FF-T (500 μM in 200 μL PBS) in T24 xenograft mice, tumor tissues were collected and fragmented into small pieces measuring 3 × overnight and treated with 1% osmium dichromate for 2 h after being rinsed thrice with PBS.
Subsequently, graded concentrations of alcohol solutions (50, 70, 80, 90, 100, 100 and 100%) were utilized for graded dehydration (with 10 minutes each).Next, tumor fragments were exposed to a mixture of alcohol/acetone (1:1) for 10 min, a mixture of acetone/EPON-812 (1:1) for 1 h and a mixture of acetone/EPON-812 (1:2) for 1 h.Pure EPON-812 was then utilized to further infiltrate the tumor fragments overnight at 4°C, after which it was cured at 37, 45 and 60°C for 24 h respectively.Finally, the morphology of NLS-FF-T in the tumor slice was observed by bio-Transmission Electron Microscope (bio-TEM) after being stained with 2% uranyl acetate and 3% lead citrate.

In vivo antitumor evaluation of IAS system in subcutaneous xenograft model
To further verify the therapeutic potency of the IAS system, a T24 xenograft mouse model was established by subcutaneously injecting T24 cells (5 × 10 6 ) suspended in 100 μL Matrigel into the right flank of BALB/c nude mice (approximately 6-8 weeks, 16-18 g).
When the average tumor volume reached 100-200 mm 3 , the T24 tumor-bearing mice were randomly divided into five groups (with N=6 mice per group) for treatments with PBS, NLS-FF, FF-T, NLS-T and NLS-FF-T (500 μM in 200 μL PBS) six times via intravenous injection on days 0, 2, 4, 6, 8 and 10, respectively.The individual tumor growth and body weight were measured every 2 d for analysis, with tumor volume calculated as follows: tumor width 2 × tumor length × 0.5.Additionally, all tumor tissues were harvested intact and weighed at the end of the treatment period.

Tumor ATP levels assay
At the end of the treatment period, T24 tumor tissues were collected and washed thrice with PBS.Subsequently, the tumor lysates were gathered by homogenization and centrifuged at 8000 g for 10 min at 4°C.The ATP levels were then measured according to the manufacturer's instructions using an ATP Content Assay Kit.

Immunofluorescence and immunohistochemistry assay
At the end of the treatment period, T24 tumor tissues were collected and fixed with 4% paraformaldehyde for analysis.Subsequently, Wuhan Servicebio Technology Co., Ltd.performed hematoxylin-eosin (H&E) staining, terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay and Ki67 immunohistochemical staining of sections of the T24 tumor tissue.

In vivo antitumor evaluation of IAS system in regrowth of residual tumor model
To establish the T24 xenograft mouse model, BALB/c nude mice (approximately 6-8 weeks, weighing 16-18 g) were subcutaneously injected with T24 cells (5 × 10 6 ) suspended in 100 μL Matrigel.The mice were randomly divided into five groups, with six mice in each group.Both body weight and tumor volume were measured for analysis.At 21 d after inoculation, tumor tissues of approximately 300-500 mm 3 were resected, leaving a residual tumor of approximately 30-50 mm 3 .After that, the T24 xenograft mice were intravenously administered with PBS, NLS-FF, FF-T, NLS-T and NLS-FF-T (500 μM in 200 μL PBS) every two days on day 21, 23, 25, 27, 29 and 31.The tumor volume was calculated using the formula: tumor width 2 × tumor length × 0.5.

In vivo antitumor evaluation of IAS system in T24-Luc orthotopic bladder cancer mice
In brief, BALB/c nude mice (approximately 6-8 weeks, 16-18 g) were anesthetized with isoflurane.The inner mucosa of bladder was slightly damaged using an IV catheter (24G), followed by the intravesical delivery of T24-Luc cells into the bladder cavity via the same catheter.After incubation for 60 min, the mice were subjected to bioluminescent imaging to confirm and monitor orthotopic tumor growth in the bladder.The mice were then randomly assigned to five groups (with N=6 mice per group) and intravenously administered with PBS, NLS-FF, FF-T, NLS-T and NLS-FF-T (500 μM in 200 μL PBS) every two days on day 0, 2, 4, 6, 8 and 10, respectively.

In vivo toxicology evaluation of IAS system
Healthy BALB/c nude mice (approximately 6-8 weeks, 16-18 g) were used to evaluate the in vivo toxicity of IAS system.NLS-FF-T was intravenously injected into four healthy mice each at doses of 1000 μM, 500 μM and 250 μM, six times on day 0, 2, 4, 6, 8 and 10, respectively.Four healthy mice treated with PBS were used as controls.On day 11, major organs including liver and kidney were excised for histological analysis by Hematoxylin and Eosin (H&E) staining, which was conducted by Servicebio Technology Co., Ltd.(Wuhan, China).Meanwhile, the mice were sacrificed for blood chemistry and blood routine analyses performed by Vital River Laboratory Animal Technology Co., Ltd.(Beijing, China).

Statistical methods
For two-group comparisons, statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test.The GraphPad Prism software 8.0 was used for all statistical analyses.Survival curves were generated using the Kaplan-Meier method and compared using the log-rank test.All values presented in this study are expressed as mean ± standard deviation (SD).Statistical significance was indicated as NS for no significance, *P < 0.05, **P < 0.01, and ***P < 0.001.

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Corning, 354248) were subcutaneously injected into the right hind of BALB/c nude mice (approximately 6-8 weeks, 16-18 g).When the average tumor volume reached around 200-400 mm 3 , Cy labeled NLS-FF-T was administered to the T24 xenograft mice at various concentrations (500 μM, 250 μM, 125 μM or 62.5 μM in 200 μL PBS) via intravenous injection.Finally, the T24 xenograft mice were sacrificed, of which the main organs and tumors were collected for ex vivo fluorescence imaging of NLS-FF-T.

Fig. S11 .
Fig. S11.(a) Representative TEM images of NLS-FF and NLS-FF transformed into nanofibrous after interaction with KPNA2 protein.Scale bars: 100 nm.(b) Particle size of NLS-FF and NLS-FF transformed into nanofibrous after interaction with KPNA2 protein measured by DLS.

Fig. S12 .
Fig. S12.(a) Representative TEM images of FF-T and FF-T interaction with KPNA2 protein.Scale bars: 100 nm.(b) Particle size of FF-T and FF-T interaction with KPNA2 protein measured by DLS.

Fig. S13 .
Fig. S13.(a) Representative TEM images of NLS-T and NLS-T interaction with KPNA2 protein.Scale bars: 100 nm.(b) Particle size of NLS-T and NLS-T interaction with KPNA2 protein measured by DLS.

Fig
Fig. S17.(a) Viability of T24 and SV-HUC-1 cells after treated with NLS-FF-T at different concentrations for 48 h.(b) Viability of T24 cells after treated with NLS-FF-T or T-ATP (pure ATP sequestration motif) at different concentrations for 48 h.