## Introduction

The increasing application of engineering nanoparticles will inevitably result in the accumulation of these engineering nanoparticles in environment and may result in potential ecological and health risks1,2,3. For example, the accumulation of silver nanoparticles (AgNPs) can inhibit embryo growth1 and cause a series of cytotoxicity such as gene mutation4, inhibition of cell proliferation5, apoptosis6,7 and necrosis8. Now in vitro cytotoxicity investigations are frequently used to explore the toxic mechanism of nanoparticles9,10,11,12. The cytotoxicity of hardly soluble nanoparticles such as AgNPs mainly caused by intracellular particles according to “Trojan-horse mechanism”5,13. Therefore, many studies have been carried out on quantitative or qualitative analysis of intracellular nanoparticles to reveal cellular uptake of nanoparticles. Qualitative methods (e.g. Transmission electron microscopy14, Scanning electron microscopy15, Light scattering microscopy16, Super-resolution fluorescence microscopy17, Atomic force microscopy18) have been fully studied to directly observe intracellular nanoparticles. However, the quantitative methods of nanoparticles entering into cell are developed slowly compared to the qualitative methods19. The main challenge is how to erase the disruption of cell surface associated nanoparticles which are hard to be differentiated from intracellular nanoparticles14,19,20,21,22. Therefore, selective removal methods of cell surface associated nanoparticles with etchants have been developed20,21. The etchant I2-KI was firstly used to selectively remove gold nanoparticle (AuNPs) from cells and the internalized mass of Au nanoparticle was successfully analyzed20. The etchant K3Fe(CN)6-Na2S2O3 was proved to effectively remove silver nanoparticles from cell surface21,23. Therefore, mass of cellular nanoparticles can be quantitatively estimated via the removal of cell surface associated nanoparticles with etchants.

Typically uptake pathway for nanoparticles are macropinocytosis and endocytosis including clathrin mediated endocytosis (CME), lipid raft/caveolae-mediated endocytosis (LME)24,25,26,27,28. The nanoparticles transported through macropinocytosis or CME into cell will be usually transferred into lysosomal where the releasing ions from the insoluble particles are occurred25,27,29, so-called “lysosomal enhanced Trojan-horse mechanism”5. However, particles entering into cell through LME would sometimes escape degradation by lysosomal and release ions into cytoplasm or reach to organelle24,26,28. Inhibitors have been widely used to reveal the uptake pathway of nanoparticles4,24,30. Now, many researches have addressed the contribution of a certain uptake pathway to the nanoparticles via the decrease of particles’ signal with the addition of inhibitor4,24,30. However, these researches generally ignore interferences of the cell surface association nanoparticles which may lead to errors. Thus, we considered the combination etching method with inhibitors to analyse uptake pathway of nanoparticles.

The physicochemical properties of nanoparticles such as size, charge, and functionalization play a key role on their cellular uptake8,25,31,32,33,34,35. Before resuspending and inhaling by human beings, AgNPs entering into environment are inevitably contacted with nature organic matters (NOMs) such as citric acid (CA), tartaric acid (TA) and fulvic acid (FA)36,37. NOMs may be absorbed on the surface of AgNPs and change the surface properties, even the size and morphology of AgNPs as our previous report37. Cellular uptake and cytotoxicity of pristine nanoparticles have been well documented1,8,19,24, therefore, more investigations should be carried out on NOMs-nanoparticles corona to reveal the mechanism of the cellular uptake of AgNPs influenced by environmental NOMs.

Human pulmonary adenocarcinoma cell (A549 cell), a common model cell strain, was usually used to explore the cytotoxicity of nanoparticles38,39. In the present study, A549 cell were exposed to polyvinyl pyrrolidone (PVP) coated AgNPs (p-nAg) with size of around 20 nm with/without the treatment of CA, TA or FA. The p-nAg treated with solution without any NOM was set as control (nAgcontrol). CA-, FA-, TA-coated nAg were marked as CA-nAg, FA-nAg and TA-nAg, respectively. A etchant (K3Fe(CN)6-Na2S2O3) was chosen to selectively remove AgNPs associated on cell surface of A549 and inhibitors of cytochalasin D (inhibiting actin involved uptake pathway), EIPA (inhibiting macropinocytosis), chlorpromazine (inhibiting CME) and filipin (inhibiting LME) were also used to investigate the uptake pathway. The aim of this study is to reveal the effects of NOMs on the cellular uptake of AgNPs.

## Results

### Characterization of NOMs-coated AgNPs and their stability in culture medium

After treated with CA, FA or TA, the size of silver nanoparticle was little changed (25.1 nm to 27.7 nm in average) as shown in Fig. 1a. This was consistent with our previous study37. Table 1 showed that DH in culture medium (CM) with 1% FBS of CA-nAg was substantially lower than DH of nAgcontrol (99 nm to 117 nm in average). CA was absorbed on the surface of AgNPs and resulted in a higher carbon contents (1.91% to 1.77%) and higher ratio of content of carbon to content of nitrogen (C:N, wt:wt) compared to nAgcontrol (6.69 to 5.10) as shown in Table 1. Moreover, Table 1 showed that zeta potential value of CA-nAg in water (− 45.0 mV) was much negative than nAgcontrol in water (− 24.2 mV) since CA absorbed on the surface of AgNPs.

Surface plasmon resonance (SPR) spectrum of NOMs-coated AgNPs suspensions in CM (20 μg ml−1) at 37 °C was shown in Fig. 1b. The maximum absorption wavelength (λmax) of nAgcontrol, CA-, FA- and TA-nAg were 402, 408, 406 and 404 nm, respectively. Figure 1c,d recorded the SPR spectrums of suspensions within 60 min and the trends of absorption value at λmax (Amax) within 60 min. Red-shift of λmax for all of suspensions depended on contact time and Amax trended to decrease. Within 60 min, λmax of nAgcontrol, CA-, FA- and TA-nAg was red-shift for 2 nm and their Amax decreased for 15.6, 8.4, 6.6 and 7.0%, respectively. The ratio of real time absorbance to initial absorbance (A/A0) at λmax over 60 min could be fitted well with the first order removal model, which is a simplified model for particles sedimentation in water solution40. The model details and fitting parameters were listed in SI (Table S1). However, Fig. 1e showed that DH of these AgNPs in CM were almost constant within 30 min. It implied that these AgNPs suspended stable in CM in dilute concentration.

### Localization of NOMs-coated AgNPs in A549 cell

Figure 2 showed the distribution of intracellular NOMs-coated AgNPs in A549 cell. The intracellular NOMs-coated AgNPs were observed when exposing to NOMs-coated AgNPs (Fig. 2). When incubated concentration was 75 μg ml−1, the size clusters of intracellular CA- and TA-nAg were larger than them of nAgcontrol and FA-nAg (Fig. 2a–d). This suggested that CA- and TA-nAg would form clusters easily. When incubated concentration was 10 μg ml−1, the observed intracellular CA-nAg clusters were made up of a few particles. CA-nAg were observed in vesicles that was reached to nucleus (Fig. 2e) and endoplasmic reticulum where was much close to nucleus (Fig. 2f), while CA-nAg in nucleus was not found. The observed intracellular TA-nAg clusters at this concentration was comparably still in larger size (Fig. 2g). The surface associated AgNPs were observed in Fig. 2a, which implied that surface associated AgNPs were hard to remove with rinsing by PBS.

### Cellular uptake kinetics of NOMs-coated AgNPs by A549 cell

Figure 3 showed time-dependent process of NOMs-coated AgNPs internalized into A549 cell. The internalizing process of nanoparticles can be described as two process14,20,41. Firstly, particles transport from CM to cell surface. This process is usually described as Langmuir absorption process which was depended the sites on cell surface to form a single layer due to association and dissociation. Secondly, surface associated particles are internalized into cell. Fig. S1 showed the schematic image of two process of kinetic mechanism. Langmuir absorption process expressed as follow17,20,41:

$$\frac{{dM_{d} }}{{dt}} = k_{a} C[M_{0} - M_{d} (t)] - k_{d} M_{d} (t)$$
(1)

where Md is the mass of AgNPs delivered to single cell which is the sum of the mass of surface associated AgNPs42 and the mass of internalized AgNPs (Mi), pg. M0 is the maximum capacity that the single cell surface could associate with AgNPs, pg. C is the concentration of AgNPs in CM, nM. ka is the association factor, μM−1 h−1. kd is the dissociation factor, h−1.

Solving the Eq. (1) got a time-dependent relationship of Md20:

$$M_{d} (t) = \frac{{k_{a} M_{0} C}}{{k_{a} C + k_{d} }}(1 - {\text{e}}^{{ - (k_{a} C + k_{d} )t}} )$$
(2)

The internalization process was expressed as follow17,20,41:

$$\frac{{dM_{i} }}{dt} = k_{i} M_{s} (t) - k_{out} M_{i} (t)$$
(3)
$$M_{d} (t) = M_{i} (t) + M_{s} (t)$$
(4)

where ki and kout are the rate constant for internalizing through cell membrane into cell via either endocytosis or other pathways and escaping from the cell via exocytosis or other pathways, respectively (h−1). Mi is the mass of surface associated AgNPs (pg).

The data of cellular uptake kinetics fitted with Eqs. (2) and (3) were shown in Fig. 3. Table 2 presented the fitting parameters of Langmuir adsorption model. The dissociation factor kd of nAgcontrol, CA-, FA- and TA-nAg was almost equal to 0. These implied that the dissociation process of AgNPs bound on A549 cell surface was weak. The larger the value of ka is, the higher the affinity of AgNPs with cell surface will be17. The association factor—ka of CA-nAg (1.006 μM−1 h−1) was similar to that of nAgcontrol (1.002 μM−1 h−1). However, ka value of FA-nAg (352.2 μM−1 h−1) was much higher than that of nAgcontrol and the value of TA-nAg (1.386 μM−1 h−1) was slightly higher than that of nAgcontrol. These implied that FA and TA absorbed on surface of AgNPs could increase the affinity of AgNPs with cell surface while CA affected very little.

Table 3 presented the fitting parameters of internalizing model. The value of ki and kout of CA-nAg was about 5.82- and 11.3-folds higher than that of nAgcontrol, respectively. The value of ki and kout of TA-nAg was about 1.69- and 1.14-folds higher than that of nAgcontrol, respectively. To FA-nAg, ki and kout was much lower than them of nAgcontrol (around 0.29- and 0.38-folds to nAgcontrol, respectively). Hence, the equilibrium time (tmax) of internalization and exocytosis of CA- and TA-nAg was shorted from 18 h of nAgcontrol to 9 h and 13 h, respectively. The value of tmax of FA-nAg (52 h) was much larger than that of nAgcontrol. The value of Mimax is frequently used in the evaluation of cellular uptake of nanoparticles with certain size or specific surface properties which could influence the association of nanoparticles on cell surface and the binding of nanoparticles with receptors14,17,41. Compared to nAgcontrol (2.54 pg per cell), Mimax of CA-nAg (1.84 pg per cell), FA-nAg (2.47 pg per cell) or TA-nAg (2.60 pg per cell) was closely related with the change of the value of ki/kout (0.47, 0.7, 1.35 and 0.92, respectively) as shown in Table 3. Definitely, the parameter of ki/kout indicated the value of dividing the rate of internalization by the rate of exocytosis. Accordingly, ki/kout was suggested to be a valuable parameter to reflect cellular uptake of nanoparticles. Table 3 also shows that the values of kout were greatly higher than those of ki for nanoparticles except TA-nAg. It was reported that Ki values were less than Kout values for citrate- and PVA-coated Au nanospheres, while Ki and Kout values for PAA-coated Au nanospheres were significantly higher than those in the former two20. The authors explained that these were mainly caused by the different amount of Au nanospheres adsorbed onto the cell surface. These were consistent with our results that internalization process of coated nanoparticles would be influenced by the different organic ligands.

### Relationship of intracellular AgNPs with AgNPs delivering from suspension

Figure 4 presented the relationship of Mi with the given mass of AgNPs in total (MT) and Md. With the increase of MT, Md exponentially increased as shown in Fig. 4a. This was different from Langmuir adsorption, since the aggregation and sedimentation happened in concentrated AgNPs suspension. Aggregation and sedimentation enhanced the delivering process of AgNPs onto cell surface and broke the rule of Langmuir association process. As a result, Md of concentrated AgNPs suspension depended on stability of AgNPs. To reach the same level of Md, CA-, FA- and TA-nAg need more given mass in CM than nAgcontrol (such as to get 20 pg per cell of Md, need MT 20, 70, 50 and 30 μg, respectively to nAgcontrol, CA-, FA- and TA-nAg). This meant that CA-, FA-and TA-nAg were more stable in CM than nAgcontrol. It was consistent with the results in Fig. 1d.

As shown in Fig. 4b, the relationship of Mi and Md was likely subject to Logistic model which was found to fit well with the uptake of magnetic iron nanoparticles by T98G and U251 cell19. The Logistic model was expressed as follow19:

$$M_{i} = M_{imaxC} - \frac{{M_{imaxC} }}{{1 + ({{M_{d} } \mathord{\left/ {\vphantom {{M_{d} } {EC_{50} }}} \right. \kern-\nulldelimiterspace} {EC_{50} }})^{p} }},$$
(5)

where MimaxC was maximum value of Mi, pg; EC50 was Md for 50% of MimaxC, pg; p was the slope factor19.

Table 4 presented the fitting parameters of Logistic model. MimaxC of CA-nAg and TA-nAg was 1.59 and 1.50 pg, respectively, higher than that of nAgcontrol (1.02 pg). MimaxC of FA-nAg was 0.85 pg, less than that of nAgcontrol. M0C of CA-nAg and TA-nAg was 6.78 and 5.39 pg, much higher than that of nAgcontrol (5.00 pg). M0C of FA-nAg (5.39 pg) was slightly higher than that of nAgcontrol.

### Cellular uptake pathway for NOMs-coated AgNPs into A549 cell

Figure 5a presented the influences of inhibitors on the internalization of AgNPs under “crowded state” cells and their influences between “rare” state cells and “crowded” state cells were show in Fig. 5b. These inhibitors (cytochalasin D, EIPA, chlorpromazine and filipin) have few influences to the stability of AgNPs suspension and little decrease in A549 cell viability incubated for 1 h under the used concentrations as shown in Fig. S2. To “crowded state” cells, the inhibition rate of internalization of AgNPs by cytochalasin D was at least 90%, except for FA-nAg (76%) (Fig. 5a). The addition of EIPA also resulted in significant inhibition of internalization of the positive control (nAgcontrol), CA-, FA- and TA-nAg at the rate of 85, 76, 73 and 81%, respectively. Chlorpromazine have also caused a large extent of decrease in internalization of nAgcontrol, CA-, FA- and TA-nAg with the rate of 58, 59, 61 and 64%, respectively. The inhibition rate of internalization of nAgcontrol by filipin was 61%, significantly higher than filipin for CA-, FA- and TA-nAg with the rate of 32, 47 and 31%, respectively. The influences of inhibitors to “rare state” and “crowded state” cells were different (Fig. 5b). The inhibition rate for “rare state” cells was significantly lower than that for “crowded state” cells, including EIPA to FA- and TA-nAg, chlorpromazine to TA-nAg, and filipin to nAgcontrol (Fig. 5b). However, some reverse phenomena were observed, including EIPA to CA-nAg, chlorpromazine to nAgcontrol, and filipin to CA-nAg and TA-nAg (Fig. 5b).

## Discussion

The size of FA-nAg was little changed as shown in Fig. 1a. Table 1 showed that carbon content of FA-nAg was 2.64%, higher than that of nAgcontrol (1.77%). This suggested that FA had been absorbed on the surface of AgNPs. FA-nAg presented more negative value of zeta potential in water (− 42.7 mV) than nAgcontrol (− 24.2 mV) (Table 1). Moreover, FA could also lightly enhance DH of FA-nAg in CM comparing to nAgcontrol in CM (121 nm to 117 nm in average) as shown in Table 1. However, carbon content of TA-nAg (1.47%) was lower than that of nAgcontrol (Table 1). This suggested that TA decreased the amounts of PVP coated on AgNPs. Moreover, C:N of TA-nAg was much higher than that of nAgcontrol (7.12 to 5.1). This suggested that TA had absorbed on surface of AgNPs replacing parts of coated PVP. DH of TA-nAg in CM was much lower than that of nAgcontrol in CM (81 nm to 117 nm in average) for shrinking hydration layer in water solution (Table 1). DH of CA-, FA- and TA-nAg in CM kept steady within 30 min as nAgcontrol in CM, implied that little aggregation happened in dilute AgNPs suspension (1 μg ml−1). However, red-shift for 2 nm observed in the SPR spectrum of CA-, FA- and TA-nAg as nAgcontrol suggested that light aggregation happened in concentrated AgNPs suspension (20 μg ml−1)43,44. The concentrated suspension of CA-, FA- and TA-nAg in CM seemed to be more stability than nAgcontrol since only 8.4, 6.6 and 7.0% decreased in Amax of them, lower than 15.6% of nAgcontrol.

The much higher affinity of FA-nAg to A549 cell than others were found as shown in Table 2. Cho et al. found that poly(allyamine hydrochloride) or PAA coated AuNPs showed higher affinity to SK-BR-3 breast cancer cells than others (ka was 10 times to others) because of the positive charge of amino function group20. FA were comprised of aromatic, carboxylic acid and amino function group according to the characterization in our previous report37. Accordingly, the amino function group in FA could make the difference.

The value of ki reflect the rate of particles internalizing into cell17,20,41. Table 3 showed that value of ki of CA-nAg and TA-nAg was larger than that of nAgcontrol. Harris et al. proved that hydrating layer hinders protein adsorption and subsequent internalization45. Table 1 showed that the thickness of hydrating layer of CA-nAg and TA-nAg was lower than that of nAgcontrol (DH of them was 99, 81 and 117 nm, respectively). Therefore, the increase of rate of CA-nAg and TA-nAg internalizing into cell were related to the decrease of hydrating layer of these AgNPs. However, the value of ki of FA-nAg was still much lower than that of nAgcontrol even though the similar DH (121 nm) with nAgcontrol as shown in Tables 1 and 3.

Table 5 recorded the Mi of AgNPs and AuNPs into cells that have reported in many literatures8,20,33,46,47,48,49,50. Mi of these AgNPs into cancer cells were about 2.1 to 10 pg at a same order of magnitude with the results in this study, despite of different size, surface functionalization or other experimental conditions8,47,50, which showed much difference from the normal cells (Mi = 47 pg for Pk 15 cells)48. Comparing with the Mi of AgNPs into cancer cells, Mi of AuNPs showed smaller value of about 0.12 to 1.23 pg (about 0.07 to 0.67 pg after converting into density of AgNPs)20,46,49. Cho et al.20 reported the kinetics of CA-nAu internalizing into SK-BR-3 cells wherein ki of CA-nAu was about 3.3 × 10–5 pg h−1, which was far below ki of CA-nAg in this study, consequently the equilibrium of CA-nAu into SK-BR-3 cells were not observed during 24 h of experiment, which could explain that the reported Mi of AuNPs lower than Mi of AgNPs.

According to the Logistic model, M0C of nAgcontrol and FA-nAg were close to the value of M0 (Table 2). However, M0C of CA- and TA-nAg were much higher than that of M0. These implied that when suspension of CA- and TA-nAg come to concentrate, the associated particles could gather together tightly and form large cluster on cell surface due to their small size of DH. The clusters made higher value of appearance M0 that was called “M0C”. As a consequence, MimaxC of CA- and TA-nAg was higher than that of nAgcontrol which was different from the situation of Mimax. Ratio of internalized AgNPs transferring from M0C (RT, RT = MimaxC/M0C × 100%) was defined to reflect the efficiency of surface association AgNPs transferring to internalized AgNPs. RT of CA-nAg and TA-nAg (23.5% and 21.2%) were higher than RT of nAgcontrol (20.4%), and RT of FA-nAg (15.8%) was lowest. Combined with the conclusion of ki (CA-nAg, TA-nAg > nAgcontrol > FA-nAg), these implied that CA and TA-nAg showed stronger ability but FA-nAg presented weaker ability of transport across plasma membrane than nAgcontrol.

Cytochalasin D is a cell permeable toxin which can disrupt actin filaments51. EIPA is a Na+/H+ ion channel blocking agent that inhibits the macropinocytosis-mediated pathway29,52. Chlorpromazine can prevent from the formation of clathrin in cells and is used to depress the uptake pathway of CME53,54. The addition of these inhibitors resulted in the suppression of internalization of the pristine AgNPs–nAgcontrol, indicated that the uptake pathway of AgNPs were mostly depended on actin and contributed a lot to both macropinocytosis and CME. Moreover, the inhibition rate of these inhibitors to NOMs-coated AgNPs was less affected by CA, FA and TA, while the inhibition rate of cytochalasin D and EIPA to FA-nAg were significantly less than them to the positive control—nAgcontrol (Fig. 5a). Accordingly, actin-involved uptake pathway or macropinocytosis would have less contribution to uptake of FA-nAg than uptake of the pristine AgNPs–nAgcontrol. It could be the reason of lower efficiency in uptake for FA-nAg than for nAgcontrol (ki and kout of FA-nAg were far below them of the pristine AgNPs–nAgcontrol as presented in Table 3).

Filipin, a drug can bind with sterol, is known as an inhibitor of LME30,55. The addition of filipin resulted in 61% decrease in the internalization of the pristine AgNP–nAgcontrol. This indicated that LME was also involved in the internalization of AgNPs. However, inhibition rate of CA-nAg, FA-nAg and TA-nAg by filipin was 32, 47 and 31%, respectively that were consistently lower than the rate to the pristine AgNPs–nAgcontrol (61%). These indicated that CA, FA or TA would change the way of uptake of AgNPs mainly through depressing the contribution of LME. The results were highly accordance with yielded ROS level by these AgNPs that nAgcontrol could arouse ROS level for twofold but 1.3-fold for FA-nAg and no significant influence for CA-nAg and TA-nAg (Fig. 6). LME is a unique pathway for nanoparticles that the intracellular vesicle could escape from lysosomal and uptake the nanoparticles into cytoplasm26,28. Huk et al. found that AgNPs caused much higher level of cytotoxicity since it could have reached into nucleus and mitochondria4. It implied that the higher dependence on LME of AgNPs would have higher opportunity to reach into nucleus or mitochondria and cause more damage to cells.

Figure 7 illuminated the reason about lower inhibition rate of “rare” state than “crowded” state. The “rare state” means that mass of cell surface associated AgNPs42 were far below M0 while “crowded state” means that mass of associated AgNPs were enough to reach M0. To the addition of inhibitor to “rare state” cells, the Ms would be higher than Ms without inhibitors, because Mi would be reduced but Md might be rarely impacted according to Eq. (4). Thus, more free sites on cell surface would be occupied and result in exceeding Mi. Therefore, the calculated inhibition rate would be lower than what it should be. This may be called as the “waning and waxing” phenomenon. To the “crowded state”, this phenomenon could be ignored since Ms was a constant.

However, some reverse phenomenon happened because of certain properties of endocytosis pathway. LME inhibited by filipin is known to be a receptor-specific uptake and usually form 50 to 80 nm caveolae in size26,28. As shown in Table 4, it can be concluded that CA-nAg and TA-nAg could form clusters on cell surface under concentrated CA-nAg and TA-nAg suspensions. Accordingly, the formed clusters were not appropriate in size to be trapped by caveolae. The inhibition rate of filipin to “crowded state” cell of CA-nAg and TA-nAg was less than the inhibition rate to their “rare state” cell.

In summary, CA treatment reduced DH and enhanced the colloidal stability of CA-nAg in CM comparing to the pristine AgNPs–nAgcontrol. Consequently, the increase for both of ki and kout but decrease in Mi were found. TA treatment reduced DH and enhanced colloidal stability of TA-nAg in CM compared to the pristine AgNPs–nAgcontrol. Consequently, the increase for ki, kout and Mi were found. FA enhanced the stability of FA-nAg in CM, but much decrease for ki, kout and Mi were found which resulted from less dependent on actin involved uptake pathway and macropinocytosis than the pristine AgNPs–nAgcontrol. In addition, intracellular mass of these AgNPs were dependent on Md, which obeyed Logistic model. According to the internalization model and Logistic model, CA and TA-nAg showed stronger ability but FA-nAg presented weaker ability of transport across plasma membrane than the pristine AgNPs–nAgcontrol. Moreover, uptake of CA-, TA- and FA-nAg was less dependent on LME comparing to the pristine AgNPs–nAgcontrol, which resulted from cell surface association state of AgNPs that affected by NOM.

## Methods

### Natural organic matters

The NOM used in this study include CA, TA and FA. CA and TA were purchased from Sinopharm Chemical Reagent Co., LTD. FA was extracted from the sediments of Xuanwu Lake at Nanjing, China, and the properties were presented in our previous report37.

### Preparation and characterization of NOM coated nAg

The pristine silver nanoparticles of 20 nm were synthesized according to the previous reports with minor modification (more details seen the Supporting Information, SI)56. The obtained silver was marked as p-nAg for the subsequent treatment.

NOMs-treated nAg was made according to our previous study37. Briefly, the obtained p-nAg suspension was treated with solutions containing citric acid, tartaric acid and fulvic acid Concentrations of CA and TA in solution were set as 10 mM and FA were 200 mg l−1 (more details seen the Supporting Information, SI).

The sizes of the pristine and NOM-coated AgNPs were characterized by Transmission Electron Microscope (TEM, JEM-2100 (HR), Japanese JEOL Corporation). The carbon and nitrogen contents of these samples were determined using element analyser (EA, CHN-O-Rapid, Germany Heraeus Corporation). The stability of the pristine and NOM-coated AgNPs suspensions in Ham’s F-12K (Kaighn’s) Medium (1×, Gibco) which is used as grow up medium (CM) for A549 cell in this study supported with 1% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin streptomycin sol, Gibco) were characterized by UV–Vis spectrometer (UH5300, Japanese Hitachi Corporation) at 37 °C. Briefly, 5 mg of the pristine and NOM-coated AgNPs was put in 5 ml 1% FBS supported CM. The suspensions were diluted with 1% FBS supported CM to a final concentration of 20 μg ml−1. The absorbance from 300 to 600 nm (step by 2 nm) of suspensions were detected at 0, 10, 20, 30, 40, 60 min, respectively. Hydrodynamic diameter (DH, nm) and zeta potential value (ZP, mV) of the suspensions (diluted to 1 μg ml−1) were monitored over 30 min at 37 °C by nanoparticle size analyzer (90Plus, Brookhaven Instruments Corporation). The initial DH was determined based on the average value of dynamic light scattering (DLS) data within 3 min.

### Cell culture

Ham’s F-12K (Kaighn’s) Medium (1×, Gibco) was used as culture medium for A549 epithelial cells after adding with 1% antibiotics (penicillin streptomycin sol, Gibco). A549 cells were cultured in 10% FBS supported CM. The cultures were incubated at incubator (37 °C, 5% CO2) and the medium was changed every two days.

### Etching AgNPs bound on the cell surface

Etching method was proposed and verified by Gray B. Braun for removing the absorbed the pristine and NOM-coated AgNPs on cell surface which disrupt the quantitative of intracellular AgNPs21. Actually, etching method failed to clean the well-plate touching side of adherent cell where solvent was hard to infiltrate while AgNPs could be transferred from top side surface due to the fluidity of cell membrane. An etching method integrated adherent and suspended cell to remove the absorbed AgNPs on cell surface for the quantitative analysis of the intracellular AgNPs was developed and the related experiments with results and discussion were described in SI. The low cytotoxicity of our used etchants to A549 cell in short etching time was shown in Fig. S3a. The high efficiency of the etching method to remove association AgNPs on A549 cell surface was also proved in Fig. S3b. Therefore, based on the quantitative analyses of particles internalized into cell.

### Kinetics of the pristine and NOM-coated AgNPs uptake by A549 cell

A549 cells were seeded in 12 well-plate for 24 h prior to exposure with the pristine and NOM-coated AgNPs. At the following day, the CM was removed, and then rinsed with PBS for twice. After the addition of 10 μg ml−1 AgNPs suspension, cells were incubated for 0, 1, 2, 4, 6, 8 and 12 h, and then treated with the etchant and collected.

### Relationship of intracellular AgNPs with AgNPs delivering from suspension

After incubation for 24 h of seeded cells in 12 well-plate, the CM were removed and cells were rinsed with PBS for twice. Designed concentrations of AgNPs suspension were added and cells were incubated for 1 h. Cells were treated with the etchant and collected. Concentrations of AgNPs were set as: 0–35 μg ml−1 for nAgcontrol, 0–50 μg ml−1 for CA-nAg, 0–40 μg ml−1 for FA-nAg and 0–60 μg ml−1 for TA-nAg. Fig. S4 showed that cell viability of AgNPs was above 80% even the incubated concentration of AgNPs up to 100 μg ml−1, which implied that concentration of AgNPs used in this experiment were harmless to A549 cell.

### Cellular uptake pathway of NOM-coated AgNPs on A549 cell

Some literatures report that cellular uptake of particles are dependent on their aggregation32 or aggregation behavior57 on cell surface. Therefore, the gathering state of AgNPs on cell surface would affect the cellular uptake pathway utilized by AgNPs. Accordingly, cellular uptake pathway for NOM-coated AgNPs to A549 cell were studied under two levels of AgNPs density on cell surface (rare state and crowded state).

#### Rare state

Rare state means the level of AgNPs associated with cell surface was much less than maximum capacity to accept AgNPs on A549 cell surface. Firstly, A549 cells were seeded in 12 well-plate. The cells were pretreated with inhibitors (their final concentrations were 5 μM for cytochalasin D, 5 μM for EIPA, 30 μM for chlorpromazine and 0.5 μg ml−1 for filipin, respectively) for 30 min in incubator. Then, cells were rinsed with PBS for 1 time and exposure to 10 μg ml−1 of pristine or NOMs-coated AgNPs suspension with inhibitor (kept the same concentration), and incubated for 1 h in incubator. Finally, cells were treated with etching method and collected.

#### Crowded state

Crowded state means the mass of AgNPs associated with cell surface was enough to reach maximum capacity to accept AgNPs on A549 cell surface. Similar to above, after pretreated with inhibitor, cells were rinsed with PBS for 1 time and exposure to pristine or NOMs-coated AgNPs suspension with inhibitor. To make 20 pg per cell AgNPs associated on cell surface before cellular uptake starting, cells were firstly incubated for 1 h at 4 °C. Then, cells were put in incubator for next 1 h. Finally, cells were treated with etching method and collected. Final concentration of nAgcontrol, CA-nAg, FA-nAg and TA-nAg for “crowded state” were 20, 70, 50 and 30 μg ml−1, respectively.

Cells exposed to the pristine silver nanoparticles with inhibitor were set as positive control (marked as nAgcontrol). Cells exposed to the pristine and NOMs-coated AgNPs without any inhibitor were set as negative control. Cells incubated with inhibitor and without pristine and NOMs-coated AgNPs were set as blank control. The experiment was run with triplicate.

### TEM observation of intracellular pristine and NOM-coated AgNPs

TEM has frequently been used to observe the localization of nanoparticles8,14. A549 cells were centrifuged and rinsed by PBS after 6 h exposure to 75 μg ml−1 nAgcontrol, CA-nAg, FA-nAg and TA-nAg or 10 μg ml−1 CA-nAg and TA-nAg. The harvested cells were prefixed in 2.5% glutaraldehyde at 4 °C overnight and washed with PBS three times. Subsequently, the cells were stained with 1% osmic acid followed by gradient dehydration with ethanol and acetone. Then, the samples were embedded in epoxy resin, sectioned, and post stained with lead citrate and uranyl acetate before TEM observation. Finally, cells were observed using the TEM.

### ROS level

The cells were seeded in 12 well-plate for 24 h prior to exposure with AgNPs. Seeding density was 5 × 105 cells per well. Cells were exposure to 1 ml 75 μg ml−1 AgNPs suspension for 24 h. Then, the plates were rinsed with PBS for twice and loaded with 10 μM DCFH-DA in CM for 20 min in incubator. Thereafter, cells were rinsed with CM for three time and treated with 0.2 ml EDTA-trypsin solution. The suspended cells were collected with PBS and the fluorescence was recorded with a flow cytometer (MoFlo XDP, Beckman Coulter) by reading 5 × 104 cells at FL1 channel (excitation 485 nm, emission 535 nm).

### Elemental analysis

The cells (cell number: 5 × 105) were digested with concentrated HNO3. Concentrations of Ag were measured using an inductively coupled plasma optical emission spectrometry (ICP-OES, Optima 5300, Perkin-Elmer SCIEX, USA). The calibration standard solutions were diluted from obtained by the dilution of the standard stock solutions (Custom Assurance Standard) purchased from SPEX CertiPreP (1000 mg l−1, Lot number: 28-232CR) with 2% HNO3 (V/V). The relative percentage differences of parallel samples were within 20%, or the experiments were repeated.

### Data analysis

The significant differences were analysed by independent-sample T tests in SPSS statistic 17.0. First-order removal model and Logistic model were fitted with the trends of AgNPs sedimentation in CM and the mass reliable internalization process, respectively in origin 9.1. Langmuir absorption model and internalizing kinetic model were fitted with cell surface association process and internalizing kinetic of AgNPs, respectively, in Matlab R2016a.