Genetically engineered T cell membrane-camouflaged nanoparticles triggered cuproptosis for synergistic bladder cancer photothermal-immunotherapy | Journal of Nanobiotechnology

Materials and reagents

Tetraethylorthosilicate (TEOS), Hexadecyl trimethyl ammonium bromide (CATB), Sodium carbonate (Na₂CO₃), Diethyldithiocarbamate-copper complex (CuET) was obtained from Aladdin Co. Ltd. (Shanghai, China). IR820 was obtained from Shanghai Bide Pharmaceutical Technology Co. Ltd. IL-4 and GM-CSF were purchased from Suzhou Novoprotein Scientific Co. Ltd. The Cell Counting Kit-8 (CCK-8) was supplied by Biosharp Life Science, and the Annexin V-FITC/PI apoptosis detection kit was supplied by Hangzhou Lianke Bio-technology Co. Ltd. Roswell Park Memorial Institute (RPMI) 1640 medium, High-glucose Dulbecco’s Modified Eagle Medium (DMEM), and phosphate-buffered saline (PBS) were sourced from Wuhan Pricella Biotechnology Co. Ltd. A 0.25% tryp-sin-EDTA solution and paraformaldehyde were obtained from BOSTER, while fetal bovine serum (FBS) was purchased from Haixing Biotechnology Co. Ltd. The Zombie NIR fixable viability kit and flow cytometry antibodies, including PERCP-CD45, APC-CD11c, BV421-CD3e, FITC-CD11b, PE-CD86, FITC-CD80, BV605-GR-1, PE-CD8a, and PE-Cy7-CD86, were obtained from BioLegend.

Cells and animals

MB49 cells were cultured in DMEM with 10% (v/v) FBS and 1% P/S at 37^°C in a 5% CO2 incubator. When cell fusion reached around 80%, the cells were treated with 0.25% trypsin for digestion, then sub-cultured or seeded onto cell plates for further experiments. C57/BL6 mic were purchased from Hubei BIONT Biological Technology Co., Ltd., and housed in a laminar flow chamber under specific pathogen-free (SPF) conditions. This study’s animal experiments were performed in line with the approved guidelines from the Committee of Animal Experimentation and the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Approval number: 202402005). CTLL2 cells (Procell Life Science & Technology Co. Ltd) and Tim3-CTLL2 cells were cultured in complete RPMI-1640 medium with IL-2 (100 U/mL) and 10% FBS at 37 °C in a 5% CO₂ incubator.

Derivation of cell membrane

CTLL2 and Tim3-CTLL2 cells were lysed in hypotonic lysis buffer for 30 min in an ice water bath. The disrupted cells were further processed by a probe ultrasonic disruptor (100 W, 5 min) on ice. The lysate was then centrifuged at 8000 rpm for 10 min to clear organelles and massive particles. The supernatant was then centrifuged again at 15,000 rpm for 30 min to isolate the cell membrane. The resulting pellet was washed once with wash buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).

Preparation and characterization of PHSM@IC, n@phsm@ic and Tim3@PHSM@IC NPs

Firstly, HMSNs were prepared based on the previous study [34]. TEOS, CTAB and Na₂CO₃ were utilized for the preparation of HMSNs. Subsequently, HMSNs (5 mg) were dispersed in PBS and added dropwise to a mixed solution of CuET (5 mg/mL) and IR820 (5 mg/mL), and stirred in the dark. After 12 h of agitation, the mixture received centrifugation and washing until the supernatant became transparent. The resulting precipitate was designated as HSM@IC, and the supernatant was collected for the determination of CuET and IR820 loading capacity. Next, HSM@IC was suspended in tris buffer (pH 8.5, 10 mM) containing dopamine hydrochloride (2 mg) and stirred in the dark for 6 h. The brown product, denoted as PHSM@IC, was obtained by centrifugation and washing. The PHSM@IC was then mixed with cell membranes isolated from 1 × 10⁸ CTLL2 and Tim3-CTLL2 cells (200 µL), sonicated (100 W, 1 min) in an ice bath, and extruded through polycarbonate membranes (pore size 400 nm) using an extruder for 40 cycles. The resulting products were labeled as n@PHSM@IC and Tim3@PHSM@IC, respectively. The size distributions and surface potentials of the particles were determined using dynamic light scattering, and the morphology was analyzed by TEM. The contents of IR820 and CuET were quantified using UV − vis spectroscopy based on a standard curve. The following equations were applied to calculate the drug loading content and encapsulation efficiency:

\(\begin{aligned}\:Loading&\:efficiency\:\left(\%\right)\cr&=\frac{loaded\:drug\:weight}{drug\:weight\:in\:synthesis}\:\times100\%\end{aligned}\)

\(\eqalign{ Loading\> & content\>\left( \% \right) \cr & = {{Loading\>weight} \over \matrix{ nanomaterials\>and\> \hfill \cr total\>loading\>weight \hfill \cr} } \times 100\% \cr} \)

Detection of Tim3 in Gene-engineered cell membranes

CTLL2 and Tim3-CTLL2 cell membrane, n@PHSM@IC and Tim3@PHSM@IC NPs with loading buffer were incubated in a water bath at boiling temperature for 15 min. The samples were then separated by SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked by QuickBlock™ Protein-Free Blocking Buffer for Western Blot for 15 min at room temperature. The membranes underwent incubation with primary antibodies, including Tim3 (1:1000) and β-actin (1:10,000), for 14 h at 4 °C. After washing the membranes three times using TBST, they were exposed to HRP-conjugated secondary antibodies (1:10,000) for 1 h at room temperature. Then they were exposed to an enhanced chemiluminescence (ECL) substrate kit for detection.

PTT effect of Tim3@PHSM@IC NPs

To measure the PTT efficacy, free IR820 and Tim3@PHSM@IC NPs solutions were irradiated with 808 nm laser, meanwhile monitoring temperature variations every 0.5 min over a duration of 5 min. A subcutaneous bladder cancer mouse model was established, and when the tumor size reached about 100 mm³, different nanoparticles were injected. 12 h later, 808 nm NIR irradiation (1 W/cm², 5 min) was applied, and the temperature change in the tumor tissue was recorded within 5 min.

Uptake of n@phsm@ic and Tim3@PHSM@IC NPs by MB49 cells

MB49 cells (2 × 10⁵/mL) were seed in a 24-well plate and treated with n@PHSM@IC and Tim3@PHSM@IC NPs for 6 h. Then, the cells were collected, and nanoparticles uptake was evaluated via FCM and confocal laser scanning microscopy.

In vivo imaging of biodistribution

Subcutaneously seeded MB49 cells (1 × 10⁶) in C57/BL6 mice (4 weeks). After 7 days, mice received an intravenous injection of n-PHSM@IC NPs and Tim3@PHSM@IC NPs. For in vivo imaging, the two types of nanoparticles were injected before 4, 12 and 24 h, respectively.

Biosafety evaluation

18 C57/BL6 mice were randomly divided to 6 groups and treated with PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, or Tim3@PHSM@IC NPs via tail vein injection, administered once every 2 days for 3 doses. After 12 days, peripheral blood was collected from all euthanized mice for routine blood and biochemical tests. The biochemical tests contained measurements of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA), while the routine blood tests assessed white blood cell count (WBC), lymphocyte ratio (LYM), granulocyte count (GR), red blood cell count (RBC), hemoglobin concentration (HGB), and platelet count (PLT). The major organs were then harvested, sectioned, and subjected to pathological examination.

Live/dead cell staining

MB49 cells (2 × 10⁵/mL) were plated in a 6-well plate, treated with PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, and Tim3@PHSM@IC NPs for 12 h. Then treatment groups received 808 nm NIR irradiation (1 W/cm², 5 minutes), stained with calcein-AM/PI, and were observed by immunofluorescence microscope.

Western blot analysis of Cuproptosis-related proteins

MB49 cells (1 × 10⁶) were plated on a 6-well plate and treated with PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, and Tim3@PHSM@IC NPs for 12 h, then treatment groups received 808 nm NIR irradiation (1 W/cm², 5 min). After 24 h, the cells were lysed with ice-cold lysis buffer for 30 min. The samples with loading buffer were boiled in a water bath for 15 min. Subsequently, the samples were separated by SDS-PAGE gel and transferred to PVDF membranes, which were blocked by QuickBlock™ Protein-Free Blocking Buffer for Western Blot for 20 min and incubated at 4 °C with primary antibodies for 12 h, included DLAT (1:1000), FDX1 (1:1500), and β-actin (1:15,000). The membranes were washed three times and exposed to secondary antibodies (1:8,000) for 1 h. Finally, they were treated with an enhanced ECL substrate kit for detection.

Intracellular level of ROS

ROS level was assessed by a detection kit (DCFH-DA). MB49 cells were plated in 12-well plates (1 × 10⁵/ml) and incubated for 12 h with PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, or Tim3@PHSM@IC nanoparticles. Cells were then received 808 nm NIR irradiation (1 W/cm², 5 minutes), followed by incubation with diluted DCFH-DA for 15 min at 37 °C. The fluorescence signal was detected by immunofluorescence microscopy and quantified by flow cytometry.

DAMPs detection in Vitro

MB49 cells (1 × 10⁶) were seeded on a 6-well plate and incubated for 24 h. PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, and Tim3@PHSM@IC NPs were added to each well (IR820: 5.0 µg/mL; CuET: 1 µg/mL) for 24 h. Besides PBS group, other groups received irradiation (808 nm; 1.0 W/cm²) for 5 min. After 24 h, MB49 cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 simultaneously retaining the supernatant. Cells subsequently underwent incubation with anti-CRT or anti-HMGB1 antibodies, proceeded by exposure to 594-conjugated secondary antibodies. Afterward, the cells were subjected to counterstaining with DAPI for nuclear visualization. Fluorescence signals were observed by immunofluorescence microscopy. Cell supernatants were obtained by centrifugation (500 g, 5 min), and HMGB1 levels in the supernatants of different treatment groups were quantified following the manufacturer’s protocol for the HMGB1 ELISA Kit (Beyotime, China). The ATP Detection Kit (Beyotime, China) was used to measure ATP release. After incubating MB49 cells with various treatments for 24 h, the supernatant was collected through centrifugation (1000 rpm, 5 min) and mixed with the ATP monitor working solution. ATP levels were then detected using a luminometer.

In vitro BMDCs stimulation

BMDCs were extracted out of 5-week-old C57/BL6 mice following an established approach [35]. BMDCs were cultured in 1640 medium supplemented with GM-CSF (20 ng/mL) and IL-4 (10 ng/mL). After 5 days of culture, BMDCs were placed in 12-well Transwell^® plate with fresh culture medium and were stimulated by pretreated MB49 cells for 24 h. Then, BMDCs were stained with APC-CD11c, PE-CD86, FITC-CD80 to assess maturation rates, and analyzed using flow cytometry.

In vivo Anti-tumor effect

A total of 1 × 10⁶ MB49 tumor cells were inoculated into C57BL/6J mice. When the tumor size reached around 100 mm³, MB49 tumor-bearing mice were randomly divided to 6 groups and received intravenous injections with PBS, PHSM@I, PHSM@C, PHSM@IC, n@PHSM@IC, and Tim3@PHSM@IC NPs (1 mg/kg CuET) on days 0, 3, 6, respectively. Besides PBS group, tumors received 808 nm NIR irradiation (1 W/cm², 5 minutes), 24 h after drug injection. The tumor size and mice weight were recorded every two days, and tumor size (mm³) was calculated by the formula V = (L×W²)/2, where L represents the length and W the width. For the bilateral tumor model, 1 × 10⁶ MB49 cells were subcutaneously injected into the right flank, and 8 × 10⁵ MB49 cells were injected into the left flank of C57/BL6 mice models. When the primary tumors (right) reached 100 mm³, the mice were divided to 6 groups and primary tumors received same treatment as mentioned above. Tumor size was recorded every 2 days. After 12 days, the tumor tissue was collected to examine the cytotoxic T cells by flow cytometric analysis.

Tim3@PHSM@IC NPs therapy Combined with αPD-1 Blockade therapy

When tumor size reached around 100 mm³, mice were randomly divided into 4 groups and administered injections of PBS, αPD-1, Tim3@PHSM@IC NPs, and Tim3@PHSM@IC NPs + αPD-1 (Tim3@PHSM@IC NPs: 1 mg/kg CuET on days 0, 3, 6; αPD-1: 10 mg/kg on days 1, 4, 7). The tumor size of each group was recorded every other day.

Cytokine level measurement

Cytokines such as TNF-α, IFN-γ, and IL-2 were considered key indicators of CTLs activation. To measure the concentrations of IL-2, IL-12, TNF-α, and IFN-γ cytokines, ELISA kits (Xin-bosheng, EMC102a, and EMC101g) were employed. Following the administration of various nanoparticles, tumor-bearing mice were euthanized, and their serum samples were collected and then subjected to centrifugation at 3000 rpm at 4 °C for 15 min to prepare for ELISA analysis. The cytokine levels were determined by detecting the optical density (OD) at 450 nm, providing insights into the immune response elicited by the nanoparticles.

Immunohistochemistry (IHC)

For immunohistochemical analysis, tumor sections were first exposed to primary antibodies against Ki-67 for 10 h. This was followed by the application of a secondary antibody for 40 min to enhance the signal. Fluorescence microscopy was applied to visualize the expression patterns of DLAT and FDX1 in the tumor tissue. Subsequently, ImageJ software was employed to quantify the mean fluorescence intensity, providing a quantitative assessment of the relative expression levels of Ki-67 in the tumor tissue. This approach offers crucial insights into the cellular and molecular dynamics within the TIME.

Histopathological analysis

On the 12th day of tumor inoculation, solid tumors were harvested from mice for histological analysis by standard H&E staining. The excised tumors and organs were treated with 4% paraformaldehyde, paraffin embedding, and sectioning, followed by H&E staining. The slides were examined under a fluorescence microscope (IX83, Olympus).

Flow cytometric analysis

Flow cytometry was used to analyze changes in immune cell populations in the tumor tissue and TDLNs. Excised tumors were processed through cutting and digestion to obtain a single cell suspension, whereas TDLNs were ground through a filter. The single cell suspensions were stained with antibodies following the manufacturer’s protocols. Supporting Information (Fig.S17-21) illustrated the gating strategies for flow cytometry.

Data analysis

GraphPad Prism 8 was used to statistical analyses. Data were shown as mean ± standard deviation (SD) from at least three independent biological replicates, unless otherwise specified in the figure legend. Comparisons between two groups were conducted by Student’s t-test. One-way analysis of variance (ANOVA) with Tukey’s post-hoc correction was applied for comparisons involving multiple comparisons. Statistical significance was defined as follows: *p < 0.05, very significant when **p < 0.01, and extremely significant when ***P < 0.001, ns: no significance.