VitC-enhanced H2O2 reduction assay
We firstly investigated whether H2O2 can act as the reductant, because this is an essential prerequisite to fulfil the function of the SEOB. Herein, VitC was used to increase H2O2 production in HepG2 tumor cells, since several studies have reported VitC-derived H2O2 elevation in tumor cells 25-27. When we treated HepG2 cells with Ag2+ alone, we observed black silver deposits due to the pre-existing H2O2 in the cells (Supplementary Fig. S1). However, many more silver deposits were observed when VitC was added prior to or after Ag+ to stimulate the production of H2O2. This confirms that H2O2 can indeed behave as the reductant to reduce Ag+ into silver nanoparticles. The level of silver nanoparticles increases as either the Ag+ concentration or the incubation time escalates, accompanied by more silver-induced HepG2 apoptosis (Supplementary Figs S2 and S3). To test the ability of VitC to induce H2O2 production in vivo, we used mice bearing HepG2-derived tumors. We injected mice intratumorally or intravenously with VitC, then monitored changes in the electrical current in the tumor, which reflect the H2O2 level. Significantly increased current signals, representing increased H2O2 levels only at the tumor site occurred after intratumoral or intravenous injection of VitC (Fig. 1c-e). This implies that VitC-derived H2O2 can reduce the Ag+-based prodrug in vivo. However, silver deposition induced by H2O2 alone is inadequate due to the low reduction efficiency. To address this, rGO was used as the catalyst.
Analysis of the catalytic activity of rGO
Despite sharing similar structures with grapheme oxide (GO) (Supplementary Fig. S4), rGO is preferred to promote electron separation and H2O2 disproportionation due to its much higher catalytic activity21-23. Compared to GO, rGO allows rapid electron transfer and favors free Ag+ deposition, as evidenced by the larger silver stripping current, shorter stable response time and higher H2O2 disproportionation current in various anodic stripping voltammetry (ASV) tests (Fig. 1f-h). Based on these extraordinary features, we selected rGO to accelerate Ag+ reduction by H2O2. Furthermore, silver deposition from an Ag+-DNA conjugate was explored via ASV testing. The results showed that the successful capture of Ag+ by DNA can concentrate Ag+ and generate the strongest silver nanoparticle-derived current signal (Fig. 1i). This implies that Ag+ chelated to DNA was efficiently reduced by H2O2 to augment the silver deposition. Therefore, it is reasonable to expect that the Apt-CS/rGO/Ag+-DNA prodrug will rapidly and efficiently produce silver nanoparticles in the presence of rGO catalyst. To understand why rGO can achieve such a high catalytic activity for silver deposition, Raman, Fourier transform infrared (FT-IR) and X-ray photoelectron spectroscopy (XPS) characterizations were carried out. Compared to GO, rGO had a higher D/G ratio and fewer oxygen-containing functional groups (Supplementary Fig. S5). The decreased oxygen content in rGO means that more sp2 hybrid orbitals are available for accommodating electrons. In this regard, rGO can undoubtedly increase electron transfer from disproportionated H2O2 to Ag+, thus favoring catalytic reduction of Ag+ and deposition of silver nanoparticles.
Synthesis of a bioreactor carrying the Ag+ prodrug
The decreased level of oxygen-containing functional groups in rGO facilitates π-π conjugation, which will enhance the binding affinity between rGO and Ag+-conjugated DNA chains (Ag+-DNA) and enable rGO stacking (Fig. 2a). When the rGO carrier is chelated with Ag+-DNA conjugates, coated with CS and modified with AS1411 aptamers, the structure of the carrier is not changed (Fig. 2b). This suggests that AS1411-CS/rGO/Ag+-DNA will retain the rGO-catalyzed silver deposition property. The presence of uniformly distributed Ag and P elements demonstrates the successful chelation of Ag+-DNA conjugates onto rGO in AS1411-CS/rGO/Ag+-DNA (Fig. 2c-e). New FT-IR characteristic peaks and changes in the zeta potential also demonstrate the successful synthesis of AS1411-CS/rGO/Ag+-DNA and its intermediate products in sequence (Fig. 2f,g). During the modification process, the particle size remains approximately constant (Supplementary Fig. S6a).
Aptamer-mediated targeting assay in vitro
AS1411 specifically targets nucleolin, which is overexpressed by many tumors30-32. Therefore, we investigated the internalization of AS1411-CS/rGO/Ag+-DNA into HepG2 cells, which overexpress nucleolin. Direct analyses by laser confocal scanning microscopy (LCSM) and flow cytometry were firstly performed. A random sequence (RS) incapable of tumor targeting was used instead of AS1411 to synthesize the control nanosystem (i.e., RS-CS/rGO/Ag+-DNA). Furthermore, the L02 cell line, which features low nucleolin expression, was used as another comparison. The results clearly showed that more AS1411-CS/rGO/Ag+-DNA than RS-CS/rGO/Ag+-DNA entered the HepG2 cells, and both systems failed to enter L02 cells (Fig. 2h). This result sufficiently validates the specific targeting of AS1411 to nucleolin-overexpressing HepG2 cells. Flow cytometry data also confirms that there is more accumulation of AS1411-CS/rGO/Ag+-DNA in HepG2 cells than RS-CS/rGO/Ag+-DNA (Supplementary Fig. S7).
SEOB unlocks the Ag+ prodrug for silver deposition and anti-tumor activity
Contributed by AS1411 targeting-enhanced AS1411-CS/rGO/Ag+-DNA accumulation, VitC-enhanced H2O2 production and rGO-catalyzed silver deposition, AS1411-CS/rGO/Ag+-DNA+VitC group exerts the most robust killing ability against HepG2 cells. (Fig. 2i) This phenomenon attributed to that silver nanoparticles birth in the AS1411-CS/rGO/Ag+-DNA+VitC group gave birth to more oxidative stress for altering or preventing cell cycle progression. However, the targeting ability of AS1411 determines AS1411-CS/rGO/Ag+-DNA failed to induce evident injures to normal liver cells with low nucleolin expression (e.g., L02) (Fig. 2k). Intriguingly, VitC-enhanced H2O2 production alone does not kill HepG2 and L02 cells (Fig. 2j), and few AS1411-CS/rGO/Ag+-DNA and RS-CS/rGO/Ag+-DNA nanoparticles accumulate in L02 cells because these systems are not specifically targeted to L02 (Fig. 2k,l). These impressive results (i.e. assessment of the levels of silver deposition and cell apoptosis) indirectly validate the targeting ability of AS1411. More significantly, laser confocal scanning microscopy (LCSM) observations of HepG2 cells after live/dead co-staining also demonstrate that AS1411-CS/rGO/Ag+-DNA+VitC induces the most cell apoptosis and results in the lowest cell density (Fig. 2m).
In-depth exploration of the anti-tumor effect of AS1411-CS/rGO/Ag+-DNA+VitC was carried out via monitoring the variation of mitochondrial membrane potential. The strongest green fluorescence of JC-1 monomers was observed in AS1411-CS/rGO/Ag+-DNA+VitC-treated HepG2 cells (Fig. 3a). This reflects the significantly decreased membrane potential in these cells, and suggests a high level of apoptosis. In contrast, the negligible level of green fluorescence in L02 cells indicates no change in membrane potential and no L02 apoptosis, which can be attributed to poor accumulation of AS1411-CS/rGO/Ag+-DNA in L02 cells (Fig. 3b). These results adequately demonstrate that AS1411-CS/rGO/Ag+-DNA in the presence of VitC can produce the most silver deposits, which triggers the greatest anti-tumor activity. Quantitative data also confirms that although VitC-enhanced H2O2 alone is safe, VitC in combination with AS1411-CS/rGO/Ag+-DNA can induce the generation of more silver nanoparticles to robustly inhibit the growth of HepG2 cells (Fig. 3c,d). Moreover, this combination has only weak effects on L02 cells (Fig. 3d). Similar results were obtained via evaluating DNA damage. AS1411-CS/rGO/Ag+-DNA+VitC treatment causes the longest tail in HepG2 cells, which means the highest degree of DNA damage (Fig. 3e,f). However, AS1411-CS/rGO/Ag+-DNA+VitC fails to induce evident apoptosis in L02 cells due to the low accumulation of AS1411-CS/rGO/Ag+-DNA in these cells (Fig. 3g,h). This tumor cell-specific effect guarantees the safety of the treatment. Furthermore, we also measured ROS levels, since ROS are directly responsible for apoptosis and ROS levels positively correlate with the deposition of silver nanoparticles. VitC alone is unable to generate sufficient ROS-induced oxidative stress for inducing HepG2 apoptosis even though the VitC concentration reached a high level (above 8 mM) (Fig. 3i). Once VitC is combined with AS1411-CS/rGO/Ag+-DNA, VitC-derived H2O2 reduces the internalized Ag+ in AS1411-CS/rGO/Ag+-DNA to produce the most silver deposits that instigate the highest ROS oxidative stress (Fig. 3j). This explains why AS1411-CS/rGO/Ag+-DNA+VitC attains the highest anti-proliferation efficiency.
Next, the in vivo targeting and anti-tumor activities of AS1411-CS/rGO/Ag+-DNA were explored. When mice with HepG2 xenografted tumors were administered with RS-CS/rGO/Ag+-DNA via intravenous (i.v.) injection, the fluorescence signal in the tumor was negligible (Fig. 4a). In contrast, in tumor-bearing mice administered with AS1411-CS/rGO/Ag+-DNA, the fluorescence signal in the tumor was high for up to 4 h. This can be ascribed to the targeting effect of AS1411, which delivers the SEOB system into the HepG2 tumor cells. Consistent with the in vitro results, high accumulation of the nanosystem favors rGO-enhanced catalytic reduction of Ag+ by H2O2, which results in abundant silver deposits, and thereby significantly suppresses HepG2 tumor growth (Fig. 4b,c). Inspiringly, the tumors were smallest in the group treated with AS1411-CS/rGO/Ag+-DNA+VitC. This result can be ascribed to the fact that VitC enhances the H2O2 supply in the tumor cells to facilitate more silver deposits, consequently resulting in the strongest inhibitory effect on HepG2 tumor growth. These results further demonstrate the feasibility of such a SEOB in unlocking the Ag+ prodrug and facilitating intratumoral accumulation of silver nanoparticles to suppress tumor growth.
Pathological examination of tumors from AS1411-CS/rGO/Ag+-DNA+VitC-treated mice showed some typical characteristics of apoptosis including cell shrinkage, nuclear density increase and nuclear rupture (Fig. 4d). TUNEL immunofluorescence staining revealed that AS1411-CS/rGO/Ag+-DNA+VitC achieves the highest level of apoptosis (Fig. 4e). In particular, biological electron microscopic observation of tumor sections indicated the presence of vacuolization and tissue necrosis, which can be regarded as direct evidence to explain the tremendously suppressed tumor growth (Fig. 4f).
General applicability of the SEOB system
The END aptamer has been documented to positively target HepG2 cells due to high expression of the endoglin receptor24. Therefore, we expected that the END-CS/rGO/Ag+-DNA system would also inhibit HepG2 tumor growth. END-CS/rGO/Ag+-DNA was easily obtained by referring to the synthesis procedure for AS1411-CS/rGO/Ag+-DNA (Fig. 2g and Supplementary Fig. S6). The targeting ability of the chelated END aptamer allows more END-CS/rGO/Ag+-DNA particles to enter HepG2 cells than RS-CS/rGO/Ag+-DNA. Macroscopic silver deposits are produced only in HepG2 cells after adding VitC, which consequently induces massive HepG2 apoptosis (Supplementary Figs S8 and S9). Video S1 clearly shows the astonishing ultra-rapid (15 s) phagocytosis of the green FITC-labeled bioreactor by HepG2 cells due to END targeting. This was followed by silver deposit production and apoptosis (represented by red PI staining), due to the potent reduction ability of rGO and VitC-enhanced H2O2. Similar to AS1411-CS/rGO/Ag+-DNA, END-CS/rGO/Ag+-DNA fails to enter other normal cells (e.g., 293T cells) with low endoglin expression, and no evident silver deposits are observed under the same conditions with HepG2 treatment (Supplementary Fig. S8 and Video S2). However, the anti-tumor outcome using END-CS/rGO/Ag+-DNA is inferior to that using AS1411-CS/rGO/Ag+-DNA in the absence or presence of VitC, probably due to differences in their accumulation. This is evidenced by the differences in membrane potential drop, cell viability, DNA damage, ROS production and in vivo anti-HepG2 tumor activity (Figs 3 and 4). Despite this, END-CS/rGO/Ag+-DNA remains preferable to other non-targeting groups.
To further demonstrate the general applicability of the AS1411-CS/rGO/Ag+-DNA SEOB prodrug, another xenograft tumor model (i.e., human pulmonary carcinoma A549) was used. The A549 model gave identical results to the HepG2 model in terms of targeting and anti-tumor effects. In detail, AS1411-CS/rGO/Ag+-DNA targets A549 cells much more effectively than RS-CS/rGO/Ag+-DNA, since A549 cells also overexpress nucleolin33 (Supplementary Fig. S10). Therefore, the Ag+ pro-drug results in more silver deposits for killing A549 cells, especially in the presence of VitC, and there is no damage to normal L02 liver cells, as evidenced by flow cytometry data (Fig. 5a), trypan blue staining and MTT data (Supplementary Fig. S11). When A549 cells were treated with AS1411-CS/rGO/Ag+-DNA+VitC in vitro, strong green JC-1 fluorescence was observed, together with a more evident DNA tailing phenomenon and more ROS production. These results indirectly demonstrate the applicability of the SEOB system to kill tumor cells (Fig. 5b-f). The in vivo targeting test reveals that the retention of AS1411-CS/rGO/Ag+-DNA in A549 tumors progressively increases and reaches a peak at 48 h post-injection, while RS-CS/rGO/Ag+-DNA fails to enter tumors (Fig. 5g). This observation validates the excellent targeting ability of AS1411-CS/rGO/Ag+-DNA towards A549. Similar to the results obtained in the anti-HepG2 tumor experiment, the AS1411-CS/rGO/Ag+-DNA prodrug performs best against A549 tumors in the presence of VitC. Tumor inhibition, tumor silver content and tumor cell apoptosis were all elevated when AS1411-CS/rGO/Ag+-DNA was combined with VitC to enhance H2O2 production (Fig. 5h-k).
Biosafety evaluation of SEOB
The biocompatibility of the SEOB-unlocked Ag+ prodrug remains a predominant concern for clinical translation. Herein, normal cynomolgus monkeys were used as a primate model to evaluate the safeties of AS1411- and End-CS/rGO/Ag+-DNA. Astonishingly, histopathological examination of the main organs in cynomolgus monkeys revealed no injuries or apoptosis, which is suggestive of excellent biosafety (Supplementary Fig. S12). This result paves a solid path to clinical translation of the SEOB-unlocked Ag+ prodrug.