Precisely Activating cGAS‐STING Pathway with a Novel Peptide‐Based Nanoagonist to Potentiate Immune Checkpoint Blockade Cancer Immunotherapy

Abstract As an essential intracellular immune activation pathway, the cGAS‐STING pathway has attracted broad attention in cancer treatment. However, low bioavailability, nonspecificity, and adverse effects of small molecule STING agonists severely limit their therapeutic efficacy and in vivo application. In this study, a peptide‐based STING agonist is first proposed, and KLA is screened out to activate the cGAS‐STING pathway by promoting mitochondrial DNA (mtDNA) leakage. To precisely activate the cGAS‐STING pathway and block the PD‐1/PD‐L1 pathway, a multi‐stimuli activatable peptide nanodrug (MAPN) is developed for the effective delivery of KLA and PD‐L1 antagonist peptide (CVR). With rational design, MAPN achieved the site‐specific release of KLA and CVR in response to multiple endogenous stimuli, simultaneously activating the cGAS‐STING pathway and blocking PD‐1/PD‐L1 pathway, ultimately initiating robust and durable T cell anti‐tumor immunity with a tumor growth inhibition rate of 78% and extending the median survival time of B16F10 tumor‐bearing mice to 40 days. Overall, antimicrobial peptides, which can promote mtDNA leakage through damaging mitochondrial membranes, may be potential alternatives for small molecule STING agonists and giving a new insight for the design of novel STING agonists. Furthermore, MAPN presents a universal delivery platform for the effective synergy of multiple peptides.


Instruments
The particle sizes and zeta potentials of GSH-PN, H2O2-PN, and MAPN were studied by Zetasizer Nano-ZS (Malvern Instruments, USA) at 25 °C.Transmission Electron Microscopy (TEM) measurements were performed on a commercial Talos F200C electron microscope at an acceleration voltage of 120 kV. 1 H-nuclear magnetic resonance ( 1 H-NMR) spectra were obtained on a Varian UNITY-plus 600 M nuclear magnetic resonance spectrometer.UV-Visible spectra were performed on a ALPHA FluorChem FC3 (Alpha, USA).Cell viability and BCA assay was reported on a Tecan Spark plate reader (USA).Flow cytometric analysis was performed on a CytoFLEX flow cytometry (Beckman, USA).CLSM images were captured with a Confocal Laser Scanning Microscopes-LSM 800 (Zeiss, Germany).
The solution was bubbled with argon gas for 45 min, then reaction system was sealed and stirred magnetically at 70 °C for 24 h.Subsequently, the reaction mixture was allowed to cool to room temperature followed by concentration, and dialysis (MWCO: 1000 Da) against methanol for 48 h.After removing the solvent by rotary evaporator, the obtained tpy-PDPA-CTA product was dried under vacuum, yielding 5.4 g (75 %).
For the better demonstration, PDPA-KLA, PDPA-CVR, and PDPA-PEG5000 were synthesized as nonresponsive polymers using similar methods as indicated in Figure S8, Supporting Information.The successful synthesis of PDPA-KLA, PDPA-CVR, and PDPA-PEG5000 were confirmed using GPC and 1 H-NMR as indicated in Figure S9 and

DLS and TEM Analysis
The average particle sizes and zeta potentials of GSH-PN, H2O2-PN, and MAPN were determined using DLS measurements.The DLS measurements were performed on a Zetasizer Nano-ZS (Malvern Instruments, USA) at 37 °C.The morphology of GSH-PN, H2O2-PN, and MAPN were observed using transmission electron microscopy (TEM, FEI Talos F200C electron microscope).For the preparation of TEM samples, GSH-PN, H2O2-PN, and MAPN were prepared as the solutions with pH 7.4 and the concentration of KLA at 4 μM.TEM samples were prepared by drop-coating of 2 μL GSH-PN, H2O2-PN, and MAPN onto carbon-coated copper grids.Droplets of samples were contacted with the grids for 15 min, then excess amount of samples was removed.

Determination of Critical Micelle Concentration (CMC)
PDPA-SS-KLA, PDPA-TK-CVR, and PDPA-TK-PEG5000 can self-assemble into micelle at pH 7.4, with PDPA as the hydrophobic inner core, PEG and peptides as the hydrophilic outer corona.The CMC values of PDPA-SS-KLA, PDPA-TK-CVR, and PDPA-TK-PEG5000 were determined by fluorescence spectroscopy using pyrene as a fluorescence probe at pH 7.4. [2]An aliquot of 1 mL acetone solution of pyrene was transferred into 20 mL vials and the acetone evaporated to dryness.Then, a series of PDPA-SS-KLA, PDPA-TK-CVR, or PDPA-TK-PEG5000 micellar solutions ranging from 1×10 -5 to 1 mg mL -1 were added into the vials to give a final pyrene concentration of 6×10 -7 M. The solution was incubated at room temperature overnight under shaking.
The measured emission wavelength was set at 395 nm, and the band width of emission was set to 3 nm.The ratios of the excitation spectra's fluorescent intensities at 337 nm and 335 nm (I337/I335) were calculated and plotted against the logarithm of polymer mass concentration.

Stability of MAPN
The stability of MAPN was evaluated using previous method. [3]Briefly, we dissolved the MAPN with a concentration of KLA at 4 μM in PBS (pH 7.4, 10 mM) and mouse serum at 37 °C, and the particle sizes of MAPN were characterized at various incubation time using DLS measurement.

Circular Dichroism (CD) Spectra
MAPN and H2O2-PN were pretreated with GSH (10 mM) to simulate the GSH-riched intracellular environment, and incubated in 10% (v/v) solution of trifluoroethanol/PBS to imitate the membrane environment. [4]CD experiments were performed with a Bio-Logic MOS-450 (Claix, France) at room temperature.The experimental condition was set as follows: speed of 50 nm/min, time response of 2s, resolution of 0.5 nm, bandwidth of 4.0 nm, and cell path length of 1.0 mm.All spectra were converted to a uniform scale after subtraction of the background.The recorded curves were smoothed with standard parameters.

Mitochondrial Swelling
MAPN and H2O2-PN were pretreated with GSH (10 mM) to simulate the GSH-riched intracellular environment.The mitochondria were isolated using a cell mitochondria isolation kit (Beyotime, China).5×10 6 B16F10 cells were collected and suspended with 2.5 mL mitochondrial separation reagent.The cell suspension was placed in ice bath for 15 min and then transferred to a glass homogenizer for 20 times' homogenate.The cell homogenate was centrifuged at 600 g for 10 min.The supernatant was carefully transferred to another centrifuge tube and centrifuged at 11000 g for 10 min.The precipitate is the isolated cell mitochondria.The isolated mitochondria were suspended in storage buffer at a concentration of 0.5 mg mL -1 .100 μL of different samples were added to above mitochondrial suspension and absorbance at 540 nm was continuously scanned within 15 min. [5]ll Culture B16F10 and BMDC cells were cultured in RAPI-1640 supplemented with 10% (v/v) heat-inactivated FBS, 100 units mL -1 penicillin, and 100 μg mL -1 streptomycin, and maintained in 5% CO2 humidified environment at 37 °C.

Cell Viability Assay
The cell viability assay was evaluated using Cell Counting Kit (CCK, ZOMANBIO Biotech, Beijing, China) as previously described. [3]Briefly, B16F10 cells were seeded in a 96-well plate at a density of 5×10 3 cells per well and incubated overnight in 0.5 mL RAPI-1640 with 10% FBS (v/v) for overnight.The cells was exposed to free KLA, H2O2-PN, and MAPN with or without H2O2 (100 μM) at various KLA concentrations (from 1 to 32 μM).Then, CCK reagent was mixed with RPMI-1640 at a volume ratio of 1/9 (freshly prepared) to achieve the CCK working solution.After 24 h incubation, the cells were rinsed using PBS (pH 7.4, 10 mM), followed by the addition of 100 μL CCK working solution for another 2 h incubation.Quantification of cell viability was determined by measuring the absorbance (λ = 450 nm) with Synergy H1 microplate reader (BIO-TEK, USA).The cell viability was calculated by referring to that of the cells without any treatment.

Cellular Internalization and Endosomal Escape
Cellular internalization of MAPN was studied through CLSM and flow cytometric assays (Beckman, USA).Briefly, B16F10 cells were seeded at a density of 1×10 4 cells per well in 35 mm confocal dish (Ф =15 mm) and incubated overnight for the cell attachment.The cells were incubated with free KLA, GSH-PN, and MAPN with a concentration of KLA at 4 μM in complete culture medium with or without H2O2 (100 μM) for 4 h.The cells were then washed twice with ice-cold PBS and fixed with fresh 4% paraformaldehyde at room temperature, and then counterstained with DAPI.All the cells were observed using a CLSM (Zeiss, Germany).For flow cytometric analysis, B16F10 cells were seeded into 6-well plates at a density of 1×10 5 cells per well.After the overnight growth, the cells were exposed to frre KLA, MAPN, and GSH-PN with a concentration of KLA at 4 μM, and incubated in complete culture medium at different conditions (with or without H2O2, 100 μM) for 2 h.After the trypsin digestion and centrifugation, the cells were collected, washed with cold PBS and fixed with fresh 4% paraformaldehyde for flow cytometric assay.All of these experiments were performed in triplicate.
The endosomal escape ability of MAPN was study by analyzing the colocolization of endosomes and KLA after internalizing into cells.Briefly, B16F10 cells were seeded at a density of 1×10 4 cells per well in 35 mm confocal dish (Ф =15 mm) and incubated overnight for cell attachment.KLA was labeled with FITC.The cells were incubated with MAPN with a concentration of KLA at 4 μM in complete culture medium with H2O2 (100 μM) for 4 h.After 1 h, 3 h, and 6 h incubation, endosomes were stained with LysoTracker probe (red) according to manufacture's instruction.The cells were then washed twice with ice-cold PBS and fixed with fresh 4% paraformaldehyde for 15 min at room temperature, and counterstained with DAPI for the easy observation of cell nucleus.All the cells were observed using a CLSM (Zeiss, Germany).

In Vitro PD-1/PD-L1 Pathway Blockade
MAPN-mediated PD-1/PD-L1 pathway blockade was evaluated with anti-PD-L1 (sc-518027, Santa Cruz, USA) staining.Briefly, B16F10 cells were seeded at a density of 1×10 4 cells per well in 35 mm confocal dish (Ф =15 mm) and incubated overnight for cell attachment.The cells were incubated with MAPN with a concentration of KLA at 4 μM in complete culture medium with or without H2O2 (100 μM) for 4 h.The cells were then washed twice with ice-cold PBS and fixed with fresh 4% paraformaldehyde at room temperature, and then stained with Alexa Fluor 594 (red) labeled anti-PD-L1 antibodies and DAPI.All the cells were observed using a CLSM (Zeiss, Germany).

The mtDNA Leakage Assay
B16F10 cells were seeded at a density of 1×10 4 cells per well in 35 mm confocal dish (Ф =15 mm) and incubated overnight for cell attachment.The cells were incubated with MAPN with a concentration of KLA at 4 μM in complete culture medium with or without H2O2 (100 μM) for 24 h.After washing with PBS, the cells were permeabilized, and incubated with Anti-TOMM20 antibodies (GB111481, Servicebio, China) and dsDNA Marker (sc-58749, Santa Cruz, USA) at 4 °C overnight, and then incubated with secondary antibodies for 2 h and counterstained with DAPI.All the cells were observed using a CLSM (Zeiss, Germany).

The qPCR Quantification of Cytosolic mtDNA
B16F10 cells were seeded in a 12-well plate at a density of 5×10 4 cells per well and incubated overnight in 1 mL RAPI-1640 with 10% FBS (v/v) for overnight.The cells was exposed to free KLA, H2O2-PN, and MAPN with a concentration of KLA at 4 μM in complete culture medium with or without H2O2 (100 μM) for 24 h.After washing with PBS, the cells were collected by centrifugation.Cytosolic mtDNA was removed using a Cell Mitochondria Isolation Kit (C3601, Beyotime, China), and the DNA was then extracted with a TIANamp Genomic DNA kit (DP304, Tiangen Biotech, China) following the manufacturer's protocol.The mtDNA D-Loop primers were synthesized by GenePharma Biotech (detailed sequences in Table S3, Supporting Information), and then quantified with Multiplex PCR Kit (PM101, Novoprotein , Shanghai, China) using Quantitative Real-Time PCR Detection System (Bio-Rad, CFX96, USA).

Caspase-9 and Caspase-3 Activity Assay
The B16F10 cells were seeded into a 6-well plate at the density of 2×10 5 cells per well.After the overnight growth, the cells were exposed to frre KLA, MAPN, and GSH-PN with a concentration of KLA at 4 μM, and incubated in complete culture medium at different conditions (with or without H2O2, 100 μM) for 24 h.After treatments, the cells were rinsed with PBS for three times and then treated with lysis buffer solution in ice bath for 1 h.Cell lysates were centrifuged at 12000 rpm for 5 min at 4 o C, and the supernatant was collected.The supernatant was incubated with caspase-9 or caspase-3 substrate at 37 o C in dark for 12 h.The activities of caspase-9 and caspase-3 were determined by measuring the absorbance (λ = 450 nm) with Synergy H1 microplate reader (BIO-TEK, USA).

Evaluation of cGAS-STING Pathway Activation
Western bolt was employed to evaluate the MAPN-mediated activation of cGAS-STING pathway.Briefly, B16F10 cells were first seeded into 24-well plates at 2×10 4 cells per well and incubated overnight in 0.5 mL RAPI-1640 with 10% FBS (v/v) for cell attachment.After the overnight growth, the cells were exposed to frre KLA, GSH-PN, and MAPN with a concentration of KLA at 4 μM, and incubated in complete culture medium with or without H2O2 (100 μM).After 24 h incubation, the cells were washed with ice-cold PBS for three times and solubilized in 1% Nonidet P-40 lysis buffer.Homogenates were clarified by centrifugation at 15000 g for 30 min at 4 °C, and protein concentrations were determined with a BCA assay kit (Solarbio, China).

MAPN-Mediated DCs Maturation In Vitro
To investigate the MAPN-mediated DCs maturation, BMDCs were first extracted from the bone marrow of 8-week-old C57BL/6 mice.B16F10 cells were pretreated with PBS, free KLA, H2O2-PN, and MAPN as described above.Afterwards, 1×10 6 immature BMDCs were co-cultured with 1×10 5 pretreated B16F10 cells for 24 h using a transwell chamber (The B16F10 cells were cultured in the upper chamber, and BMDCs were seeded in the lower chamber).After staining with anti-CD45, anti-CD11c, anti-CD80, and anti-CD86 antibodies according to the manufacturer' protocols, the DCs maturation was examined by flow cytometric measurement.

In Vivo Distribution of MAPN
To investigate the tumor accumulating ability of MAPN, tumor-bearing mice were established by subcutaneous injection of B16F10 cells (1×10 6 for each mouse) in the mammary fat pad.The mice were randomly divided into three groups (n = 3).When the tumor volume was about 500 mm 3 , the mice were intravenously injected with free KLA, GSH-PN, and MAPN containing Cy5.5 labeled KLA (100 µM ; 150 µL ).All experimental protocols were conducted within Anhui Medical University guidelines for animal research and were approved by Institutional Animal Care and Use Committee (LLSC20230877).At 24 h post-injection, the mice were imaged by IVIS Lumina imaged system (IVIS, Spectral Instrumen, USA).The mice were then sacrificed, and major organs and tumors were collected for ex vivo imaging.The fluorescence images were analyzed using Version 4.0.0 (Spectral Instrumen).

Tumor Inhibition with MAPN
To investigate the anti-tumor effects of MAPN treatment, B16F10 tumor-bearing mice was established as described above.When primary tumors reached a size of about 30-60 mm 3 , the mice were randomly divided into five groups (n = 6), and intravenously injected with PBS, free KLA, GSH-PN, H2O2-PN, and MAPN (KLA: 100 μM, 150 μL) every four days for four times.The body weights and tumor volumes were measured every four days.Tumor volumes were calculated with the formula: tumor volume = 0.5 × length × width 2 .The mice were sacrificed when the volume of tumor exceeded 2 cm 3 .About one-half of the mice were sacrificed on day 15 after tumor implantation, the blood and major organ of mice were collected and analyzed.Survival was evaluated from the first day of tumor implantation until day 42.To examine the immune responses against primary tumor, CD45 + CD3 + CD8 + T cells, CD45 + CD11c + CD86 + CD80 + DCs, ki67 + CD8 + T cells, and IFN-γ + CD8 + T cells in tumors and LNs were analyzed using flow cytometry.All anti-bodies for flow cytometric analysis were purchased from Biolegend (USA).IFN-β, IFN-γ, TNF-α, IL-12, and IL-6 in tumors were also examined with ELISA kits (Jianglai biology, Shanghai).In apoptotic cells, the double or single                    Table S1.Sequences of peptides.

Figure S13 .
Figure S13.a) Schematic illustration of transwell experiment.The B16F10 cells were cultured in the upper chamber, and BMDCs were seeded in the lower chamber.b) Representative plots of matured BMDCs (gated on CD11c + CD80 + CD86 + cells) in each group analyzed by flow cytometry.

Figure S14 .
Figure S14.H&E analysis of the major organs (heart, liver, spleen, lung, and kidney) from the mice in each treatment group.The scale bars are 100 μm.

Figure
Figure S16.a) Representative flow cytometric plots of IFN-γ + CD8 + CTLs in tumor tissues after different treatments.b) Representative flow cytometric plots of CD8 + CTLs infiltration in spleens after different treatments.

Figure S17 .
Figure S17.Individual tumor growth curves of B16F10 tumor-bearing mice after different treatments.

Figure S18 .
Figure S18.TUNEL (a) and H&E (b) staining of tumor tissues from the mice after different treatments.Scale bars are 100 μm.