Synthesis and Characterization of PB NPs
Citric acid capped PB NPs were fabricated by a hydrothermal synthetic method [28]. The hydrodynamic diameter and polydispersity (PDI) of PB NPs were measured by DLS as 88.15 ± 1.15 nm and 0.105 ± 0.015, respectively. The Zeta potential of PB NPs was measured as -37.87 ± 1.72 mV. Under TEM observation, the PB NPs showed uniform cubic morphology with a diameter around 40 nm (Fig. 1a). The prepared PB NPs showed characteristic absorption in the near infrared (NIR) region (Fig. 1b). PB NPs showed the distinctive diffraction peaks under X-ray irradiation (2θ values of 17.45, 24.77, 35.30, 39.62, 43.68, 50.86, 54.07 and 56.87°) (Fig. 1c). In addition, Distinctive bands of 2084 cm− 1 and 498 cm− 1 that corresponding to the vibration of the FeII-CN-FeIII and C ≡ N group were observed in the FTIR spectrum of PB NPs (Fig. 1d). All the results indicated the successful fabrication of PB NPs.
General toxicity studies
General toxicity studies were performed after i.v. injection of PB NPs into mice at the dose of 5, 10 and 20 mg/kg, respectively. The control group was i.v. injected with saline. No mice death occurred in all the treated groups during the experiment period. It was observed that, different from other treated groups, the mice treated with 20 mg/kg of PB NPs exhibited dyspnea and bradykinesia at the initial stage post-injection, while the symptoms were temporary and could recover within 30 min. The body weight changes of mice were monitored as shown in Supplementary Fig. S1. For mice treated with 5 or 10 mg/kg of PB NPs, the increase in body weight was similar to the saline group within 30 days, while mice treated with 20 mg/kg of PB NPs showed obvious loss of weight at initial 2 days, though the body weight increased similar to control group subsequently. Furthermore, we monitored the food intake of mice with different treatments. Compared with other treated groups, mice treated with 20 mg/kg of PB NPs showed a loss of appetite and decrease in food intake (Supplementary Fig. S2), this may account for the body weight loss at initial 2 days. To further uncover the potential toxicity of PB NPs, evaluations on the hematological and biochemical parameters, organ coefficient indices and histopathology were carried out. Compared with mice treated with saline and low dose of PB NPs (5 and 10 mg/kg), mice treated with 20 mg/kg of PB NPs showed similar hematological and biochemical parameters (Fig. 1e). For mice treated with 20 mg/kg of PB NPs post-injection 1st day, the livers and lungs presented dark colors, PB NPs were observed distinctly in the livers and lungs tissue sections, while this dark color and accumulation gradually faded with time (Fig. 2). Though discernible high accumulation of PB NPs was found in the liver and lungs after injection, the coefficients and histopathology of the main organs showed no changes compared with the saline group and the other two treatment groups (Fig. 1f, Supplementary Fig. S3-S6).
Pharmacokinetic Profile and Protein Corona Component of PB NPs
PK investigation was performed to understand the fate of PB NPs in mice after i.v. injection. The blood clearance and biodistribution of PB NPs were determined by quantitative detection of the Fe content derived from PB NPs in the blood and the main organs via ICP-OES after i.v. injection of PB NPs into mice at the dose of 20 mg/kg. As shown in Fig. 3a, compared with the control group, mice treated with PB NPs showed elevated blood Fe level post injection, while the high blood Fe level gradually decreased with time and was similar to that of control group at 4 h post injection, indicating that PB NPs undergo the fast clearance from blood post injection. By deducting the background blood Fe level, the PK parameters of Fe from PB NPs in the blood including AUC0−∞, T1/2 and MRT were calculated as 1365.89 ± 206.79 µg/g∙h, 1.00 ± 0.41 h and 1.70 ± 0.44 h, respectively (Supplementary table S1). The biodistribution of PB NPs was investigated by measurement of PB NPs-derived Fe content in the tissues. For each tissue, PB NPs-derived Fe content was calculated by deducting the endogenous tissue Fe content from the total Fe content of PB NPs contained tissue. For accurate detecting the tissue Fe content, the mice tissues were perfused with saline to remove residual blood (Supplementary Fig. S7) to eliminate the interference from endogenous Fe in the blood. As shown in Fig. 3b, by deducting the endogenous Fe content, the liver and lungs of treated mice showed increased Fe content at 24h after injection, while the heart, spleen and kidneys did not. The results indicated that PB NPs mainly accumulated in the liver and lungs after fast clearance from the blood.
It was reported that protein corona is a key factor that can affect the biodistribution of NPs [42, 43], thus we investigated the formation and components of the protein corona of PB NPs after incubation with fresh mice plasma. After incubation, the particle size of PB NPs increased to 141.6 ± 2.09 nm and the Zeta potential of PB NPs decreased to -15.56 ± 3.42 mV (Fig. 3c, 3d), indicating the formation of protein corona around PB NPs. The components of protein corona were determined by iTRAQ-based proteomic analysis. It was found that the protein corona of PB NPs contained abundant opsonin proteins such as the complement family members (C3, C5, C9, C4b, Cfh, Cfb, Cfd, Cfi, C1ra, C4bpa, C8a, C8b), immunoglobulin, laminin, fibronectin, C-reactive protein (CRP) and collagen (Supplementary table S2). Opsonin proteins could activate the complement system and perform opsonin functions, leading to increased phagocytosis by phagocyte [42, 43].
Degradation of PB NPs in vitro and in vivo
Mice organs were harvested at different time points after injection of PB NPs to investigate the in vivo clearance of PB NPs. On 1st day post-injection, the livers and lungs of treated mice presented dark colors due to the high accumulations of PB NPs, while this dark color gradually faded with time (Supplementary Fig. S8). On 60th day after injection, the livers and lungs of the treated mice presented similar color to that of untreated mice. The tissue clearance of PB NPs was studied by detecting the changes of PB NPs-derived Fe content in the tissues, which was calculated by deducting the endogenous tissue Fe content from the total Fe content of PB NPs contained tissues. By deducting the endogenous Fe content, we found that the Fe levels in the hearts, spleens and kidneys of treated mice were similar to untreated mice at each time point, the accumulation and the clearance of PB NPs in these tissues were unobservable (Fig. 3e, 3h, 3i). By contrast, the clearance of PB NPs was observable in the livers and lungs after the deduction of the endogenous tissue Fe content (Fig. 3f, 3g). The PK parameters of Fe from PB NPs in the livers and lungs were further calculated (Supplementary table S3). The AUC0-∞, T1/2 and MRT of Fe from PB NPs in the liver were evaluated as 9688.83 ± 2534.41 µg/g∙day, 20.71 ± 2.21 days and 29.35 ± 2.54 days, respectively. The AUC0-∞, T1/2 and MRT of Fe from PB NPs in the lungs of mice were evaluated as 13671.92 ± 4118.49 µg/g∙day, 17.33 ± 4.99 days and 25.48 ± 6.49 days, respectively. Above results suggested that PB NPs undergo slow clearance from the livers and lungs of mice.
Then, in vitro simulated degradation of PB NPs was investigated in simulated body fluid (SBF) with pH 7.4 (simulating tissue fluid) and pH 4.5 (simulating acidic environment of intracellular endosome), respectively. After 48 h of incubation, both the UV-vis absorption and the color of colloidal PB NPs showed attenuation in the two SBFs (Fig. 3j-l), indicating the degradation of PB NPs. XRD and FTIR analyses further proved the degradation of PB NPs (Fig. 3m-n). It was found that the diffraction peaks and the vibration of FeⅡ-CN-FeⅢ band of PB NPs showed attenuation after incubation (Fig. 3m, 3n), while vibration ascribed to Fe-O band emerged in the FTIR spectrums (Fig. 3n), especially obvious in pH 7.4 SBF, implying the collapse of the lattice of PB NPs. According to the changes in UV-vis absorptions, XRD and FTIR peaks and the colors of PB NPs, it can be found that PB NPs undergo a faster degradation in pH 7.4 SBF than in pH 4.5 SBF. Afterward, FeCl3 solution was added to the supernatant solutions from the incubated SBFs, and blue colloid was only generated in the supernatant of pH 7.4 SBF (Fig. 3o). FTIR analysis showed that the blue colloid presents the specific vibration of PB NPs in the FTIR spectrum (Supplementary Fig. S9), indicating that [Fe(CN)6]3- existed in the supernatant of incubated pH 7.4 SBF. This result suggested that, in pH 7.4 SBF, the FeⅡ-CN-FeⅢ bond of PB NPs was broken into the [Fe(CN)6]3- and Fe3+ in the presence of hydroxide ion. Instead of [Fe(CN)6]3-, CN- was detected in the supernatant of incubated pH 4.5 SBF (Supplementary Fig. S10), this is likely that CN- can be released from FeⅡ-CN-FeⅢ bonds in acidic condition. [44] Thus, our results indicated PB NPs undergo different degradation patterns in different pH conditions.
Integrated Proteomics and Metabolomics Analysis of Mice Lungs
An integrated omics analysis was used to further uncover the response of mice lungs on molecules level to the PB NPs exposure. By proteomics analysis, a total of 4676 proteins were identified in lung samples by LC-MS/MS. The differential expression proteins were screened with standard of 1.5-fold change and P value < 0.05. Compared with the control group, 80 and 122 differentially expressed proteins were identified in the lungs on 7th and 60th day after PB NPs injection, respectively (Fig. 4a). The differentially expressed proteins in the lungs on the 7th and 60th day after i.v. injection of PB NPs were analyzed through KEGG database. Here, the main related KEGG pathways enriched are discussed. The main related pathways enriched are shown in Fig. 4b and 4c. The proteins related to the main enrichment pathways on the 7th and 60th day after exposure were shown in table S4 and S5, respectively. Adrenaline signal pathway and smooth muscle contraction were both enriched in the lungs on the 7th and 60th day after exposure (Fig. 4b and 4c). The pathway enriched alone on the 7th and 60th day after exposure is tight junction and leukocyte transepithelial migration, respectively.
In metabolomics analysis, the parameters Q2 > 0.5 for the OPLS-DA means the model was effective (Fig. 4d), and 167and 136 significantly changed metabolites were identified in mice lungs on 7th and 60th day post injection of PB NPs, respectively (Fig. 4e). Based on the KEGG database, the main metabolites related with KEGG pathway enrichment information on the 7th and 60th day after exposure were shown in table S6 and S7, respectively. Shikimic acid pathway, alkaloid synthesis, tyrosine metabolism, flavonoid biosynthesis, nicotinic acid and nicotinamide metabolism and linoleic acid metabolism were affected in varying degrees on the 7th and 60th day after exposure (Fig. 4f and 4g). Arachidonic acid metabolism and phenylalanine metabolism pathways were enriched in the lungs on the 7th day after exposure (Fig. 4f). Glutathione metabolism, pyruvate metabolism, thioacetone metabolism, cysteine and methionine metabolism (Fig. 4g) were the metabolic pathways that are enriched in the lungs on the 60th day after exposure. These pathways are mainly related to inflammatory response and antioxidation.
Integrated Proteomics and Metabolomics Analysis of Mice Livers
We also used the integrated omics analysis to uncover the toxicological response of mice livers to the exposure of PB NPs. By proteomics analysis, a total of 5059 proteins were identified in livers samples by LC-MS/MS. 18 and 15 differentially expressed proteins were identified in mice livers on 7th and 60th day after injection of PB NPs, respectively (Fig. 5a and 5b). based on the KEGG database, the proteins related to the main enrichment pathways on the 7th and 60th day after exposure were shown in Table S8 and S9, respectively. LDH and CDO1 were up-regulated in liver on the 7th day after exposure (Table S8), mainly enriched pathway was cysteine and methionine metabolism (Fig. 5b). CYP2A5 and CYP2A12, members of cytochrome P450 (CYP) subfamily, were significantly up-regulated in the liver on the 7the day after exposure (Fig. 5b), mainly enriched in the retinol metabolic pathway.
In metabolomics analysis, the parameters Q2 > 0.5 for the OPLS-DA, and 125 and 148 significantly changed metabolites were screened at 7th and 60th day post injection of PB NPs (Fig. 5c), suggesting that the accumulation of PB NPs caused the change of metabolites in the liver. The main metabolites related with KEGG pathway enrichment information on the 7th and 60th day after exposure were shown in Table S10 and S11, respectively. Purine metabolism, pantothenic acid and CoA biosynthesis, biotin metabolism, tyrosine metabolism and phenylalanine metabolism (Fig. 5e and 5f) were affected in varying degrees on the 7th and 60th day after exposure. Ascorbic acid and aldose glycol metabolism were enriched on the 7th day after exposure (Fig. 5e). Cysteine and methionine metabolism was enriched in the liver at 60 days after exposure (Fig. 5e). The above pathways were mainly related to inflammatory response and antioxidation.