Chiral polymer modified nanoparticles selectively induce autophagy of cancer cells for tumor ablation
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.
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.
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.
KeywordsNanomaterials Chiral polymer Cytotoxicity Autophagy Tumor ablation
triple-negative breast cancer
cell counting Kit-8
transmission electron microscopy
dynamic light scattering
reactive oxygen species
white blood cells
Breast cancer is the most frequent carcinoma in females and the second common cause of cancer-related mortality in women. Moreover, about 63,410 cases of carcinoma in situ of the female breast will be newly diagnosed in the United States in 2017 . Particularly, approximately 10–20% of breast cancer cells are triple-negative breast cancer (TNBC), which do not express estrogen receptor, epidermal growth factor receptor 2 and progesterone receptor . Until now, TNBC is regarded as an aggressive type of breast cancer with drug resistance, high metastatic and tumor relapse rates [3, 4, 5]. The treatment for TNBC is extremely difficult due to the lack of validated molecular targets, prognostic and therapeutic markers. Even more worse, the current treatment for the TNBC including chemotherapy  and radiation therapy  are severe short and long term side effects due to the short of efficacious selectivity of differentiating between cancerous and normal cells. Therefore, there is an urgent need for development of alternative effective therapies for treatment of TNBC to overcome the drawbacks above.
Autophagy is a catabolic process, and plays critical roles for cytoplasmic quality control including maintaining cellular homeostasis and contributing to cellular defense . Generally, through autophagy, endogenous as well as foreign materials are sequestered in vesicles (e.g., autophagosomes), and finally degraded upon fusion of these autophagic vesicles with lysosomes . Recently, scientists have attempted to manipulate autophagy and inhibit further tumor progress. Compared with present cancer therapy strategies including chemo-, immuno-, gene, photothermal, photodynamic and radiation therapies, the autophagy-based conceptual pilot studies for cancer therapy are of particular-and growing-interest in autophagy-deficient cancers, such as breast, ovarian, and prostate cancers. Therefore, effective control of the autophagy is expected to be used for treatment of TNBC.
The advancement of nanotechnology allows the fabrication of materials at the nanoscale, which provides these materials with the potential ability to modulate/influence cellular systems in unforeseen ways [10, 11, 12]. Compared with the traditional drugs such as growth factors and proteins/peptides, nanomaterials possess some unique properties including excellent photo, thermal and magnetic performance, and advantages including easy to synthesize, lower risk of drug resistance and cheaper [13, 14, 15]. Recently, nanoparticles (NPs), considered as foreign materials for cells, have been reported that could influence autophagy process . However, to the best of our knowledge, few examples have been reported to effectively control autophagy process and ultimately achieve therapeutic potential. Thereby, alternative NPs or surface modifications are urgently required to achieve the above objectives.
Chirality is an important and common phenomenon in living systems. For instance, almost all of amino acids (except glycine) in proteins are “left-handed” (l-), while all sugars in DNA and RNA are “right-handed” (d-) . Our and other previous studies have demonstrated chirality-dependent nature of cellular uptake , cell adhesion , cell differentiation [19, 20], protein adsorption [21, 22] (i.e., amount and affinity) and cytotoxicity  on flat substrates or nanoparticles coated with chiral molecules. More recently, two studies observed that NPs with surface-anchored chiral molecules could modulate the autophagy [24, 25]. However, the conclusions, interestingly, are diametrically opposed, which was possibly due to the observed weak chiral effects on autophagy. To employ the chirality-dependent activation of autophagy in cancer treatment, there are two important issues need to be understand. Firstly, the efficiency of regulation of autophagy for tumor eradication especially in vivo needs to be largely improved. Secondly, the NP coated with chiral molecules should possess the ability to selectively induce autophagy between normal cells and cancer cells.
Herein, to address these challenges, we describe the development of autophagy-inducing gold nanoparticles (AuNPs) coated with chiral poly (acryloyl-l, d and racemic valine) (l-PAV-AuNPs, d-PAV-AuNPs and l/d-PAV-AuNPs) for highly efficient chiral selectivity induction of autophagy in MDA-MB-231 cells (triple-negative breast cancer cells) and eradication of TNBC in vitro and in vivo. 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. In contrast, the PAV-AuNPs exhibit autophagy inactivation in two model normal cells, 3T3 fibroblasts and HBL-100 cells (normal breast epithelial cells), regardless of surface chirality. Moreover, the PAV-AuNPs possess excellent biocompatibility in vitro and in vivo. To the best of our knowledge, we for the first time reported that simultaneously employing of the chirality-dependent and chirality-selectivity activation of autophagy could be used in tumor ablation.
Gold(III) chloride hydrate (HAuCl4) and sodium citrate (C6H5Na3O7·2H2O) were purchased from Sinopharm group Co. Ld. Poly(acryloyl-l-valine) (l-PAV) (Mw 4926 Da, polydispersity 1.24), poly(acryloyl-d-valine) (d-PAV) (Mw 4997 Da, polydispersity 1.15) were synthesized and were characterized according our previous works [17, 23]. High glucose dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were obtained from HyClone (USA). Cell counting Kit-8 (CCK8) assay was purchased from DOJINDO Company (Japan). Acridine Orange (AO), 3-methyladenine (3-MA) and primary antibody (Anti-LC3B) (antibody produced in rabbit, L7543) were obtained from Sigma-Aldrich (USA). Annexin V-FITC/PI Kit was bought from BestBio (Shanghai, China). Cyto-ID Green Kit was purchased from ENZO (USA). 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA), radio immunoprecipitation assay (RIPA) lysis buffer penicillin–streptomycin and secondary antibody (Anti-Rabbit IgG (H + L), A0208) were obtained from Beyotime (China). The Milli-Q water was used throughout the experiments.
Synthesis and characterization of L(D)-PAV-AuNPs
The synthesis of poly(acryloyl-l, d and racemic valine) coated gold nanoparticles (l-PAV-AuNPs, d-PAV-AuNPs, l/d-PAV-AuNPs) with diverse surface chirality can be found in previous studies [17, 23]. Briefly, for the modification of AuNPs with PAV, AuNPs were mixed with excess l-PAV or d-PAV or a mixture of l-PAV and d-PAV with equal quantities. The stock solutions of PAV-AuNPs were obtained by centrifugation at 9000 rpm for 5 min to remove nondispersive aggregates from the suspensions, and dialyzed with odium phosphate solution. The mass concentration (μg/L) of l(d)-PAV-AuNP solution was determined using inductively coupled plasma mass spectrometry (ICP-MS, XSENIES, USA). The nanoparticle (NP) geometry was characterized by transmission electron microscopy (TEM, JEM-1400PLUS). The hydrodynamic diameters of PAV-AuNPs were obtained using dynamic light scattering (DLS) with a high performance particle size analyzer (Zetasizer Nano, Malvern) at room temperature (25 °C) with a fixed detector angle of 173°. The UV–Vis and circular dichroism (CD) spectra were measured with a UV–Vis-NIR spectrophotometer (Shimadzu UV-3600) and a Jasco J-810 spectropolarimeter at room temperature according our previous works , respectively.
In vitro experiments
MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells were obtained from Cell Bank of Chinese Academy of Sciences, Shanghai Branch (Shanghai, China), and routinely cultured in DMEM containing 10% of FBS and 1% penicillin–streptomycin. Cells were cultured at 37 °C with environment of 95% air and 5% CO2.
Cell viability assay
Cell viability was determined using a CCK-8 assay according to the manufacturer’s specification. Briefly, MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells were seeded on a 48-well plate at a density of 2 × 104 cells/cm2 and allowed to attach for 24 h. Then, the culture medium was replaced with 10% FBS/DMEM containing PAV-AuNPs with various Au concentration (5–200 μg/mL), respectively. After being co-cultured for another 24 h, the plates were washed 3 times with phosphate buffered saline (PBS). The CCK-8 reagent was added to each well and incubated for another 2 h, and then the absorbance at 450 nm was measured using a microplate reader (Varioskan Flash, Thermo Scientific, USA).
Cellular uptake of PAV-AuNPs
The amount of PAV-AuNPs internalized by MDA-MB-231 cells was determined by ICP-MS according to our previous reported protocol . The Au amount per 104 cells from ICP-MS analysis was presented as the mean ± standard deviation.
For Annexin V-FITC/propidium iodide (PI) assays, cells were stained and analyzed using the flow cytometry (FCM, NovoCyte, ACEA Biosciences, USA) in line with the manufacturer’s instruction .
The cells were seeded on a 6-well plate at a density of 2 × 105 cells/cm2 to attach for 24 h. On the one hand, the cells were pre-treated with or without 3-MA (5 mmol/L) for 1 h before changing the medium containing PAV-AuNPs with an Au concentration of 100 μg/mL. After being co-cultured with PAV-AuNPs for 24 h, the cell viability was determined using CCK-8 assay. On the other hand, after being incubated with PAV-AuNPs for 24 h, cells were stained using Cyto-ID Green Kit according to the manufacturer’s instruction. Then, the cells were imaged using fluorescence microscope (DM 6000 B, Leica, Germany). For quantitatively study, cells were tested using FCM to assess the level of autophagy.
Reactive oxygen species (ROS) generation
The intracellular generation of ROS was measured with a procedure similar to the apoptosis assay. After being treated with PAV -AuNPs (100 μg/mL), then cells were washed with PBS for three times and loaded with 1 mL of 10 μM DCFH-DA for further 20 min incubation. Next, cells were washed for three times with DMEM without FBS to get rid of the DCFH-DA outside cell membrane. Finally, the cells were measured using FCM (excitation at 488 nm and emission at 525 nm).
Lysosomal membranes assay
To examine the release of AO, from lysosomes into the cytosol, the procedure was performed identically to the protocol of ROS generation. Briefly, after being treated with PAV-AuNPs for 24 h, cells were then incubated with 5 µg/mL of Acridine Orange (AO, Sigma) for additional 15 min at 37 °C. After being washed with PBS three times, the cells were then harvested by trypsinization and measured using FCM (excitation at 488 nm and emission at 525 nm).
Western blotting analysis
MDA-MB-231 cells, 3T3 fibroblasts and HBL-100 cells were seeded in 6-well plates at a density of 1 × 106 cells/mL in the presence of PAV-AuNPs with an Au concentration of 100 μg/mL at 37 °C for 24 h. Following, the cells were treated as described . In the present study, anti-LC3B (1:1000, Sigma, USA) was used for primary antibodies, subsequently with an appropriate secondary antibody (1:5000, Beyotime, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control.
Animal care and use were conducted according to the Third Military Medical University (Army Medical University) for the Care and Use of Laboratory Animals. BALB/C mice and nude mice (6–8 weeks old), ranging from 20 to 25 g, were purchased from BEIJING HFK BIOSCIENCE CO.LTD.
In vivo treatment study
After 7 days, mice were randomly assigned to 3 groups (5 animals per group) upon tumors reached a diameter of 4 to 6 mm  and intratumorally injected with 50 μL of 1 mg/mL l(d)-PAV-AuNPs every other day. For control groups, mice were treated with the same volume of physiological saline. The tumor sizes were measured by a caliper every day and calculated as the volume (V, tumor length × (tumor width)2/2). The data was presented as relative tumor volume which was calculated as V/V0 (V0 is the tumor volume before the treatment).
The in vivo biodistribution of NPs was analyzed by testing the Au content in main organs (liver, kidneys, spleen, heart, and lung). The mice were injected with 0.5 mL of L(D)-PAV-AuNPs (1 mg mL) via the tail vein. After being treated for a certain time (1 or 30 days), organs were collected and washed with PBS buffer and lyophilized for 1 day. Subsequently, the dried tissues were pulverized and dissolved in aqua regia (HCl/HNO3 = 1:3, volume ratio) for 7 days. Then, the Au content was analyzed using ICP-MS.
Transmission electron microscopy (TEM) analysis
After being treated with 100 µg/mL of l(d)-PAV-AuNPs for 24 h, cells were collected, centrifuged, and then fixed with 2.5% (v/v) glutaraldehyde. After fixation for 2 h at 4 °C, the samples were washed with PBS three times. Then, the samples were fixed with 1% perosmic oxide, and dehydrated in an alcohol series, embedded, and sliced with the thickness between 50 and 70 nm. Finally, the images were obtained by using the TEM analysis (JEM-1400PLUS).
In vivo toxicity assay
After being injected with 0.5 mL of l(d)-PAV-AuNPs, the mice were sacrificed at 1 or 30 days. Afterwards, main organs (liver, kidneys, spleen, heart, and lung) were gathered, washed with PBS, and immobilized in 4% paraformaldehyde overnight. Then, the samples were embedded with paraffin, sliced into 4 μm, and stained with hematoxylin and eosin (H&E). Simultaneously, blood samples were subjected for the measurement of hematology and biochemistry. The organ (Spleen) index defined in our research as the weight percentage of the spleen to the body was calculated.
The experimental data are expressed as mean ± standard deviation, and the significant difference between groups was analyzed by using one-way analysis of variance (ANOVA) (for two groups) and two-way ANOVA (for more than two groups) in the Origin software. The statistical significance was set as p < 0.05 and p < 0.01, respectively.
Characterization of chiral PAV-modified gold nanoparticles
Diameter (TEM, nm)
Diameter (DLS, Z-average, nm)
Zeta potential (mV)
15.1 ± 0.6
20.2 ± 1.7
23.7 ± 2.3
− 29.1 ± 1.9
− 8.8 ± 3.6
15.2 ± 0.2
20.3 ± 1.4
23.8 ± 3.6
− 29.2 ± 3.2
− 8.9 ± 3.2
15.1 ± 0.7
20.3 ± 0.8
23.8 ± 1,1
− 29.1 ± 2.4
− 8.9 ± 2.2
These caused us keen interest how the chirality elicited the nanotoxicity leading to MDA-MB-231 cells death. As we all know, the process of cell death is very complex, and during its progress, damaged cells typically present various markers representative of different cell death pathways, mainly including apoptosis and autophagy . Apoptosis is an intrinsic self-killing process that is necessary for maintaining tissue homeostasis. Autophagy is a cytoprotective mechanism that responds to various stresses conditions such as starvation, radiation, hypoxia and chemotherapeutic drugs to adapt to environmental changes . In several studies, NPs have been found to be capable of inducing cellular responses of autophagy and apoptosis [35, 36, 37], so we further systematically explore the underlying mechanism of PAV-AuNPs-induced nanotoxicity.
Apoptosis is commonly identified as programmed cell death-1, while an autophagic cell death is generally deemed as programmed cell death-2 . Thus, apoptosis of PAV-AuNPs treated cells was measured firstly by flow cytometry (FCM). As shown in Fig. 2b and Additional file 1: Figure S2, relative to the untreated control group, only few apoptotic cells (early and late apoptosis) were detected in MDA-MB-231 cells, 3T3 fibroblasts or HBL-100 cells with PAV-AuNPs treatment, and the activation of apoptosis was not chirality-independent. In short, it was deduced that apoptosis might not be the principal reason giving rise to the nanotoxicity of PAV-AuNPs in MDA-MB-231 cancer cells. Analogously, Wu et al. reported that cytotoxicity induced by NPs was through autophagy rather than apoptosis . Therefore, we speculate that the autophagy may play a major role in the process of PAV-AuNPs-induced nanotoxicity in MDA-MB-231 cancer cells.
Oxidative stress is believed to be one of the most important mechanisms of nanotoxicity, and the levels of intracellular ROS serve as reliable indicators of oxidative stress, which is essential for the induction of autophagy . Therefore, the ROS levels in MDA-MB-231 cells treated with PAV-AuNPs were evaluated. Compared with the untreated control, there is a remarkable increase of ROS generation in MDA-MB-231 cells treated with PAV-AuNPs. Moreover, the level of ROS generation was higher in cells treated with d-PAV-AuNPs than in ones treated with l-PAV-AuNPs (Fig. 4b). It was observed that the interaction of NPs with cell surface receptors could result in activating of intracellular signaling cascades that induce formation of ROS . Hence, we speculated that the surface chirality-variant ROS generation was largely due to the chirality-dependent cellular uptake as well as the specific interaction between receptors and biomolecules in cells and chiral molecules. It was known that ROS could be attenuated by pretreatment with antioxidant agents such as N-acetyl-cysteine (NAC). To further evaluate whether the autophagy was caused by the overproduction of ROS, the cancer cells were pretreated with 5 mM of NAC. As shown in Fig. 4b, the level of ROS in the cells pretreated with NAC was largely attenuated. Subsequently, the degree of transforming of LC3 I to LC3 II was examined, which also be reduced by the pretreatment of NAC (Fig. 4c). Overall, these results above strongly supported that the autophagy induced by PAV-AuNPs in cancer cells was largely attributed to the ROS overproduction.
Considering the differential biological effects of NPs could also be linked to their differential cellular uptake [13, 24, 47], quantitative measurements of internalized PAV-AuNPs were carried out by examining the element of Au content using the ICP-MS. The internalized amount of d-PAV-AuNPs was significantly larger than that of l-PAV-AuNPs in MDA-MB-231 cells (Fig. 4d), which is correlated well with our previous study that used the HepG2 cells due to the chirality-dependent interaction with the feature of cell membrane . In contrast, internalized amount of PAV-AuNPs in 3T3 fibroblasts and HBL-100 cells was significantly smaller than that of PAV-AuNPs in MDA-MB-231 cells, and exhibited chirality-independent cellular uptake (Fig. 4d). Our previous work also reported that the level of ROS produced was associated with the amount of cellular uptake in lung adenocarcinoma A549 cells . Therefore, the observed differential effects of surface chirality at the nanoscale on the autophagy of cancer cells, and the selectivity autophagy activation between cancer cells and normal cells are dependent to some extent, if not entirely, on the differential cellular uptake.
To determine whether PAV-AuNPs could induce autophagy in vivo, an immunohistochemical method was used to detect the expression of LC3-II in tumor tissues . Compared with the untreated control group, there was stronger immunofluorescence of LC3-II in the PAV-AuNPs treated group. In addition, the d-PAV-AuNPs treated group presented more immunofluorescence of LC3-II than l-PAV-AuNPs treated one (Fig. 5e). These results suggested that the in vivo activation of autophagy is chirality-dependent, with d-PAV-AuNPs are more effectively inducing autophagy in vivo. Overall, PAV-AuNPs with different chiral forms have shown the possibility of autophagy induction, which can be used as an effective regulator for tumor eradication in vivo.
The central point of our work is that the chiral PAV-AuNPs could distinguish the normal cells and cancer cells, and show a chirality-dependent anticancer effects. Chirality, as one of the most distinctive biochemical signatures of life, has great influence on many biological events, for instance, the maintenance of normal functions of living cells [49, 50]. Pioneering works have revealed that cells can sense surface chiral signals and show differential interactions with enantiomorphous surfaces [18, 51]. Among these chiral molecules, amino acids have been widely used for studying the interaction between cells and chiral surface due to their versatility and biocompatibility . For example, Gammon et al. investigated sequence-specific cell uptake characteristics of Tat basic domain and related permeation peptides with an emphasis on residue chirality, length, and modified side chains . It was observed that the length, sequence and types of chelation domain impacted peptide uptake into cells. More importantly, with all the other factors are same, once the chirality of the peptide sequence was changed from l to d, uptake values increased up to 13-fold. Furthermore, the eight essential amino acids showed stronger chirality-dependent cell uptake effect and would appear to optimize the permeation sequence for both Tat basic domain and poly-Arg peptides . Valine is one of the eight essential amino acids for the human body, and plays essential roles in a wide variety of physiological processes [53, 54, 55]. Sun et al. reported stereo selective cell behaviors on a pair of chiral brush films, which were composed of a chiral unit of acryloyl derivatives of l(d)-valine (AV) . A fibroblast-like cell line—COS-7 cells were cultured on l(d)-valine based films (l(d)-PAV). It was found that the adhesion, spreading, growth and assembly processes of cells were significantly different on two films. The cells preferred to connect to each other and spread on the l-PAV film as interlinked clusters, whereas those on the d-PAV film tended to remain isolated stacks with lower spreading extent . More recently, they further studied the influence of the molecular structure of the chiral units on this chiral effect by substituting the l(d)-Val units with other two kinds of aliphatic amino acids, l(d)-alanine (Ala), and l(d)-leucine (Leu) . The only difference among these three amino acids is the size of the hydrophobic side groups. It was observed that the smallest Ala units led to the weakest chiral effect in which differential cell behaviors with statistical significance could only be observed after long periods of cell incubation. However, for polymer films based on the Leu units, a more distinct chiral effect was found compared with those based on Val units (as reported above) . The above results revealed that the amino type could influence the chiral effects on biological systems. Our previous works also prepared chiral surface based on the valine and studied stereoselective interactions between cells and chiral interface materials [13, 20, 23, 56]. We observed that cancer cells (e.g., A549 cells and HepG2 cells) prefer to internalize the d-PAV-AuNPs through the possible preferable interaction between the l-phospholipid-based cell membrane and the d-enantiomers , while the bone marrow mesenchymal stem cells uptake more l-PAV-AuNPs than the d-PAV coated ones . Such effects were attributed to the different cell types. More interestingly, the PAV-AuNPs could discriminate model tumor cells (MDA-MB-231 cells) from normal cells (3T3 fibroblasts and HBL-100 cells). Upon contact with the complex biological systems, the proteins will be progressively and selectively adsorbed on the NPs surface unless they have been designed to do otherwise, which was defined as “protein corona” [55, 57, 58, 59]. Increasing evidence suggest that the protein corona define the biological interactions of NPs [60, 61]. Thus, we make assumptions that the protein corona in PAV-AuNPs could selectively interact with cancer cells from normal cells and exhibit chirality-selective. For example, transferrin receptor is an ubiquitous human cell surface glycoprotein related to cell proliferation , and it is expressed more abundantly in malignant tissues than in normal tissues . The affinity of transferrin to the surface of cancer cells is 10–100 times greater than that of normal cells . Furthermore, the abundance of transferrin receptor in malignant tissues of human breast has been demonstrated . Li et al. reported that the transferrin–transferrin receptor-mediated cellular uptake of gold nanoparticles is six times of that in the absence of this interaction . More recently, Wang et al. showed that l(d)-penicillamine coated gold nanoparticles could strongly interact with the transferrin and realize the tumor targeting . For d-penicillamine modified AuNPs, the binding transferrin-related residues are positioned outward, which facilitates the interaction between transferrin and transferrin-receptor, while for the l-penicillamine coated AuNPs, they face inward and hinder the interaction. Meanwhile, transferrin undergoes different degrees of changes in its secondary structures after its interaction with different chiral surfaces of AuNPs . Moreover, our previous work also observed that the albumin showed secondary structures change after being adsorbed on l(d)-PAV-AuNPs . As expected, some proteins from serum such as transferrin adsorbed on PAV-AuNPs could strongly and selectively interact with breast cancer cells. Moreover, the proteins adsorbed on d-PAV-AuNPs had a stronger binding to cancer cell receptors than on l-PAV-AuNPs. The chiral molecule interact with other biomolecules (e.g., receptor or proteins) via hydrogen bonding (H-bonding), hydrophobic interaction, electrostatic attraction, and dipole–dipole interaction, etc. . Thus, the molecular functional groups, charges and hydrophobic units in chiral molecules may affect their chiral effects.
In addition, another insight was that NP could induce cancer cellular injury through autophagy pathway, which exhibited chirality-dependent. The possible mechanism of the chirality-dependent activation autophagy in cancer cells was attributed to the larger intracellular accumulation of the d-PAV-AuNPs, which leads to higher ROS production. According to our results (Fig. 3c), we speculate that l(d)-PAV-AuNPs induced cancer cell autophagy may occur through the phosphatidylinositol 3-kinase (PI3K) signaling pathway (an important pathway in autophagy regulation) , because it could be efficiently inhibited by 3-MA, which is considered to be a specific inhibitor of PI3K activity. Taken together, it is probable that the ROS induced by the l(d)-PAV-AuNPs can trigger autophagy both directly and indirectly via inhibition of the classical autophagy signaling pathway, phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR).
Finally, our anticancer depot especially the d-PAV-AuNPs could be used as an efficient anticancer agent for future clinic application, which was demonstrated by the largely tumor suppression and negligible toxicity in vitro and in vivo.
In summary, this work provides new insights into NP induced cancer cellular injury by demonstrating differential autophagy associated with the chirality of PAV coating. The cytotoxicity induced by PAV-AuNPs in model breast cancer cells (MDA-MB-231 cells) was mainly through autophagy pathway, which was chirality-dependent, with d-PAV-AuNPs exhibiting efficient autophagy-inducing activity in vitro and in vivo. In contrast, the PAV-AuNPs presented autophagy inactivation in the model normal cells (3T3 fibroblasts and HBL-100 cells), regardless of the surface chirality. Compared with l-PAV-AuNPs, d-PAV-AuNPs exhibited excellent internalization, a crucial first step in obtaining the intracellular accumulation of d-PAV-AuNPs. The larger intracellular accumulation of the d-PAV-AuNPs can lead to higher ROS production and more serious lysosome function interfered, which dramatically, if not entirely, gave rise to the occurrence of chirality-dependent autophagy. Additionally, what’s exciting is that stereospecific d-PAV-AuNPs could act as a high efficient artificial autophagy-inducing agent for tumor autophagy induction, which was demonstrated by the largely tumor suppression and negligible toxicity in vitro and in vivo. Identification of this chirality-dependent autophagy of NPs provides an important insight that chiral effect can act as a novel strategy for designing bio-interface materials for cancer cell therapy especially TNBC.
LY and FZ carried out experiments, analyzed data and wrote the paper. JJ and JD designed the study and supervised the project. YJY, and CY drew the figures and assisted in the synthesis and characterizations of the l(d)-PAV-AuNPs. YJY assisted to the in vitro experiments. XWQ participated to animal experiments. All authors read and approved the final manuscript.
We thank the kind assistance from Mr. Guangchao Qing (Medical Research Center, Southwest Hospital, Third Military Medical University, China) for the in vivo experiments.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional file.
Consent for publication
Ethics approval and consent to participate
All the animal experiments were conducted in accordance with the guidelines and the ethical standards of the Institutional Animal Care and Use Committee of the Third Military Medical University (Army Medical University).
This work is supported by the Science and Technology Innovation Plan of Southwest Hospital (SWH2016JCYB-04, SWH2016YSCXZD-10), Third Military Medical University Foundation (2016XPY12) and the Natural Science Foundation of China (51703243, 30700814).
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