Ethical statement
All the animal experiments were performed according to the guidelines and ethical regulations and approved by the Laboratory Animal Welfare and Ethics Committee of Southern University of Science and Technology (SUSTech-SL2022051501). Mice were maintained in a specific pathogen-free (SPF) barrier environment with controlled temperature (20–22 °C) and humidity (30–70%) under a 12 h light/12 h dark cycle, food and sterile water were available ad libitum. When the maximum diameter of the tumour was greater than 15 mm, experiments would be terminated, the mice were euthanized, and the survival rate was statistically calculated based on tumour size greater than 15 mm.
Cells
Human SKBR-3 (ATCC, HTB-30), human MDA-MB-231 (ATCC, HTB-26), human MCF7 (ATCC, HTB-22), human SKHep-1 (ATCC, HTB-52), human SKOV-3 (ATCC, HTB-77), mouse 4T1 (ATCC, CRL-2539), mouse DC2.4 (EK-bioscience, CC-Y2117), mouse LLC (ATCC, CRL-1642), human HFF (ATCC, SCRC-1041), and human HUVEC cells (ATCC, CRL-1730) were purchased and cultured using DMEM (Gibco, c11995500bt), RPMI-1640 (Gibco, C11875500BT), or McCoy’s 5 A (EpiZyme, CB008) medium containing 10% fetal bovine serum (FBS; Gibco, A3161001C) and 1% penicillin/streptomycin (Beyotime, C0222), as appropriate, at 37 °C in a humidified atmosphere containing 5% CO2. No mycoplasma contamination was detected in all cell lines by mycoplasma genus-specific polymerase chain reaction (PCR) assay. Murine CD8+ T cells were isolated from spleens of healthy female Balb/c mice and prepared as a single-cell suspension.
Animals
Female Balb/c mice (aged 6–8 weeks; weight, 18–22 g) were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd. 4T1 cells (1 × 106) and LLC cells (1 × 106) were subcutaneously inoculated into the right flank of each mouse to establish subcutaneous models of 4T1 breast cancer and LLC pulmonary cancer, respectively. Generally, a subcutaneous tumour was observed on day 5 after tumour cell inoculation.
Synthesis and characterization of peptide monomers
Peptide nanoparticles were synthesized according to the standard solid-phase peptide synthesis technique44. In brief, the amino protection group (Fmoc) of the main chain on Rink amine resin (GL Biochem, 49001-20g) was removed with 20% (v/v) piperidine/N,N-Dimethylformamide (DMF; Titan, 68-12-2) to expose the N-terminal amino acid, which was then acetylated with acetic anhydride. Amino acid sequences of TPM1, TPM2, and TPM3 were synthesized via condensation reaction. Afterwards, the amino protection group (Dde) of the last lysine (K) of these peptides was deprotected by 2% hydrazine hydrate for 20 min and Ce6 (Macklin, 19660-77-6) was decorated to the exposed side chains of the last lysine (K) of TPM1, TPM2 and TPM3 monomers. Finally, TPM1, TPM2 and TPM3 monomers were cleaved from the resin by using the cocktail of trifluoroacetic acid (TFA; Macklin, 76-05-1)/triethylsilane (Macklin, 6485-79-6)/H2O (95%/2.5%/2.5%, V/V/V). The peptide mass was confirmed by MALDI-TOF MS (Xevo G2-XS QTOF, Waters, USA). After 5 mg peptide was dissolved in 500 μL dimethyl sulfoxide (DMSO; Macklin, 67-68-5), its chemical structure was analyzed by 1H NMR spectroscopy (AVANCE III 500 M with Prodigy Platform, Bruker, USA).
Synthesis and characterization of peptide nanoparticles
Rapid precipitation was used to assemble polypeptide monomers into spherical nanoparticles. In brief, TPM1, TPM2, and TPM3 monomers were dissolved in DMSO and then rapidly added to MilliQ water to self-assemble into peptide nanoparticles. Rapid ultrasound, oscillation, and vortex are used to disperse the peptide nanoparticles evenly.
UV-vis absorption (Cary 60 UV-Vis, Agilent, USA) and fluorescence spectra (Infinite E Plex, TECAN, Switzerland) were measured to validate the nanoparticle formation in the TPM1 solution with different water content (20%, 40%, 60%, 80%, 90%, and 99.5%). The particle sizes and zeta potentials of TPM1, TPM2 and TPM3 nanoparticles (200 μM) were monitored by DLS (Zetasizer Nano ZS90, Malvern, UK). A fluorescence spectrophotometer (F-4600, HITACHI, Japan) was used to measure the CACs using pyrene (Macklin, 129-00-0) as a hydrophobic probe. Briefly, different concentrations (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 30, and 50 μM) of TPM1, TPM2 and TPM3 solutions were incubated with 0.1 mM pyrene acetone solution at 37 °C for 2 h. The fluorescence intensity of pyrene (excitation, 335 nm) was recorded, and the fluorescence intensity ratio (I1 (373 nm)/I3 (384 nm)) of pyrene was calculated to evaluate the CACs. TEM was used to detect the morphology of nanoparticles. Briefly, TPM1, TPM2 and TPM3 solutions (50 μM) were deposited on a holey carbon film on a 200-mesh copper grid, which was then dyed with 2% uranyl acetate (ACMEC, U25690) for 3 min after the droplet was completely dried. Then the samples were observed under a TEM microscope (Talos L120C, Thermoscientific, USA) operated at 120 kV.
PD-L1-induced fibrillar transformation of peptide nanoparticles in aqueous solution
The human (Sino Biological, 10084-H08H) and mouse (Sino Biological, 50010-M02H-100) PD-L1 recombination proteins were respectively added to TPM1 solution (50 μM) at different molar concentration ratios of 1:100, 1:500, 1:1000, 1:2000 and 1:5000 and incubated for 0.5 h, 4 h, and 24 h. Samples were prepared for TEM to observe the morphology transformation. CD spectroscopy (Chirascan, Applied Photophysics, UK) and FTIR spectrometer (Bruker Vertex 70, Bruker, Germany) were used to detect the formation of β-sheet after TPM1, TPM2 and TPM3 nanoparticles incubated with or without human and mouse PD-L1 recombinant protein. For CD spectroscopy, the peptide nanoparticles (50 μM) were incubated with human or mouse PD-L1 at molar concentration ratios of 1000:1 for 24 h, then CD spectra were recorded. For FTIR analysis, the mixture was lyophilized and mixed with dehydrated KBr crystals for the measurement.
All-atom molecular dynamics simulation
The three-dimensional molecular model of the TPM1 monomer was built by using Gaussian software (version A.03, https://gaussian.com/gaussian16/). The molecular docking of TPM1 and PD-L1 was analyzed using molecular operating environment (MOE) software (version 2020.09, https://www.chemcomp.com/). The PD-1/PD-L1 complex derived from the Protein Data Bank (PDB) database (https://www.rcsb.org/; PDB ID of the PD-1/PD-L1 complex: 3BIK) was used to analyze the interaction between PD-1 and PD-L1 proteins. The binding sites between TPM1 and PD-L1 protein (PDB ID: 3BIS) were predicted through Gromacs software (version 2022.4, https://www.gromacs.org/). The dynamics of the SASA and the number of hydrogen bonds in the binding model of TPM1 and PD-L1 complex were evaluated by Gromacs software.
Western blot assay of cells
The expression level of PD-L1 protein in SKBR-3, MCF7, MDA-MB-231, 4T1, SKHep-1, SKOV-3, LLC, HFF, HUVEC and DC2.4 cells were analyzed. Cells were collected and washed with PBS (biosharp, BL302A) three times, then lysed by the radio immune precipitation assay lysis buffer (EpiZyme, PC101) containing 1% protease inhibitor (YEASEN, 20124ES03) on the ice, followed by 13523 × g centrifugation for 3 min at 4 °C. Total cellular proteins were estimated using BCA kit (Epizyme, ZJ101L). The rest cell lysates were heated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis sample loading buffer (Beyotime, P0287-10ml) for 10 min at 100 °C and subjected to analysis. The PVDF membranes were blocked in 5% (wt/v) non-fat dry milk (Epizyme, PS112L) for 1 h at room temperature, and then incubated with primary anti-PD-L1 antibody (1:1000, Abmart, M033179S) overnight at 4 °C. After that, the PVDF membranes were washed thrice with Tris-buffered saline with tween buffer (Epizyme, PS103S) for 5 min each, and then incubated with secondary antibody (1:10,000, goat-anti-mouse, Abbkine, A21010) for 1 h at room temperature. Lastly, the PVDF membranes were visualized by chemiluminescence on imaging equipment (AniView SE, BLT, China). The band density was analyzed and quantified by using Image J software (https://imagej.en.softonic.com/).
Flow cytometry assay of cells
The surface levels of PD-L1 were analyzed. After cells were washed with PBS three times, cells were incubated with BV421-labelled human (1:200, BD Pharmingen, 563738) or mouse anti-PD-L1 antibodies (1:200, BD Pharmingen, 564716) for 15 min at 4 °C. Then cells were washed with PBS for three times. The expression levels of PD-L1 on the cell membrane were detected using a fluorescence-activated cell sorting (FACS) cytometer (BD Biosciences, USA). To determine the expression levels of PD-L1 protein in SKBR-3 and 4T1 cells after incubation with peptide nanoparticles (50 μM) or anti-PD-L1 antibody (68 nM; Selleck, A2004), cells were washed with PBS to remove the complete culture medium and then cultured using the culture medium without FBS and penicillin/streptomycin. After TPM1, TPM2 and TPM3 nanoparticles and anti-PD-L1 antibodies were added and incubated for 4 h at 37 °C in a humidified atmosphere containing 5% CO2, cells were incubated with BV421-labelled anti-PD-L1 antibody for 15 min at 4 °C. After that, cells were analyzed using the FACS cytometer (BD Biosciences, USA).
Cellular distribution of peptide nanoparticles
To observe the cellular distribution, the peptide nanoparticles (50 μM) were incubated with the cells in 24-well plates (1 × 105 cells/well) for 4 h. After the cells were fixed with 4% paraformaldehyde (biosharp, BL539A) solution at room temperature for at least 15 min and washed thrice with PBS for 5 min each, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542-1MG) for 3 min at room temperature to stain cell nuclei, and then washed thrice with PBS for 5 min each. Cells were imaged using a CLSM microscope (LSM 980, ZEISS, Germany) with 63× oil immersion objective lens.
Endocytosis of peptide nanoparticles
To determine the endocytic pathway of peptide nanoparticles, 5 mM β-CD (Macklin, C6289-25g), 2 mM amiloride (YUANYE, S82129-5mg), and 450 mM hypertonic sucrosex (Macklin, S818046-500g) were added to cells to inhibit clathrin-, caveolae- or macropinocytosis-dependent endocytosis. To determine the intracellular distribution pattern of internalized TPM2 and TPM3 nanoparticles, cells were incubated with the peptide nanoparticles for 4 h, 24 h and 48 h. To determine the intracellular distribution pattern of internalized anti-PD-L1 antibodies, cells were incubated with anti-PD-L1 antibodies (0.25 mg/mL; Selleck, A2004) for 1 h, 4 h and 8 h. LysoTracker Green probe (Solarbio, L7400) was used to stain the lysosomes for 30 min and Hoechst-33342 (Beyotime, C1028) was used to stain cell nuclei for 15 min. Then, cells were analyzed using a CLSM microscope (LSM 980, ZEISS, Germany). 405 nm laser was chosen to excite Ce6 and DAPI or Hoechst-33342 to obtain red and blue fluorescence, respectively.
PD-L1-induced fibrillar transformation of TPM1 nanoparticles on cell membrane
The fibrillar transformation of TPM1 nanoparticles on the cell membrane induced by PD-L1 was detected by SEM and TEM assays. Cells were incubated with TPM1 nanoparticles (50 μM) for 4 h, and then fixed with 2.5% glutaraldehyde (Macklin, BL539A) overnight at 4 °C. After cell fixation, the specimens were dehydrated in a series of gradient alcohol (Macklin, E821483-500ml) solutions (30%, 50%, 70%, 80%, 90%, and 99.5%), and then further washed in hexamethyldisilazane (Macklin, H810965-100ml) thrice for 1 min each and finally kept in a desiccator for drying. Before SEM observation, the cell specimens attached to the double-sided conductive adhesive were coated with gold at 15 mA for 60 s and imaged using an SEM microscope (SU8100, HITACHI, Japan). For TEM observation, samples of cells were processed through multiple steps, including fixing, washing, dehydrating in gradient alcohol, embedding, slicing, and dyeing. The sample was observed under a TEM microscope (Talos L120C, Thermoscientific, USA) operated at 120 kV. To determine the retention of the nanofiber networks on the cell membrane, cells were incubated with TPM1 nanoparticles (50 μM) for 4 h, then the supernatants containing the TPM1 nanoparticles were discarded, and the fresh complete medium was added to continually culture for 24 h. Cells were analyzed using an SEM microscope (SU8100, HITACHI, Japan) and a CLSM microscope (LSM 980, ZEISS, Germany).
Co-location of TPM1 nanoparticles and PD-L1 on cell membrane
The co-location of TPM1 nanoparticles and PD-L1 after their binding on the cell membrane was detected by immunofluorescent staining against PD-L1. Briefly, SKBR-3 cells were incubated with TPM1 nanoparticles for 4 h, then were fixed in 4% paraformaldehyde overnight at 4 °C. After the cells were blocked in 5% (wt/v) non-fat dry milk for 1 h at room temperature, cells were incubated with primary anti-PD-L1 antibody (1:200, Abmart, M033179S) overnight at 4 °C. After that, the cells were washed thrice with PBS for 5 min each and incubated with FITC-labelled secondary antibody (1:1000, goat-anti-mouse, Abbkine, A23210) for 1 h at room temperature. Then, cell nuclei were stained with DAPI and were imaged using a CLSM microscope (LSM 980, ZEISS, Germany). The co-localization was analyzed using MATLAB software (version 9.6.0.1072779, MATLAB, MathWorks, USA, https://www.mathworks.com).
Competitive binding of TPM1 nanoparticles and PD-1 with PD-L1 on cell membrane
Immunofluorescent staining was conducted to determine the competitive binding of TPM1 nanoparticles and PD-1 with PD-L1. Briefly, 1 × 105 SKBR-3 cells were incubated with biotinylated PD-1 protein (183 nM; novoprotein, CY18) alone or together with TPM1 nanoparticles for 4 h. Then, cells were fixed in 4% paraformaldehyde overnight at 4 °C and incubated with 488-conjugated streptavidin (YEASEN, HB170614) for 1 h and stained with DAPI. After covering with an anti-fade reagent (Invitrogen&trade, P36965), cells were analyzed by CLSM microscope (LSM 980, ZEISS, Germany).
PD-L1 capture mediated by peptide nanoparticles
Western blot and flow cytometry assays were used to determine whether TPM1 nanoparticles could bind to the newly generated PD-L1 proteins for overcoming immune resistance. In brief, human (100 ng/mL; PeproTech, 315-05-20) or mouse IFN-γ (100 ng/mL; PeproTech, 300-02-100) was added to the complete culture medium of cells and incubation for 24 h to upregulate PD-L1 expression. After that, western blot and flow cytometry analyses were performed to assess the upregulation of PD-L1 expression in cells induced by IFN-γ, as mentioned above. To determine whether TPM1 could capture newly generated PD-L1 proteins, supernatants of the cells after incubation with IFN-γ for 24 h were discarded, and cells were continuously incubated with TPM1 nanoparticles (50 μM) for 4 h. Then, cells were analyzed by CLSM (LSM 980, ZEISS, Germany).
PD-L1 aggregation mediated by peptide nanoparticles
To determine the ability of TPM1 nanoparticles to aggregate PD-L1 protein on the tumour cell membrane, SKBR-3 cells were transfected with pGIPZ-PD-L1-EGFP plasmid (Addgene, 120933) to label newly generated PD-L1 protein on the tumour cell membrane. In brief, 2 μg pGIPZ-PD-L1-EGFP plasmid was diluted with 200 μL jetPRIME® buffer (Polyplus Transfection, PT-114-07) and then incubated with 4 μL jetPRIME® reagent (Polyplus Transfection, PT-114-07) for 10 min at room temperature. Afterwards, the pGIPZ-PD-L1-EGFP plasmid mixture was added to SKBR-3 cells that were seeded in 6-well plates (2~4 × 105/well) with McCoy’s 5 A complete medium. After cells were incubated for 6 h, the supernatants containing the pGIPZ-PD-L1-EGFP plasmid were discarded and fresh McCoy’s 5 A complete medium was added to continual culture for 24 h. Then, cells were analyzed by CLSM (LSM 980, ZEISS, Germany) at different time points.
Effect of fibrillar transformation of TPM1 nanoparticles on PGRMC1
CLSM and western blot analyses were performed to determine whether PGRMC1 protein was encapsulated into TPM1 nanofiber networks and whether its expression was affected on the tumour membrane, respectively. For CLSM observation, SKBR-3 cells were incubated with TPM1 nanoparticles for 4 h, then were fixed in 4% paraformaldehyde overnight at 4 °C. After the cells were blocked in 5% (wt/v) non-fat dry milk for 1 h at room temperature, cells were incubated with primary anti-PGRMC1 antibody (1:500, Santa Cruz Biotechnology, sc-393015) overnight at 4 °C. After that, the cells were washed thrice with PBS for 5 min each and then incubated with FITC-labelled secondary antibody (1:1000, goat-anti-mouse, Abbkine, A23210) for 1 h at room temperature. The cell nuclei were stained with DAPI. Then, cells were observed on a CLSM microscope (LSM 980, ZEISS, Germany). For the western blot assay, cells were incubated with TPM1 nanoparticles (50 μM) for 4 h. After that, the sample was prepared and the PVDF membranes were visualized by chemiluminescence on an imaging equipment (AniView SE, BLT, China).
Effect of PD-L1 expression on fibrillar transformation of TPM1 nanoparticles
To determine the effect of the PD-L1 expression level on TPM1 fibrillar formation on the cell membranes, the PD-L1 expression level was decreased and increased by changing cell membrane tension via cytomechanical agents. Cells were treated with Y-27632 (APE×BIO, B1293) for 10 min to decrease cell membrane tension. After the cells were completely attached, the cells were incubated with TPM1 nanoparticles (50 μM) and Y-27632 (50 μM) together for 4 h. Cells were transfected with RhoA V14 plasmid for 24 h by using JetOptimus chemical transfection reagent following the recommendations of manufacturer to increase cell membrane tension. Then cells were incubated with TPM1 nanoparticles for 4 h and imaged using a CLSM microscope (LSM 980, ZEISS, Germany).
Effect of the fibrillar transformation of TPM1 nanoparticles on cellular mechanics
Cellular mechanical characteristics were assessed using nanopillar arrays traction force microscopy. In brief, nanopillars were cast in a clean mould with a liquid adhesive (NOA73, Norland Products), the main steps included degassing, UV cure, demolding, and sterilization. To enable cell adhesion to the top of nanopillars, fibronectins (10 μg/mL; Roche, 11080938001) were applied to coat the nanopillar arrays at room temperature for 2 h. Cells (7500 cells/cm2) were seeded on the nanopillar arrays, which were allowed to adhere and spread at least for 2 h. Then, the nanopillars were placed under a microscope (Eclipse Ts2, Nikon, Japan) to record the movements and forces of nanopillars arrays until the cells completely spread. Subsequently, TPM1 nanoparticles (50 μM) were added to treat the cells, and the movements and forces on the nanopillars arrays continued to be recorded through microscopy.
Cytotoxicity of peptide nanoparticles
CCK-8 colorimetric assay was used to assess the in vitro cytotoxicity of peptide nanoparticles. In brief, cells seeded in 96-well plates with 8 × 103 cells per well were incubated with different concentrations (1, 10, 50, 100, and 200 μM) of TPM1, TPM2 and TPM3 nanoparticles in DMEM, RPMI-1640, or McCoy’s 5 A complete medium. After 24 h incubation, 10 μL of CCK-8 solution (Abbine, BS350B) was added to each well and incubated for another 2 h. The optical density (OD) 450 of treated wells (OD 450 treated), blank wells (OD 450 blank), and control wells (OD 450 control) were measured by using a microplate reader of a multi-wavelength measurement system (Infinite E Plex, TECAN, Switzerland). Cell viability was calculated by measuring OD 450 of CCK-8 formazan, and the equation is as follows: cell viability (%) = [(OD 450 treated – OD 450 blank)/(OD 450 control – OD 450 blank)] × 100.
In vitro immunotherapeutic effects of TPM1 on 4T1 cells
CCK-8 and calcein-AM staining assays were used to determine the in vitro killing effect of CD8+ T cells on TPM1-treated 4T1 cells. Mouse CD8+ T cells were isolated from spleens of female Balb/c mice aged 6–8 weeks (n = 6) using the MojoSort Mouse CD8+ T cell isolation kit (Biolegend, 480007) and activated with the Dynabeads Mouse T-Activator CD3/CD28 for activation of mouse T cells (Gibco, 11456D) following the manufacturer’s recommendation. For the CCK-8 assay, 4T1 cells were seeded on 96-well plates with 6 × 103 cells per well. Afterwards, 4T1 cells were treated with TPM1 nanoparticles, TPM2 nanoparticles, TPM3 nanoparticles, PD-L1-targeted peptides, or anti-PD-L1 antibody (68 nM; Selleck, A2004) for 4 h, followed by washing with PBS thrice, and finally co-cultured with activated CD8+ T cells at a ratio of 1:10 for 24 h. After co-culture for 24 h, the supernatant was removed and a fresh complete medium with CCK-8 assay was used to determine the cell viability of 4T1 cells. For the calcein-AM staining assay, 4T1 cells were firstly seeded at a density of 2 × 104 cells per well in 8-well plates for 24 h. Then, cells were respectively incubated with TPM1 nanoparticles, TPM2 nanoparticles, TPM3 nanoparticles, PD-L1-targeted peptides, and anti-PD-L1 antibodies (68 nM; Selleck, A2004) for 4 h. Afterwards, 4T1 cells were co-incubated with activated CD8+ T cells for 24 h and 4T1 cells were co-incubated with only activated CD8+ T cells for 24 h were used as controls. After discarding the medium containing CD8+ T cells, 4T1 cells were stained with Calcein-AM (YEASEN, 40747ES76) for 20 min, then washed thrice with PBS. Cells were imaged using a CLSM microscope (LSM 980, ZEISS, Germany). 488 nm laser was chosen to obtain green fluorescence of Calcein-AM.
ELISA assay
ELISA was performed to measure IFN-γ, IL-2, TNF, and GZMB contents in the supernatant collected from the CCK-8 assay experiments by using the IFN-γ (BIOESN, BES0211K), IL-2 (BIOESN, BES0032K), TNF (BIOESN, BES0087K), and GZMB (BIOESN, BES1497K) ELISA Kits, respectively. Before the tumour cell killing assay, the co-culture cell samples were made and observed on an SEM microscope (SU8100, HITACHI, Japan) to validate that CD8+ T cells could recognize tumour cells after nanofiber network formation on cell membranes.
Plasma pharmacokinetics of peptide nanoparticles
To assess the plasma pharmacokinetics of TPM1, a single dose of TPM1 nanoparticles (13 mg/kg) was intravenously injected into normal mice via the tail vein (n = 3 for each time point, a total of 21). At a predetermined time, 20 uL of blood sample was collected and diluted 1:10 in PBS. To obtain the supernatant, the samples were centrifuged at 3378 × g for 5 min. The concentration of Ce6 derived from TPM1 was detected by a fluorescence spectrometer (excitation wavelength: 405 nm).
In vivo inflammatory effect of peptide nanoparticles
To assess the effect of peptide nanoparticles on inflammation in vivo, ELISA was used to determine the pro-inflammatory cytokines secretion in serum of mice bearing 4T1 breast cancer. In brief, the serum of 4T1 tumour-bearing mice (n = 5 for each group, a total of 25) was collected 21 days after a single dose of PBS (200 μL per injection via tail vein injection), TPM1 nanoparticles (13 mg/kg per injection via tail vein injection), TPM2 nanoparticles (13 mg/kg per injection via tail vein injection), TPM3 nanoparticles (13 mg/kg per injection via tail vein injection) and anti-PD-L1 antibody (5 mg/kg per injection; Selleck, A2004; via intraperitoneal injection). Then, pro-inflammatory cytokines including IL-6, TNF, and IL-1β were detected in serum using ELISA kits for TNF (BIOESN, BES0087K), IL-6 (BIOESN, BES0086K), and IL-1β (BIOESN, BES0085K) according to the manufacturer’s instructions. Detection was measured by absorbance in an ELISA reader (Infinite E Plex, TECAN, Switzerland).
Bio-distribution of peptide nanoparticles
Ex vivo fluorescence imaging was used to assess the bio-distribution of TPM1 nanoparticles. The mice bearing 4T1 tumour were randomly divided into 3 groups (n = 3 for each group at each time point, a total of 75) to respectively receive intravenous injections of a single dose of TPM1 (13 mg/kg), TPM2 (13 mg/kg) and TPM3 nanoparticles (13 mg/kg) via tail vein. After 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 48 h, 72 h, and 168 h post-injection, the tumour, heart, liver, spleen, lung, kidney, intestine, and muscle were harvested for ex vivo imaging using a fluorescence imaging system (AniView SE, BLT, China).
In vivo immunotherapeutic effects of peptide nanoparticles
To assess the immunotherapeutic effects of peptide nanoparticles, the mice bearing 4T1 tumour or LLC pulmonary tumour were randomly divided into 5 groups (n = 5 for each group, a total of 50) to respectively receive intravenous injection of PBS (200 μL per injection) via tail vein, intravenous injection of TPM1 nanoparticles (13 mg/kg per injection) via tail vein, intravenous injection of TPM2 nanoparticles (13 mg/kg per injection) via tail vein, and intravenous injection of TPM3 nanoparticles (13 mg/kg per injection) via tail vein, and intraperitoneal injection of anti-PD-L1 antibody (5 mg/kg per injection; Selleck, A2004) every two days for nine days. During the treatment period, tumour volume and body weight were measured with a calliper every other day for 21 days, and tumour volume was calculated by using the following formula: 1/2 × length × width2. On day 21 after treatments, mice were euthanized by using isoflurane (RWD, R510-22-10) inhalation followed by cervical dislocation, and then the tumour tissues were harvested for histologic examinations including HE staining, immunohistochemistry analysis of Ki67 (1:200, Servicebio, GB111499-50), CD4 (1:200, Servicebio, GB15064-50), CD8 (1:200, Servicebio, GB15068-50), GZMB (1:200, Proteintech, 13588-1-AP), IFN-γ (1:200, BOSTER, A00393-3), IL-2 (1:200, Servicebio, GB11114-50) and TNF (1:200, Servicebio, GB11188-50), immunofluorescent staining for CD4 (1:200, Servicebio, GB15064-50), CD8 (1:200, Servicebio, GB15068-50) and immunofluorescent staining for TUNEL using Fluorescein (CF488) TUNEL Cell Apoptosis Detection Kit (Servicebio, G1501-100T). The tumour tissues were also processed for flow cytometry and real-time reverse transcription PCR analysis to the underlying therapeutic mechanism of peptide nanoparticles. Major organs including the heart, liver, spleen, lung and kidney were collected for HE staining, and whole blood was collected for routine biochemistry examinations.
Survival analysis
Mice bearing 4T1 tumour were randomly divided into 5 groups (n = 8 for each group, a total of 40) to respectively receive intravenous injection of PBS (200 μL per injection) via tail vein injection, intravenous injection of TPM1 nanoparticles (13 mg/kg per injection) via tail vein injection, intravenous injection of TPM2 nanoparticles (13 mg/kg per injection) via tail vein injection, and intravenous injection of TPM3 nanoparticles (13 mg/kg per injection) via tail vein injection, and intraperitoneal injection of anti-PD-L1 antibody (5 mg/kg per injection; Selleck, A2004) every two days for nine days. The survivals of mice were observed up to 90 days after the five treatments. Survival analysis was plotted using a Kaplan–Meier Curve.
Flow cytometry assays of tumour tissues
After the subcutaneous tumours were excised and cut into small pieces, the tumour tissues were digested with the mixed solution (1 mg/mL) of collagenase I (Solarbio, C8140-100mg) and IV (Solarbio, C8160-100mg) for 15 min on constant temperature shaker (225 × g, 37 °C). After digestion, the single-cell suspension was obtained by filtering through 70 μm cell strainers, followed by a staining procedure with fluorescence-labelled antibodies. &&&In brief, the cells were stained with anti-CD45 (1:200, BD, 557659), anti-CD3 (1:200, BD, 553061), anti-CD8 (1:200, BD, 551162), anti-CD4 (1:200, BD, 553051), fixable viability (1:200, BD, 564406), anti-Foxp3 (1:200, BD, 562996), anti-IFN-γ (1:200, BD, 563376), anti-IL-2 (1:200, BD, 554429), anti-TNF (1:200, Biolegend, 506345), or anti-GZMB (1:200, Biolegend, 372207) antibodies. The fluorescence of cells was quantitatively analyzed by FACS (BD Biosciences, USA). All staining steps were protected from light. The data was analyzed using the FlowJo software (FlowJo, USA).
Real-time reverse transcription PCR assay of tumour tissues
Total RNA was extracted from the 4T1 or LLC tumour tissues using TRIzol reagent (Invitrogen, 15596026) according to the manufacturers’ instruction. Synthesis of cDNA was conducted according to the protocol of the RNA PCR kit (TaKaRa, RR024A). Then, cDNAs were used for PCR (IFN-γ, IL-2, TNF, GZMB, and β-actin) with SYBR Green reagents (Macklin, S917731-100μL) under the reaction conditions. The primer sequences (from Sangon Biotech) are shown in Supplementary Table 1. The relative value of mRNA expression was calculated by the comparative ΔΔCt method using β-actin as a reference gene.
Statistical analysis
All experiments were independently performed at least three times, and a minimum five independent biological replications was conducted for immunological assay. Data were expressed as mean ± standard deviation (SD) or as median [interquartile range: IQR] when appropriate. One-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey’s post hoc analysis was used for multiple group comparison. Log-rank test was used for testing statistical significance in the survival assay. All statistical analyses were conducted using GraphPad Prism (version 9, GraphPad Software, La Jolla, CA, USA, https://www.graphpad.com/). P < 0.05 was considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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- Source: https://www.nature.com/articles/s41467-024-54081-9