Synthesis and characterization of PEG-FA-Ni NTs
We have clarified detailed formation mechanism of this FA-Ni NTs in our published works18 that is the intricate coordination of FA, Ni2+ and hydrazine. Meanwhile, there were still some free and active carboxyl groups site from FA on the surface of FA-Ni NTs, which provided the binding position for the further modification of the naked FA-Ni NTs (Scheme 1).
The TEM image showed that the FA-Ni NTs tend to be heavily agglomerated, although single-tube structures can still be seen at the edge (Fig. 1A). After dendritic PEG modification, the agglomeration of the nanotubes was reduced and the dispersibility was greatly improved. In addition, due to the large space occupancy of dendritic PEG, the surface of the modified nanotubes was obviously wrapped by dendritic PEG. Individual tubular structures were relatively ambiguous, but tubular contours were still visible at the edge indicated by red arrows (Fig. 1B).
Compared with linear PEG, dendritic PEG has been widely used as versatile excipients in drug research and production owing to its outstanding advantages on maintaining drug activity, hydrophilicity, numerous active site and spatial effect. Hence, we propose that the amine groups could covalently linked to the carboxyl groups suspended on the outer wall of FA-Ni NTs, which will substantially improve the water solubility of this type of nanotubes. The sedimentation experiment result shows that the better dispersion of PEG-FA-Ni NTs than that of FA-Ni NTs (Figs. 2A). The Ni 2p XPS spectrum of PEG-FA-Ni NTs is shown in Fig. 2B. The Ni 2p3/2 XPS spectrum presented an asymmetric doublet peak, with the main peak located at 854.43 eV, a minor peak at 855.72 eV, and a satellite peak showing broad features at higher binding energy of 861 eV. The curve fitting by G/L peak revealed that satisfactory fitting of peaks required at least five peaks of 2p3/2 portion. The main peak could be assigned to the nickel ion with divalent oxidation state. The satellite peak generally arises due to multiple splitting in the energy levels of the transition metals, and is typically used for confirming the existence of divalent nickel. TGA was used to determine the amount of coupled dendritic PEG (Fig. 2C). The initial weight loss in the TGA curve up to 300°C is assigned to the loss of free and coordinated water molecules present in the sample. The further changes weight in the temperature range of 300–400°C in the TGA curve is due to the loss of dendritic PEG27–28. By comparing the weight loss rates of PEG-FA-Ni NTs and FA-Ni NTs in the range of 300–400°C, we calculate the coupling rate of dendritic PEG was 9.2%. The zeta-potential values of FA-Ni NTs and PEG-FA-Ni NTs were measured (Fig. 2D). Due to the presence of some carboxyl groups on the surface of the nanotube that is not involved in coordination, the zeta potential of FA-Ni NTs exhibits a negative charge, which is about − 12.86 mV. The resulting zeta potential of PEG-FA-Ni NTs modified with dendritic PEG reversed and rose to + 0.97 mV, which was mainly due to the positive charge displayed by amino groups at the end of the dendritic PEG. Thus, the exposure of positive charges is conducive to enhancing cellular uptake of PEG-FA-Ni NTs. The results showed that the FA-Ni NTs have been modified with hydrophilic dendritic PEG, which could further reduce agglomerate, improve dispersity.
In vitro antitumor activity of PEG-FA-Ni NTs
Based on previous in vitro cell viability experiments that FA-Ni NTs have good antitumor activity against FR overexpressed HeLa cells and FR low-expressed A549 cells even without loading any positive drugs18, here we further assess the antitumor activity of PEG-FA-Ni NTs. HeLa cells, HepG2 cells, KB cells, MCF-7 cells, A549 cells and L-O2 cells lines were treated with PEG-FA-Ni NTs for 72 h. Cells incubated with cisplatin or DOX were used as the positive controls and cell viability was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) assay. The cytotoxicity experimental results showed that PEG-FA-Ni NTs have good antitumor activity against HeLa cells, HepG2 cells, KB cells, MCF-7 cells and A549 cells compared to the positive drugs DOX and cisplatin. Among them, the IC50 values of HeLa cells, HepG2 cells, and A549 cells are depending on their FA expression levels. The cytotoxicity of PEG-FA-Ni NTs against LO2 was significantly less than that of cisplatin (5.32 µg/mL) and DOX (3.40 µg/mL), indicating that this type of nanotubes is much safer and indeed valuable for further application (Table 1). In general, the PEGylation of FA-Ni NTs, PEG-FA-Ni NTs, can still maintain excellent in vitro antitumor activity.
Table 1
The IC50 values of HeLa, HepG2, KB, MCF-7, A549 and L-O2 after co-incubation with PEG-FA-Ni NTs, DOX and cisplatin, respectively, for 72 h.
IC50 (µg/mL) |
| HeLa | HepG2 | KB | MCF-7 | A549 | L-O2 |
PEG-FA-Ni NTs | 2.91 | 2.99 | 3.21 | 5.13 | 6.56 | 20.30 |
DOX | 1.16 | 3.41 | 1.66 | 2.82 | 1.43 | 3.40 |
Cisplatin | 1.11 | 2.37 | 1.78 | 1.89 | 0.65 | 5.32 |
Pharmacological activity test of folate nanosheets and free nickel ions on different cells
In order to investigate the anti-tumor mechanism of this kind of folate-nickel complex nanotubes, under the same conditions, we tested the pharmacological activities of free Ni2+ and folate nanosheets formed by solvothermal method of pure folic acid without coordinating with Ni2+ by MTT method. The concentration of Ni2+ in each group was set according to the content of Ni2+ in FA-Ni NTs. The results showed that the inhibition rates of folate nanosheets and Ni2+ on different tumor cells and normal hepatocytes were no more than 20%, which was consistent with the known viewpoint that isolated folate and nickel ions have no obvious antitumor activity (Fig. 3). Hence, we conclude that folate molecules and nickel ions exhibit certain tumor-inhibiting activity in the formation of folate-nickel complex nanotubes, and their nano-sized morphology may be critical to the anti-tumor activity.
Uptake of PEG-FA-Ni NTs by tumor cells
To demonstrate that the PEG-FA-Ni NTs were internalized to the cell mediated by the FR, we chose the Hela cell (a kind of human cervical carcinoma cell with a high growth rate and over-expression of FR) and HepG2 cell (a human liver carcinoma cell line with a high-expression of FR), with FR low-expression A549 cells (adenocarcinomic human alveolar basal epithelial cells with a low-expression of FR) served as control29. Fluorescein isothiocyanate (FITC) was used as a marker to track PEG-FA-Ni NTs. As shown in Fig. 4, after incubation with FITC-labelled PEG-FA-Ni NTs, the fluorescence brightness was sequentially Hela > HepG2 > A549, which was consistent with their corresponding FR expression level. The results suggested that PEG-FA-Ni NTs could easily bind to the tumor cells overexpressing folate receptor, indicating their targeting delivery capacity. Meanwhile, PEG-FA-Ni NTs are not inclined to bind to A549 cells with low expression of folate receptor, resulting inefficiently uptake by the A549 cells.
The endocytosis process and the distribution of PEG-FA-Ni NTs in cytoplasm could be observed by bio-TEM imaging (Fig. 5). Firstly, part of PEG-FA-Ni NTs could be found near the cell membrane, the morphology is consistent with TEM images of PEG-FA-Ni NTs (Fig. 5A-1). Then, the PEG-FA-Ni NTs are phagocytized by the cell and trapped into the intracellular endosomes (Figs. 5A-2), and then the process of escaping from the endosomes into the cytoplasm is observed in Fig. 5B. Finally, the PEG-FA-Ni NTs are degraded and discharged outside the cell (Fig. 5C). The enlarged image shows the morphology change of nanotubes, implies the PEG-FA-Ni NTs are gradually degraded. In addition, it is worth mentioning that after the PEG-FA-Ni NTs incubation, the nuclear membrane of the HeLa cells becomes shrunk and the nucleoli basically disappear (Figs. 5D). This indicates that the nanotubes induced cell death after entering the cell.
Ex vivo imaging of PEG-FA-Ni NTs in normal mice
The biodistribution of PEG-FA-Ni NTs was estimated from the ex vivo images of the organs retrieved after the systemic administration of Cy5.5-labeled PEG-FA-Ni NTs into the normal mice. Mice were injected with the Cy5.5-labeled PEG-FA-Ni NTs via tail vein, to circulate, and then sacrificed at pre-determined time points and major organs were harvested for imaging. Organ images were obtained as soon as feasible after sacrifice to minimize degradation of the Cy5.5-labeled PEG-FA-Ni NTs. The major organs distribution of PEG-FA-Ni NTs after different time points post-injection are shown in Figs. 6A, 6B. At 1 h, the fluorescence intensity of the kidney is the strongest, followed by the liver and lung. When the time was extended to 48 h, the fluorescence intensity of the liver and lungs almost disappeared, and the fluorescence intensity of the kidneys also gradually decreased. This showed that after blood circulation, most of the nanomaterials may be cleared by the kidneys, and there is almost no accumulation in other major organs. In addition, it was found that PEG-FA-Ni NTs displayed significantly stronger fluorescence intensities than FA-Ni NTs (Fig S1). These results may suggest that PEGylation of nanoparticles reduces major organs uptake and allows prolonged circulation in the bloodstream.
Study on the damage mode of tumor cells by PEG-FA-Ni NTs
According to the literature, most of the inorganic metal anti-tumor drugs mainly achieve the inhibition by inducing apoptosis of tumor cells30–31. Therefore, in order to explore whether folate-nickel complex nanotubes can cause apoptosis, we incubated PEG-FA-Ni NTs and FA-Ni NTs with HeLa cells for 48 h, then double stained with PI and Annexin V-FITC, and the percentages of early and late apoptosis population in treated and untreated cells were calculated from the flow cytometric data. The results showed that almost no apoptosis was observed in HeLa cells of control group (Fig. 7A). However, after PEG-FA-Ni NTs and FA-Ni NTs treatment, the early and late apoptosis rate is 14.4% and 16.8%, 10.5% and 17.2%, respectively. The results of flow cytometric analysis clearly establish the efficient induction of apoptotic in HeLa cells by PEG-FA-Ni NTs.
In view of the above results, we went on investigating whether PEG-FA-Ni NTs cause apoptosis by damaging DNA. As we all know, DNA damage usually results in the cell cycle arrest and then affects cell division. We performed a qualitative study on the expression of intracellular DNA damage marker γ-H2AX after PEG-FA-Ni NTs and FA-Ni NTs were incubated with HeLa by immunofluorescence experiments, and cisplatin was set as a positive control (Fig. 7B). The experimental results showed that compared with the blank group, the red fluorescence signal of the DNA damage marker γ-H2AX was significantly enhanced after HeLa cells were treated with PEG-FA-Ni NTs, indicating the expression level of γ-H2AX was significantly increased, while the fluorescence intensity of PEG-FA-Ni NTs was comparable to that of the positive drug cisplatin group. Meanwhile, the red fluorescence intensity of the PEG-FA-Ni NTs group was slightly higher than that of the FA-Ni NTs group, which further proved its superior antitumor activity. Actually, some works have proved that PEGylation can protect nanomaterials from degradation by lysosomes, promote nanomaterials to enter the nucleus, and exacerbate DNA damage and cell cycle arrest32–35. The above results can be inferred that PEG-FA-Ni NTs induce apoptosis by damaging DNA.
Next, we studied the cell cycle arrest induced by PEG-FA-Ni NTs. After 48 h of PEG-FA-Ni NTs treatment, double-stranded DNA was stained with PI, and then the intracellular DNA content was determined by flow cytometry. According to the fluorescence intensity of PI, the content of DNA and the distribution of cells in different cell cycles determine whether the cell cycle is stagnant. The cell cycle test results were analyzed by Modfit software and it was found that the cell cycle of the drug group changed significantly. Quantitative analysis on the cell cycle distribution results showed that the proportion of G0/G1 phase cells in the blank control group, FA-Ni NTs group and PEG-FA-Ni NTs group was 67.92%, 46.28% and 38.73%, the proportion of cells in S phase was 27.44%, 45.21% and 49.73%, and the proportion of cells in G2/M phase were 4.64%, 8.52% and 11.54%, respectively. Compared with the blank control group, the proportion of G0/G1 phase cells in the FA-Ni NTs group and the PEG-FA-Ni NTs group decreased significantly, and the proportion of cells in the S phase and G2/M phase increased (Fig. 7C). It indicated that FA-Ni NTs could arrest HeLa cells in the S phase or G2/M phase, and the arrest of cell cycle caused by PEG-FA-Ni NTs was more obvious. Therefore, we speculate that the folate-nickel complex nanotubes may achieve optimal anti-tumor effects by damaging DNA, which causes cell cycle arrest and ultimately leading to apoptosis.
In vivo Tumor Inhibition of PEG-FA-Ni NTs
Overcoming biological barriers in turn is the basis for effective tumor therapy in vivo. We have already demonstrated that PEG-FA-Ni NTs have good biocompatibility and inhibitory activity against a variety of tumor cells in vitro. To further verify the antitumor effects of the nanotubes in vivo, the HeLa, HepG2 and A549 tumors xenograft model of Balb/c mice were established. When the mean tumor volume reached to 100 mm3, the tumor-bearing mice were treated with PBS, free cisplatin or DOX, FA-Ni NTs and PEG-FA-Ni NTs. After 15 days of treatments, mice were sacrificed and tumors were excised. The digital photos of excised tumors were presented in Fig. 8A, Figure S1A and S1B), where PEG-FA-Ni NTs exhibited the strongest tumor inhibition effect among all groups and the anti-tumor effect exceeded that of the positive drug cisplatin and DOX, suggesting that PEG-FA-Ni NTs had an excellent antitumor growth activity, which was attributed to good dispersion, better targeting ability, and excellent cellular uptake. Figure 8B, Figure S1C and S1D showed the change in tumor volume during the experiment. A similar tumor inhibition effect was also observed in PEG-FA-Ni NTs group. The final tumor volume of PEG-FA-Ni NTs group was the smallest among all groups, indicating its prominent therapeutic effect. Meanwhile, the body weight of mice in experimental groups was not significantly different from that in the control group, implying that the treatments did not induce systemic toxicity (Fig. 8C, Figure S1E, S1F).
Systematic toxicity assay
To further evaluate the general toxicity of folate-nickel complex nanotubes, we examined the major organs of tumor-bearing HepG2 (Fig. 9), HeLa (Fig. S2A) and A549 (Fig. S2B) mice after administration by histopathological biopsy technique. The results showed that after treatment with PEG-FA-Ni NTs and FA-Ni NTs, the histological manifestations of major organs were not significantly different from those in the PBS group, indicating that NTs had no obvious toxic and side effects on normal tissues. In addition, H&E staining of tumor tissue showed that compared with the PBS group, DOX group, cisplatin group, and FA-Ni NTs group, the nuclear damage in the PEG-FA-Ni NTs group was more severe: the nuclei contracted and some nuclei were ruptured. The above results indicate that PEG-FA-Ni NTs have excellent antitumor activity and low side effects.