Nanoparticles targeting mutant p53 overcome chemoresistance and tumor recurrence in non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) shows high drug resistance and leads to low survival due to the high level of mutated Tumor Protein p53 (TP53). Cisplatin is a first-line treatment option for NSCLC, and the p53 mutation is a major factor in chemoresistance. We demonstrate that cisplatin chemotherapy increases the risk of TP53 mutations, further contributing to cisplatin resistance. Encouragingly, we find that the combination of cisplatin and fluvastatin can alleviate this problem. Therefore, we synthesize Fluplatin, a prodrug consisting of cisplatin and fluvastatin. Then, Fluplatin self-assembles and is further encapsulated with poly-(ethylene glycol)-phosphoethanolamine (PEG-PE), we obtain Fluplatin@PEG-PE nanoparticles (FP NPs). FP NPs can degrade mutant p53 (mutp53) and efficiently trigger endoplasmic reticulum stress (ERS). In this study, we show that FP NPs relieve the inhibition of cisplatin chemotherapy caused by mutp53, exhibiting highly effective tumor suppression and improving the poor NSCLC prognosis.

Fig. 1. Mechanistic validation of a vicious accumulation between cisplatin and p53.

a Kaplan-Meier plot of the correlation between the mutation of p53 and the survival of patients with NSCLC (Log-rank Mantel–Cox test). Cisplatin (Cis), fluvastatin (Flu). b Outline of the assays of cisplatin treatment (low dose, 10 μM; moderate (mod) dose, 25 μM; high dose, 50 μM). Cholesterol (Cho). Heatmap of the number of mutations and RNA-seq analysis (c), ROS levels (d), Cho levels (e), and DNA damage (f) for wtp53-expressing cells treated with cisplatin for 30 days. g High-throughput sequencing before and after fluvastatin sodium (4 μM) treatment in A549 cells. h Genome-wide analysis. The volcano plot depicts the significance and magnitude of difference (Fold Change). The dashed line indicates the threshold of the Fold Change > 2 and adjusted p < 0.05. Some of the cancer related genes are labeled by dark colors. i GO enrichment analysis of differentially expressed genes (DEGs). The advanced bubble chart shows GO enrichment of DEGs in signaling pathways. The x-axis label represents the gene ratio, and the y-axis label represents GO terms. j GSEA analysis. The normalized enrichment scores (NES) and p values are indicated in each plot. k Heatmap analysis of mevalonate pathway genes from RNA-seq data. The color scale indicates the fold change in genes expression. l Schematic illustration of treatment with cisplatin. b, g created with BioRender.com. Data are shown as the mean ± SD; n.s. = no significance. Source data are provided as a Source Data file.

Fig. 2. Design and characterization of FP NPs.

a CI value in different ratios of physical mixture, and the synthesis route of Fluplatin. b Particle size, zeta potential, PDI, and TEM image of the F NPs. Scale bars, 50 nm. c Particle size, PDI, and encapsulation efficiency of nanoparticles formed by different proportions of Fluplatin and PEG–PE (n = 3 independent samples). d Particle size, zeta potential, PDI, and TEM image of the FP NPs. Scale bars, 200 nm. e Release profile of the F NPs (i) and FP NPs (ii) in phosphate solution of 10% plasma at pH 7.4. f Release profile of the F NPs (i) and FP NPs (ii) in phosphate solution of 1% Tween 80 (n = 3 independent samples). g Mechanism for the F NPs (i) and FP NPs (ii) (n = 3 independent samples). h SEM image and EDS element mapping of FP NPs. Scale bars, 3 μm. (n = 3 independent samples) i Confocal images of H1975 cells after treatment with 2 μM Dil@FP NPs (n = 3 independent samples). Scale bars, 20 μm. j Uptake Pt in H1975 cells after treatment with 2 μM different formulations (dosage based on Fluplatin) detected by ICP‒MS (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). k Confocal images of Dil@FP NPs and PEG–PE in H1975 cells after treatment with 2 μM Dil@FP NPs. Scale bars, 5 μm. (n = 3 independent samples) l Uptake efficiency of 2 μM FP NPs treatment for 4 h in H1975 cells in the presence of various endocytosis inhibitors was detected by ICP‒MS (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). m Uptake of Pt in organelles after 8 h treatment with 2 μM different formulations (dosage based on Fluplatin; n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). n Major intracellular distribution of FP NPs. Data are shown as the mean ± SD. Source data are provided as a Source Data file.

Fig. 3. Antitumor activity of FP NPs in vitro.

a Cytotoxicity and IC₅₀ of various formulations as determined by the MTT assay in H1975, A549, and A549/DDP cells after 24 h incubation (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). FI is defined as IC₅₀ (Cis)/IC₅₀ (FP NPs) b Inverted microscopy images of H1975, A549, and A549/DDP cells calcein AM/PI double staining after 6 h treatment with 4 μM different formulations (dosage based on Fluplatin; n = 3 independent samples). Scale bars, 100 μm. c The Apoptosis rate of H1975 cells after incubation with 4 μM different formulations (dosage based on Fluplatin) for 6 h was determined by flow cytometry (FACS) analysis. d The fold rate of caspas3/9 enzymatic activities was detected after 6 h treatment with 4 μM different formulations (dosage based on Fluplatin; n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). After Pifithrin-α and STI571 treatment for 24 h, the cytotoxicity and IC₅₀ of cisplatin treatment in H1299 cells transfected with expression constructs containing wtp53 (e), with vector (f), and expression constructs containing the R273H variant (g) were measured by the MTT method, n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test for (e, f, g). STI571 (STI), pifithrin-α (pif). Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 4. Mechanism of p53-independent antitumor activity of FP NPs in vitro.

a Schematic summary of the p53-independent antitumor mechanism of FP NPs. b Confocal images of Dil and ER-Tracker in H1975 cells treated with 2 μM Dil@FP NPs. Their colocalization determined by Pearson’s correlation coefficient was quantified (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. c Confocal images of Dil and Fluo-4 in H1975 cells treated with 2 μM Dil@FP NPs. Their fluorescence intensity was quantified (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. d Confocal images of Dil and MitoTracker in H1975 cells treated with 2 μM Dil@FP NPs, and their colocalization determined by Pearson’s correlation coefficient was quantified (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. e Confocal images of JC-1 in H1975 cells treated with 4 μM different formulations (dosage based on Fluplatin) for 6 h, and their fluorescence intensity was quantified (j), (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. Western blotting analysis of p-elF2α, elF2α, CHOP, and ATF4 in H1975 cells (f) and A549 cells (g) after treatment with 4 μM of different formulations (dosage based on Fluplatin; n = 3 independent samples). h Western blotting analysis of p-elF2α, elF2α, CHOP, and ATF4 in H1975 cells after treatment with different concentration of FP NPs for 12 h (n = 3 independent samples). i Confocal images of IF staining against γ-H2AX in H1975 cells treated with 4 μM FP NPs for 6 h, and their fluorescence intensity was quantified (k), (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Scale bars, 10 μm. a Created with BioRender.com. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 5. Mechanism of p53-related antitumor activity of FP NPs in vitro.

a Western blotting analysis of p53 in cells after treatment with 4 μM of different formulations (dosage based on Fluplatin) for 12 h. Their grayscale values were quantified (b–e) (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). f Western blotting analysis of p53 in cells after treatment with different concentrations of FP NPs for 12 h. Their grayscale values were quantified (g, h) (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). i Western blotting analysis of p53 in cells after treatment with 4 μM of FP NPs for 12 h. And their grayscale values were quantified (j) (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). k Western blot of cells treated with 4 μM FP NPs or not for 12 h and cultured in medium containing 10 μM MG132 or 10 mM 3-MA. Their grayscale values were quantified (l) (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). Western blot of H1975 (m), H2087 (n), H2342 (o), and A549 (p) cells treated with 2 μM FP NPs or not for 12 h and then treated with 100 μg/mL CHX at the indicated time points. n  =  3 biologically independent samples, relative p53/actin ratios are shown. q Western blotting analysis of total ubiquitination (Ub) of immunoprecipitated (IP) p53 in cells after treatment with 2 μM FP NPs with or without MG132 (n = 3 independent samples). r HMGR inhibition assay under different formulations (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). s The volcano plot shows differential expression of FP NPs treated and untreated H1975 cells. t Schematic summary of the p53-related antitumor mechanism of FP NPs. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 6. Antitumor efficacy of intravenously injected FP NPs in an H1975 tumor-bearing mouse model.

a Schematic illustration of the experimental design. b Images of tumor and tumor weight/volume on day 19 of each group (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). c Tumor volume changes for each mouse in each group over 21 days of treatment (n = 5 mice per group). d The tumor volumes of mice during treatments with each group (n = 5 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). e The body weight of mice during treatments with each group (n = 5 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). Confocal images of ROS (f) and TUNEL (g) in the tumor tissues of mice after treatment with each group (n = 3 independent samples). Scale bars, 100 μm. IHC of cleaved caspase-3 (h) and H&E staining (i) in the tumor tissues after treatment with each group (n = 3 independent samples). Scale bars of cleaved caspase-3, 20 μm; Scale bars of H&E, 100 μm. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 7. Biodistribution and anticancer mechanism of FP NPs in the H1975 tumor-bearing mouse model.

a Ex vivo biodistribution imaging of the main organs in mice bearing H1975 xenografts of the DiR@FP NPs at various time points post injection. Scale bars, 5 mm. b ICP‒MS measurement of Pt accumulation in individual organs at 1, 6, 12, and 24 h after treatment with cisplatin (i) or FP NPs (ii) (n = 3 mice per group). c ICP‒MS measurement of Pt accumulation in the tumor tissues at 1, 6, 12, and 24 h after treatment with cisplatin or FP NPs (n = 3 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). d TC levels in tumor tissues and TC, TG, HDL, LDL levels in serum after treatment with each group (n = 3 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). Confocal images of ATF4 (e) and γ-H2AX (f) in the tumor tissues of mice after treatment with each group. Scale bars, 100 μm. g Western blotting analysis of p53^R273H in tumor tissues after treatment with each group. Their grayscale values were quantified (h) (n = 3 independent samples; one-way ANOVA followed by Tukey’s HSD post hoc test). i IHC of p53 in the tumor tissues after treatment with each group. Scale bars, 20 μm. j Kaplan-Meier survival curve of mice treated with each group over 70 days (n = 8 mice per group; Log-rank Mantel–Cox test). k The body weight of mice during treatments with each group over 70 days (n = 8 mice per group;two-way ANOVA followed by Tukey’s multiple comparisons post test). l IHC of Ki67 in the tumor tissues after treatment with each group. Scale bars, 20 μm. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 8. Antitumor efficacy of intravenously injected FP NPs in H1975-luc and A549-luc orthotopic lung tumors.

a, g Schematic illustration of the experimental design. b, h The progression of orthotopic lung tumors (n = 5 mice per group) in BALB/c nude mice. A representative bioluminescent image from each group is shown. c, i The final anatomical picture and H&E staining of the lungs. Scale bars, 5 mm. d, j Average tumor nodule. Tumor nodules of 2–10 mm³ in volume were counted using harvested lungs from the control and treated groups, and the average number of tumor nodules was determined. Each dot represents a tumor from an individual mouse. The tumor nodules in the lungs are indicated by arrows (one-way ANOVA followed by Tukey’s HSD post hoc test). e, k Quantitative bioluminescence analysis in mice bearing orthotopic lung tumors; P/S = photons/second (two-way ANOVA followed by Tukey’s multiple comparisons post test). f, l Mouse body weight in the orthotopic lung tumor mouse model (two-way ANOVA followed by Tukey’s multiple comparisons post test). Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.

Fig. 9. The tumor recurrence and metastasis inhibition of FP NPs in H1975-luc tumor-bearing mice.

a Schematic illustration of the experimental design. b The tumor volumes of mice during treatments with each group (n = 7 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). c The body weight of mice during treatments with each group (n = 7 mice per group; two-way ANOVA followed by Tukey’s multiple comparisons post test). d Tumor volume changes for each mouse in each group (n = 7 mice per group). e In vivo bioluminescence imaging of tumor-bearing mice receiving various treatments after surgery. Three representative mice in each treatment group are shown. Images of day 0 were taken on the day of surgery. f The final anatomical picture and H&E staining of the lungs (n = 3 independent samples; Scale bars, 5 mm). g IHC of p53, E-cadherin, MMP-2, MMP-9, Vimentin in the primary tumor tissues of cisplatin or FP NPs treatment, and recurrent and metastatic tumor tissues of cisplatin treatment (n = 3 independent samples). Scale bars, 20 μm. h Recurrence tumor weights of different groups on day 42 after surgery (n = 7 mice per group; one-way ANOVA followed by Tukey’s HSD post hoc test). i Kaplan-Meier survival curve of mice treated with each group over 65 days (n = 7 mice per group; Log-rank Mantel–Cox test). j Final weights of the heart, liver, spleen, lungs, and kidneys (n = 7 mice per group;two-tailed unpaired t test). k Heatmap of TNF-α, VEGF, IL-10 and IL-6 expression profiles in serum (n = 3 mice per group). l Indicators of routine blood examination (n = 3 mice per group; two-tailed unpaired t test) of mice. Data are shown as the mean ± SD; n.s. no significance. Source data are provided as a Source Data file.