Dual Synergistic Tumor‐Specific Polymeric Nanoparticles for Efficient Chemo‐Immunotherapy

Abstract Chemo‐immunotherapy has made significant progress in cancer treatment. However, the cancer cell self‐defense mechanisms, including cell cycle checkpoint and programmed cell death‐ligand 1 (PD‐L1) upregulation, have greatly hindered the therapeutic efficacy. Herein, norcantharidin (NCTD)‐platinum (Pt) codelivery nanoparticles (NC‐NP) with tumor‐sensitive release profiles are designed to overcome the self‐defense mechanisms via synergistic chemo‐immunotherapy. NC‐NP remains stable under normal physiological conditions but quickly releases 1,2‐diaminocyclohexane‐platinum(II) (DACHPt, a parent drug of oxaliplatin) and NCTD in response to the tumor acidity. NCTD inhibits protein phosphatase 2A (PP2A) activity to relieve cell cycle arrest and downregulates the tumor PD‐L1 expression to disrupt the programmed cell death‐1 (PD‐1)/PD‐L1 interaction, synergistically enhancing Pt‐based chemotherapy and immunogenic cell death‐induced immunotherapy. As a result, NC‐NP exhibits potent synergistic cytotoxicity and promotes T cell recruitment to generate robust antitumor immune responses. The dual synergism exhibits potent antitumor activity against orthotopic 4T1 tumors, providing a promising chemo‐immunotherapy paradigm for cancer treatment.

1,2-diaminocyclohexane-platinum (II) (DACHPt).PEG-PLL/NCTD or PEG-PLL/SA were dissolved in DI water(C COOH = 5 mM), and the solution pH was adjusted to 8.5 using NaOH (0.1 N), followed by the addition of DACHPt (C Pt : C COOH = 1: 1).The reaction was performed at 37°C for 72 h with shaking.The product was purified by dialyzing (MWCO 3,500) against DI water to remove the free platinum drug, and the resulting PEG-PLL/NCTD-DCHAPt (NC-NP) and PEG-PLL/SA-DCHAPt (SA-NP) nanoparticles were obtained.The size distributions were measured by dynamic light scattering (DLS) (Malvern Instrument Ltd., UK).The morphology of the nanoparticles was observed using a transmission electron microscope (Hitachi h-7000 TEM system, Kyoto, Japan).The Pt content was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer Optima 3100XL).

pH responsiveness of nanoparticles
The hydrolysis of PEG-PLL/NCTD was monitored using 1 H-NMR spectroscopy.
Briefly, PEG-PLL/NCTD was dissolved in D 2 O at a concentration of 10 mg/mL, and the solution pH was adjusted to 5.0 using DCl.The mixture was incubated at 37°C with shaking (200 rpm).At timed intervals, 600 μL of the solution was taken out for 1 H NMR characterization after the pH was adjusted to 8.5.PEG-PLL/NCTD or NC-NP was dissolved in HEPES solution (10 mM) at pH 7.4, 6.5, or 5.0 and incubated at 37°C with shaking (200 rpm).At timed intervals, the solution was sampled for zeta potential measurement using DLS.
The stability of NC-NP in response to acidic conditions was measured by monitoring the size change.NC-NP was incubated in PBS at pH 7.4 or 5.0.at 37°C for 72 h, and the size distributions were detected using DLS.

In vitro drug release
The in vitro drug release from the polymeric nanoparticles was evaluated via a dialysis method.NC-NP or SA-NP were loaded into dialysis bags (MWCO 3500) and immersed in 50 mL PBS (10 mM) at pH 7.4 or 5.0 in a 37°C shaker (200 rpm).At timed intervals, 100 µL dialysate was withdrawn and replaced with an equal volume of fresh medium.The platinum content was measured by ICP-MS, and the NCTD content was measured by HPLC.

Cellular uptake
The cellular uptake rate was quantified by measuring the intracellular Pt contents.
4T1 cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well, incubated for 24 h, and then treated with free OXA, NC-NP, or SA-NP for 1 h, 2 h, or 6 h at a Pt-eq.dose of 3 μg/ml.The cells were washed three times with cold PBS and isolated to detect the Pt content by ICP-MS.
We further determine the cell internalization pathway of NC-NP.4T1 cells were seeded in 12-well plates at a density of 2×10 5 cells/well overnight.Then the cells were incubated under 4 °C or treated with endocytosis inhibitors chlorpromazine (50 μM), wortmannin(5 μM), cytochalasin D (5 μM), and filipin (7.5 μM) at 37 °C for 2 h and Cy5.5 NC-NP was added into wells.After 4 h incubation, the cells were washed with PBS three times, and the cellular uptake rate was examined by flow cytometry (BD FACSCalibur™, San Jose, CA)

In vitro cytotoxicity
In vitro cytotoxicity of polymeric nanoparticles was assessed using the 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) method.The cells were cultured in 96-well plates with a density of 5,000 cells per well for 24 h, followed by the addition of each formulation at a series of concentrations.After 48 h incubation, 20 μL of MTT solution (5 mg/mL) was added to incubate with cells for 4 h.The medium in each well was removed, and 100 μL of DMSO was added to dissolve the purple formazan crystals.The absorbance of the sample at 562 nm was detected using a microplate spectrophotometer (SpectraMax M2E, Molecular Device, USA).The cell survival rate is calculated by dividing the absorbance value of the experimental group by that of the control group.Each sample concentration was performed in triplicate, and three independent experiments were performed.

Live/dead cell analysis
4T1 cells were cultured in a 12-well plate at a density of 1 x 10 5 cells per well for 24 h.Then cells were treated with PBS, NC-NP, SA-NP, NCTD, OXA, and NCTD + OXA (NC-NP and SA-NP were pretreated at pH 7.4 or 5.0 for 24 h) for 48 h at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL.Afterward, the culture medium was removed, and the cells were washed with PBS three times and stained with Live and Dead Cell Double Staining Kit (Calcein-AM and Propidium Iodide) at 37 °C for 15 min.Finally, the live and dead cells were observed under confocal fluorescence microscopy with 490 nm excitation.

PP2A phosphatase activity assay
4T1 cells were seeded in 6-well plates at a density of 2 × 10 5 cells per well and cultured overnight.After 6 h-incubation with PBS, NC-NP, SA-NP, NCTD, OXA, or NCTD +OXA at an NCTD-eq.dose of 5.2 μg/mL and OXA eq.dose of 3 μg/mL, the cells were washed twice with cold PBS and lysed in RIPA buffer containing protease inhibitor for 20 min (ice).Then the cell lysates were ultrasonic for 10 s and centrifuged for 15 min (1.5 × 10 4 g).The supernatants were collected and analyzed using the PP2A Immunoprecipitation Phosphatase Assay Kit.

Flow cytometric analysis of cell cycle
4T1 cells were seeded in 6-well plates at a density of 1.5 x 10 5 cells per well and cultured overnight, followed by treatment with different formulations at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL.After 48 h, the cells were harvested and fixed with 70% ethanol overnight at 4°C.The fixed cells were incubated with RNase A (100 μg/mL) in PBS for 30 min at 37 °C and stained with PI (50 μg/mL) for 6 30 min in the dark before flow cytometry analysis.Each sample concentration was performed in triplicate, and three independent experiments were performed.

CRT analysis
4T1 cells were cultured in the 12-well plates (1 × 10 5 cells/well) overnight and then incubated with each formulation at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL for 4 h.Then the cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min.Afterward, the cells were washed with PBS and incubated with the anti-CRT antibody (ab196158, 1: 500) for 30 min.The CRT-positive cells were analyzed using flow cytometry.

ATP detection
Extracellular secretion of ATP was detected using the ATP detection kit.Briefly, 4T1 cells were cultured in 12-well plates (1 × 10 5 cells/well) for 12 h and then incubated with each formulation at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL for 4 h.Afterward, the cell culture supernatant was collected, and the ATP contents were measured by the ATP detection kit according to the manufacturer's protocol.

HMGB1 analysis
4T1 cells were cultured overnight in the glass-bottomed Petri dishes at a density of 1 × 10 5 cells per dish and then incubated with each formulation at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL for 24 h.The cells were washed with PBS three times and fixed in 4% paraformaldehyde for 15 min.The cells were permeabilized with 0.3% Triton X-100 for 15 min, followed by PBS washing and incubation with 5% BSA for 30 min.The cells were incubated with anti-HMGB1 for another 30 min and stained with DAPI for 15 min before CLSM observation.

DC maturation
Bone marrow-derived dendritic cells (BMDCs) were collected from the bone marrow of BALB/c mice and cultured in 1.5 mL 1640 medium containing GM-CSF (20 ng mL −1 ) and IL-4 (10 ng mL −1 ).4T1 cells (1 x 10 5 cells/well) and BMDCs (5 × 10 5 cells/well) were seeded in the transwells and 12-well plates, respectively, and cultured overnight.4T1 cells were cultured with different treatments for 24 h and then co-incubated with BMDCs for 24 h.Afterward, the BMDCs were harvested for antibody staining, and the matured DCs (CD11c + CD80 + CD86 + ) were analyzed using flow cytometry.

Flow cytometric analysis of PD-L1
4T1 cells were cultured in the 12-well plates at a density of 1 × 10 5 cells per well overnight and then incubated with each formulation at a Pt-eq.dose of 3 μg/mL and NCTD-eq.dose of 5.2 μg/mL for 24 h.Then the cells were washed with PBS and incubated with the anti-PD-L1 antibody (1: 500) for 30 min.The PD-L1-positive cells were analyzed using flow cytometry.

Western blotting analysis
For western blotting analysis, the cells with different treatments were lysed with RIPA cell lysis buffer.The protein concentration was determined by the BCA protein detection kit, and samples containing 40 μg protein were loaded onto an SDS-PAGE gel (8% separating gel).The proteins in the cell lysates were separated and electrotransferred to a PVDF membrane.The membrane was then blocked with 5% non-fat powdered milk in TBST buffer for 1 h, followed by incubation with primary antibodies: β-Catenin (1: 1000), PD-L1 (1: 1000), or GAPDH (1: 1000) at 4 °C overnight.The membrane was washed with TBST three times and incubated with horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse secondary antibody (1: 1000) at room temperature for 1 h.Afterward, the membrane was washed with TBST three times and visualized with a chemiluminescence imaging system (CLiNX Science instruments, China).The grey value ratios of the bands to the internal reference were analyzed by ImageJ software to obtain the relative protein expressions.

Blood Clearance
Female ICR mice (6-8 weeks) were intravenously (i.v.) injected with free OXA, NC-NP, or SA-NP at a Pt-eq.dose of 1 mg/kg (n=3).At timed intervals, 50 μL blood samples were collected from the orbital venous plexus of the mice, heparinized, and centrifuged (5000 rpm, 10 min) to obtain the plasma.The samples were decomposed in aqua regia, and the Pt contents were detected by ICP-MS.

Biodistribution
Female BALB/c mice (6-8 weeks) were subcutaneously injected with 5 × 10 5 4T1 cells into the mammary fat pad.When the tumor volume reached ~ 80 mm 3 , the mice were randomly divided into 3 groups (n=3) and i.v.injected with free OXA, NC-NP, or SA-NP at a Pt-eq.dose of 1 mg/kg.After 24 h postinjection, the mice were sacrificed, and tumors and main organs, including the heart, liver, spleen, lung, and kidneys, were collected.The Pt contents were measured by ICP-MS.

In vivo antitumor activity
Female BALB/c mice (6-8 weeks) bearing orthotopic 4T1 tumors of ~ 100 mm 3 were randomly divided into six groups (n=7), and i.v.injected with PBS, OXA, NCTD, OXA+NCTD, SA-NP, or NC-NP at a Pt-eq.dose of 1 mg/kg and NCTD-eq.dose of 1.7 mg/kg every two days three times.The body weight and tumor volume were recorded individually.On day 22 post-treatment, the mice were sacrificed according to animal ethical requirements, and tumors and major organs were collected.
Tumor volume was calculated according to the formula: V = 0.5 × LW 2 (L: tumor length, W: tumor width).The tumor inhibition rate (TIR) was calculated following the formula: TIR = 100% × (mean tumor weight of control group -mean tumor weight of experimental group)/ mean tumor weight of control group.

In vivo antitumor immune response
Female BALB/c mice (6-8 weeks) bearing orthotopic 4T1 tumors of ~ 100 mm 3 were randomly divided into six groups (n=3), and i.v.injected with PBS, OXA, NCTD, NCTD + OXA, SA-NP, or NC-NP at a Pt-eq.dose of 1 mg/kg and NCTD-eq.dose of 1.7 mg/kg every two days for 3 times.The mice were sacrificed 7 days after the last administration, and blood, tumors, and tumor-draining lymph nodes (TDLNs) were collected.Blood samples were used to analyze the systemic cytokines, including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), interleukin 10 (IL-10), and interleukin 12 (IL-12) using the corresponding ELISA kit according to the manufacturer's protocol.4-μm-thick slices.Then the slices were treated with Ki-67 Detection KIT according to the manufacturer's protocol and observed using fluorescent microscopy.

Statistical analyses
Statistical analysis was performed using GraphPad Prism and Excel.The two-tailed, unpaired Students' t-test was utilized to calculate the significance.Data are presented as mean ± SD.P < 0.05 was regarded as statistically significant.

Figure S4 .
Figure S4.The TEM images of NC-NP after incubation at pH 7.4 or 5.0 for 72 h.scale bar = 200 nm.

Figure S5 .
Figure S5.The hemolysis result of NC-NP at pH 7.4.

Figure S6 .
Figure S6.The relative cellular uptake of NC-NP in 4T1 cells in the presence of endocytic inhibitors.

Figure S7 .
Figure S7.The in vitro cytotoxicity of NC-NP against A549, Hela, B16, or HepG2 cells at pH 7.4 or 5.0 determined by MTT assay.

Figure S11 .
Figure S11.Quantitative analysis of cell cycle distribution of 4T1 cells after each treatment in Figure 2h (n=3).

Figure S13 .
Figure S13.Quantification of the western blotting results in Figure 3i.

Figure S14 .
Figure S14.Quantification of the western blotting results in Figure 3j,l.

Figure S18 .
Figure S18.Gating strategies for flow cytometry.(a) Gating strategy to identify mature DCs and cytotoxic T lymphocytes presented in Figures 5a, c, k, and m; (b) Gating strategy to identify regulatory T cells presented in Figure 5c and o.