Combination of an autophagy inhibitor with immunoadjuvants and an anti-PD-L1 antibody in multifunctional nanoparticles for enhanced breast cancer immunotherapy

Abstract

Background: The application of combination therapy for cancer treatment is limited due to poor tumor-specific drug delivery and the abscopal effect.

Methods: Here, PD-L1- and CD44-responsive multifunctional nanoparticles were developed using a polymer complex of polyethyleneimine and oleic acid (PEI-OA) and loaded with two chemotherapeutic drugs (paclitaxel and chloroquine), an antigen (ovalbumin), an immunopotentiator (CpG), and an immune checkpoint inhibitor (anti-PD-L1 antibody).

Results: PEI-OA greatly improved the drug loading capacity and encapsulation efficiency of the nanoplatform, while the anti-PD-L1 antibody significantly increased its cellular uptake compared to other treatment formulations. Pharmacodynamic experiments confirmed that the anti-PD-L1 antibody can strongly inhibit primary breast cancer and increase levels of CD4+ and CD8+ T cell at the tumor site. In addition, chloroquine reversed the "immune-cold" environment and improved the anti-tumor effect of both chemotherapeutics and immune checkpoint inhibitors, while it induced strong immune memory and prevented lung metastasis.

Conclusions: Our strategy serves as a promising approach to the rational design of nanodelivery systems for simultaneous active targeting, autophagy inhibition, and chemotherapy that can be combined with immune-checkpoint inhibitors for enhanced breast cancer treatment.

Keywords: Anti-PD-L1 antibody; Autophagy response; Immuno-chemotherapy; Multifunctional nanoparticles.

Fig. 1

Schematic illustration of the combined application of chemotherapy, immunotherapy, and PD-L1 blockade therapy in tumor-bearing mice. CpG, immunopotentiator; CQ, chloroquine, DC, dendritic cell; HS15, Solutol HS15®; OVA, ovalbumin; PEI-OA, polyethyleneimine-oleic acid polymer; PTX, paclitaxel

Fig. 2

Construction and characterization of multifunctional nanoparticles (MNPs). a Chemical structure of the polyethyleneimine-oleic acid (PEI-OA) polymer. b Nanoparticles 1 were prepared by the thin-film hydration method. Encapsulation of an immunopotentiator (CpG) and ovalbumin (OVA) into 1 by electrostatic interactions, followed by surface coating with atezolizumab and chondroitin sulfate generated nanoparticles 3. c Transmission electron micrographs (TEM) of nanoparticles 1–3. Scale bar, 200 nm. d Confocal microscopic images of DiD-N/A, FAM-CpG-N/A, and DiD+FAM-CpG-N/A. Scale bar, 200 nm. e Agarose gel electrophoresis of DiD+FAM-CpG-N/A. f Western blot analysis of DiD-N/A, FITC-OVA-N/A, and DiD+FITC-OVA-N/A. Free FITC-OVA solution was used as control. g Flow cytometry of DiD-N/A, FITC-OVA-N/A, and DiD+FITC-OVA-N/A. h Representative size distribution of nanoparticles 3, as determined by dynamic light scattering. i Size, polydispersity index (PDI), and zeta potential of nanoparticles 1–3. Data are shown as mean ± SD (n = 4). DiD, 4-chlorobenzenesulfonate salt; FAM, fluorescein amidite; FITC, fluorescein; N, nanoparticles; S, solution

Fig. 3

In vitro characterization of multifunctional nanoparticles (MNPs). a Confocal microscopy images of 4T1 cells incubated with various formulations for 2 and 4 h. Scale bar, 100 nm. b, c Number of b DiD-positive and c FITC-positive 4T1 cells after incubation with DiD+FITC-OVA-N/A and DiD+FITC-OVA-S for 2 and 4 h (DiD, 1 μg/mL). Student’s t-test was performed. *P < 0.05. d Cell viability of 4T1 cells after incubation with different formulations for 24 h, as determined by the MTT assay. Blank nanoparticles were used as control. e Apoptosis of 4T1 cells stained with FITC-Annexin V and propidium iodide (PI) after incubation with different formulations for 24 h. f Percentage of cells in the apoptotic or necrotic stage after treatment with different formulations. Data are shown as mean ± SD (n = 3). Cells without any treatment were set as control group. CpG, immunopotentiator; CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; DiD, 4-chlorobenzenesulfonate salt; FAM, fluorescein amidite; FITC, fluorescein; N, nanoparticles, N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution

Fig. 4

Effect of multifunctional nanoparticles on autophagosome formation. a Signals of LC3B puncta detected in 4T1 cells cultured in RPMI-1640 medium with different formulations for 4 h (a 5% glucose solution was used as control), as determined by immunofluorescence assay with an anti-LC3B antibody, followed by confocal microscopy. LC3B-positive puncta were detected in the cytoplasm by fluorescein-conjugated ImmunoPure goat anti-rabbit IgG (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Enlarged boxes highlight LC3B signals. Scale bar: 5 μm. b Number of LC3B-positive puncta per cell quantified from ~20 cells treated with different formulations. The means ± SD are from 3 independent experiments. One-way ANOVA was performed. *P < 0.05; **P < 0.01. (n = 3 independent experiments.) c Transmission electron micrographs of autophagosomes in the cytoplasm of HeLa cells treated with CpG+OVA+PTX-N/A. Scale bar, 500 nm. d Confocal microscopy images of HeLa cells transfected with the autophagy dual fluorescent reporter mCherry-GFP-LC3B and cultured with different formulations in Dulbecco’s modified Eagle medium for 4 h. Scale bar: 5 μm. e Number of autophagosomes and autolysosomes in HeLa cells treated with different formulations (>15 cells/experiment). Co-localized dots were counted. Data are presented as means ± SD. * P < 0.05, ** P<0.01 (n = 3 independent experiments). f Representative Western blots for LC3B-II and SQSTM1. g Relative protein levels of LC3B-II and SQSTM1, normalized to levels of GAPDH. Cells without any treatment were set as the control group. Data are shown as mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01. CpG, immunopotentiator; CQ, chloroquine; N, nanoparticles; N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution

Fig. 5

In vivo targeted delivery of multifunctional nanoparticles (MNPs). a Fluorescence imaging of 4T1 breast tumor-bearing mice at 2, 4, 8, 24, and 48 h post-administration of DiD-loaded MNPs (N) or a solution of free DiD (S). b Nanoparticle distribution in tumors and major organs at 48 h post-injection of DiD-loaded MNPs (N) or a solution of free DiD (S). c Semiquantitation of total radiant efficiency in isolated tumors and major organs at 48 h post-injection of DiD-loaded MNPs (N) or a solution of free DiD (S). Data are shown as mean ± SD (n = 3). Student’s t-test was performed. ^*P < 0.05. d, e Confocal imaging of frozen tumor sections. Tumor cells were stained with d anti-CD44 antibody (green) or e anti-PD-L1 antibody (green). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Red fluorescence (DiD) indicated MNPs. Mice injected with 5% glucose solution were set as control group. CQ, chloroquine; DiD, 4-chlorobenzenesulfonate salt

Fig. 6

In vivo antitumor efficacy of multifunctional nanoparticles in 4T1 breast tumor-bearing Balb/c mice. a Establishment of a subcutaneous 4T1 breast cancer model of Balb/c mice. A 5% glucose solution was used as control. b Change in tumor volume over time after treatment with different formulations. Data are shown as mean ± SD (n = 5). c Weight of excised tumors after the completion of the experiment. Data are shown as mean ± SD (n = 5). d Body weight of 4T1-bearing mice treated with different formulations for up to 15 days. Data are shown as mean ± SD (n = 5). e Survival rates of mice over time after treatment with different formulations. Data are shown as mean ± SD (n = 10). f Tumor sections collected on day 15 after the indicated treatments and visualized by TUNEL labeling. Scale bar, 50 μm. Student’s t-test was performed. ^*P < 0.05 vs control. Mice injected with 5% glucose solution were set as control group. CpG, immunopotentiator; CQ, chloroquine; DAPI, 4′,6-diamidino-2-phenylindole; N, nanoparticles; N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end-labeling

Fig. 7

Autophagy inhibition in the tumor enhances the anticancer activity of MNPs. a LC3B-positive puncta, as detected by immunofluorescence assay with an anti-LC3B antibody, followed by confocal microscopy. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 μm. b Number of LC3B-positive puncta per cell quantified from ~20 cells treated with different formulations. c Representative Western blots for SQSTM1 and LC3B-II. d Relative protein levels of LC3B-II and SQSTM1, normalized to levels of GAPDH. Data are shown as mean ± SD (n = 3). One-way ANOVA was performed. ^*P < 0.05, ^**P < 0.01. Mice injected with 5% glucose solution were set as the control group. CpG, immunopotentiator; CQ, chloroquine; N, nanoparticles; N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution

Fig. 8

CpG+OVA+PTX+CQ-N/A induce antitumor immune responses in vivo. a Levels of CD8+ and CD4+ T cells, as determined by flow cytometry on day 15 after the indicated treatments. b Representative flow cytometric plots of CD8+ and CD4+ T cells in tumors. c, d Immunohistochemistry on tumor biopsies. Brown regions indicate the presence of c CD8+ T cells and d CD4+ T cells. Scale bar, 100 μm. e, f Levels of e CD8+ T cells and f CD4+ T cells in the spleen of tumor-bearing mice, as determined by flow cytometry on day 15 after the indicated treatments. g Representative flow cytometric plots of CD8+ and CD4+ T cells in the spleen. h Immunohistochemical staining of the spleen. Brown regions indicate the presence of CD8+ T cells. Scale bar, 100 μm. i, j Serum levels of i tumor necrosis factor-α (TNF-α) and j interferon-γ (IFN-γ) on day 15 after the indicated treatments. Data are shown as ± SD (n = 5). One-way ANOVA was performed. ^*P < 0.05. CpG, immunopotentiator; CQ, chloroquine; N, nanoparticles; N/A, nanoparticles coated with atezolizumab; OVA, ovalbumin; PTX, paclitaxel; S, solution