Chiral polymer modified nanoparticles selectively induce autophagy of cancer cells for tumor ablation

Abstract

Background: Autophagy regulation through exogenous materials has aroused intensive attention to develop treatment protocols according to diverse human diseases. However, to the best of our knowledge, few examples have been reported to selectively control autophagy process and ultimately achieve efficient therapeutic potential.

Results: In this study, monolayers of poly (acryloyl-L, D and racemic valine) (L-PAV-AuNPs, D-PAV-AuNPs and L/D-PAV-AuNPs) chiral molecules were anchored on the surfaces of gold nanoparticles (PAV-AuNPs), and the subsequent chirality-selective effects on autophagy activation were thoroughly studied. The cytotoxicity induced by PAV-AuNPs towards MDA-MB-231 cells (Breast cancer cells) was achieved mainly through autophagy and showed chirality-dependent, with D-PAV-AuNPs exhibiting high autophagy-inducing activity in vitro and in vivo. In contrast, the PAV-AuNPs exhibited autophagy inactivation for normal cells, e.g., 3T3 fibroblasts and HBL-100 cells. The chirality-selective autophagy activation effect in MDA-MB-231 cells was likely attributed to the chirality-variant ROS generation, cellular uptake and their continuous autophagy stimulus. Furthermore, the intratumoral injection of D-PAV-AuNPs could largely suppress the tumor growth but exhibit negligible toxicity in vivo.

Conclusions: As the first exploration on stereospecific NPs for autophagy induction, this work not only substantiates that chiral polymer coated NPs can selective induce autophagy-specific in cancer cells and achieve a high tumor eradication efficiency in vivo, but also opens up a new direction in discovering unprecedented stereospecific nanoagents for autophagy-associated tumor treatment.

Keywords: Autophagy; Chiral polymer; Cytotoxicity; Nanomaterials; Tumor ablation.

Fig. 1

Characterizations of l-PAV-AuNPs, d-PAV-AuNPs l/d-PAV-AuNPs. a Schematic illustration of the synthesis of poly (acryloyl-l, d and racemic valine) (l-PAV-AuNPs, d-PAV-AuNPs l/d-PAV-AuNPs) and their conjugation to the AuNP surface. b Preventative TEM images of PAV-AuNPs. c UV–Vis-NIR spectra of PAV-AuNPs in PBS. d The curves of the size distribution by in intensity of PAV-AuNPs. e Circular dichroism (CD) spectra of PAV-AuNPs

Fig. 2

The toxicity study of l-PAV-AuNPs, d-PAV-AuNPs. a Dose- and chirality-dependent cytotoxicity of PAV-AuNPs in MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells, respectively. b Apoptosis rates of the MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells treated with PAV-AuNPs. FCM analysis was tested via Annexin V-FITC and PI as probes. c Expression levels of LC3 in MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells with PAV-AuNPs treatment. GAPDH was used as a loading control

Fig. 3

Activation of autophagy induced by l-PAV-AuNPs and d-PAV-AuNPs, respectively. a Cyto-ID Green dye labeled dots formed in MDA-MB-231 cells incubated with l- and d-PAV-AuNPs. Scale bar is 50 µm. b TEM images of the MDA-MB-231 cells incubated with PAV-AuNPs. The untreated cells were considered as control. The PAV-AuNPs form distinct, dark aggregates in the cells (green arrows). The PAV-AuNPs were localized in lysosomes (blue arrows) and autophagosomes (yellow long arrows), consisting of double layered membranes containing cellular debris. c The viability of MDA-MB-231 cells was measured after the cells treated with pre-treatment with or without 3-MA (5 mM) for 1 h. * and ** present p < 0.05 and p < 0.01, respectively

Fig. 4

The possible chirality-dependent mechanisms of the PAV-AuNPs on autophagy-inducing activity. a l- and d-PAV-AuNPs reduced lysosome stability in MDA-MB-231 cells. b Generation of ROS in MDA-MB-231 cells exposed to l- or d-PAV-AuNPs with an Au concentration of 100 µg/mL in the presence or absence of antioxidants (NAC), respectively. ROS up is regarded as the positive control. The mean fluorescence intensity (MFI) is quantified using FCM. c The level of LC3-II in MDA-MB-231 and treated without (Control) or with d-PAV-AuNPs in the presence or absence of NAC for 24 h is determined by western-blotting, GAPDH served as loading control. d Cellular uptake of l(d)-PAV-AuNPs. Quantification of the Au amount per 104 cells is presented as the mean ± standard deviation (n = 4). * and ** present p < 0.05 and p < 0.01, respectively

Fig. 5

Antitumor efficacy on the tumor-bearing nude mice model. a Tumor tissues. b Relative tumor growth curve. c Pathological features of tumor tissues in nude mice treated with physiological saline or PAV-AuNPs. Tumor issues were H&E stained. d Changes in body weight during the course of treatment. e Immunohistochemical representative images for cryosections of MDA-MB-231 tumor tissues after intratumoral injection with physiological saline, l- and d-PAV-AuNPs, respectively. Stained with LC3-II; the entire nucleus was stained with DAPI. * and ** present p < 0.05 and p < 0.01, respectively

Fig. 6

The blood chemistry analysis after injection of PAV-AuNPs in vivo. a Blood analysis of healthy Balb/c mice intravenous injected with PBS (PH = 7.4), l-PAV-AuNPs or d-PAV-AuNPs sacrificed at day(s) 1 and 30, including a red blood cells, b white blood cells, c platelet(PLT), d hemoglobin, e blood urea nitrogen (BUN), f alanine aminotransferase (ALT), g aspartate aminotransferase (AST), h organ index

Fig. 7

In vivo toxicity of PAV-AuNPs evaluation. H&E staining in main organs (liver, kidneys, spleen, heart, and lung) of the mice sacrificed at 1 (a) or 30 days (b) after intravenous injection of PBS or PAV-AuNPs. Scale bar stands for 100 μm

Fig. 8

Schematic diagram of the mechanism of the chirality-dependent activation autophagy and their application in tumor therapy