Characterization of CdTe QDs and CdTe/ZnS QDs
The absorption and emission maximum peaks for the QDs are centered at 526 nm, 566 nm (CdTe QDs) and 550 nm, 588 nm (CdTe/ZnS QDs) (Fig. S1). A well-resolved absorption maximum of the first electronic transition and narrow fluorescence spectrum were observed, pointing to a monodispersed and homogeneous size distribution of the prepared CdTe QDs and CdTe/ZnS QDs. The particle-size distribution and morphology of CdTe QDs and CdTe/ZnS were measured by transmission electron microscopy (TEM) (Fig. S2). The estimated diameters obtained for CdTe QDs and CdTe/ZnS QDs were of 2.91 nm and 3.24 nm according to calculate method in literature, respectively.[23] This gave the thickness of the ZnS shell as ∼0.3 nm. Regarding the zeta potential, the CdTe/ZnS QDs (-56.6±4.2 mV) are more stable than the CdTe QDs (-41.2±3.6 mV).
QDs induced derangement of coagulation balance
At days 1, 3, 7 and 14 post-exposure of CdTe QDs and CdTe/ZnS QDs, PT, APTT and TT were measured to characterize the effects of CdTe QDs and CdTe/ZnS QDs on coagulation function in rats. As shown in Fig. 1, PT and APTT were prolonged in a dose-dependent manner at day 1, indicating that CdTe QDs and CdTe/ZnS QDs activated both intrinsic and extrinsic coagulation cascade pathways. The greater effects on APTT in 1.5 µmol/kg bw group and 5 µmol/kg bw group of CdTe/ZnS QDs than CdTe QDs. Nevertheless, the effects on PT, APTT and TT disappeared in a short period of time (shorter than 3 days) for both CdTe QDs and CdTe/ZnS QDs (Fig. S3). These results illustrated that exposure of CdTe QDs and CdTe/ZnS QDs caused coagulation function disorders in rats in a dose-dependent manner and time-dependent manner.
For further elucidating how coagulation function disorders were initiated by CdTe QDs and CdTe/ZnS QDs, factors screening was also carried out to identify the changes in the expression level of coagulation factors (FIB, TAT, TF, FXa), anticoagulation factors (TFPI, AT-III) and fibrinolysis factors (PLG, t-PA, D-dimer) in rats. As shown in Fig. 1, the interference of QDs with coagulation factors and fibrinolytic factors not only activated the coagulation cascade (e.g. FIB, TAT and TF obviously increased), but also exceeded the regulation ability of the natural coagulation and anticoagulation system.[24] Extensive production of thrombin and large consumption of coagulation factors (e.g. FXa significantly decreased) subsequently stimulated secondary fibrinolytic hyperactivity (e.g. D-dimer and t-PA remarkably increased). Notably, the effects of CdTe/ZnS QDs on coagulation factors and fibrinolysis factors were stronger and lasted longer than CdTe QDs at same dosage (Fig. S4, S5). For anticoagulation factors, no significant changes were observed for TFPI and AT-III in each dosage group of CdTe QDs and CdTe/ZnS QDs at each point in time of observation (Fig. S6). These results illustrated that exposure of CdTe QDs and CdTe/ZnS QDs induced derangement of coagulation factors and fibrinolytic factors, that eventually caused coagulation function disorders. FIB, PLG and PTM, as initial and primary factors in the coagulation and fibrinolytic systems, their interaction with QDs may be key contributors for interference with coagulation balance.
CE-ICP-MS to monitor the formation of QDs-protein conjugates
CE-ICP-MS is well known not only as an efficient separation technique but also as a viable tool to characterize the formation of metal-containing nanoscale materials upon interaction with biomolecules.[25–28] As shown in Fig. 2 and Fig. 3, 50 µM CdTe QDs and CdTe/ZnS QDs were incubated respectively with different concentrations of FIB, PLG and PTM for 1 h, and the mixture was injected into the capillary. While the concentration of FIB reached 1 µM, CdTe QDs emerge out of the capillary (moving toward the anode) at a notably shorter time and a new peak appeared. Along with higher FIB concentrations (≥2 µM), the change in mobility was no longer observable, and a broader peak formed. For interaction with PLG, the peak slightly shifted to the left and the peak area of new peak increased with PLG concentration from 1 µM to 4 µM. For interaction with PTM, the migration time of CdTe QDs shortened quickly with the concentration of PTM increased. A new peak did not appear until the concentration of PTM reached 4 µM. These results suggested that the interaction of proteins with CdTe QDs formed QDs-protein conjugates, that bring in changes in charge and size of CdTe QDs, thus changing their mobility. The reduction in migration time of CdTe QDs after incubation with proteins was probably due to the increase in CdTe QDs size after being bound with proteins. The numbers of CdTe QDs-protein conjugates increased with protein concentration until conjugates formed enough and the dissociation rate was slower than the CE speed, while the stable QDs-protein conjugates were resolved from the free that caused the appearance of a new peak. The further aggregation of CdTe QDs-protein conjugates may contribute to a broader peak when the concentration of protein is very large.
Compared to CdTe QDs, the migration time of the whole peak shortened and the peak broadened for CdTe/ZnS QDs when the concentration of three proteins increased, but no new peak of QDs-protein conjugates appeared. These phenomena may be due to that thermodynamically stable CdTe/ZnS QDs-protein conjugates may form and attain an equilibrium state more rapidly even at a low concentration of proteins on account of higher affinity of CdTe/ZnS QDs with proteins than CdTe QDs. As the concentration of proteins increased continuously, the peak of CdTe/ZnS QDs-protein conjugates broadened gradually, indicating the aggregation of conjugates may occur. Besides, it is worth noting that a new Cd peak and a new Zn peak appeared at 2 µM and 4 µM PLG, suggesting the probable degradation of CdTe/ZnS QDs.
Kinetic and thermodynamic study of QDs-protein interactions
Typical BLI assays were established for individual binding kinetics of the interaction of QDs with FIB, PTM and PLG, respectively (Fig. 4). During the association period from 0 s to 180 s, QDs bound to the protein immobilized on the SA biosensor tips and induced the increase of thickness of the bio-layer. This indicated a concentration dependent binding behavior of QD and protein that caused the positive shifts of interference wavelength as the QDs concentration increased. During the dissociation period from 180 s to 360 s, partial QDs dissociated from the protein, and the interference wavelength showed small negative shifts for dissociation of FIB with QDs whereas slightly positive shifts for dissociation of PLG and PTM with QDs. The inconsistency of dissociation curves and corresponding fitted curves illustrated that the interaction of QDs with PLG and PTM may result in the conformational changes of proteins and aggregation of QDs that caused the increase in thickness of the bio-layer and subsequent positive shift of interference wavelength. The Ka and Kdis for interaction of QDs and protein were obtained by fitting the response of association and dissociation on concentration of proteins and summarized in Table 1. The Ka for interaction of both CdTe QDs and CdTe/ZnS QDs with proteins followed the order of PLG>PTM>FIB. For the same protein, the Ka of CdTe/ZnS QDs was larger than CdTe QDs. Nevertheless, the accurate Kdis for interaction of both CdTe QDs and CdTe/ZnS QDs with PLG and PTM could not be acquired as the dissociation is too slow (Kdis<10−7). Besides, no interaction of CdTe QDs and CdTe/ZnS QDs with uncoated streptavidin (SA) biosensor tips during the association period were confirmed and the streptavidin itself showed no cross reactivity with CdTe QDs and CdTe/ZnS QDs (data not shown).
Table 1
Kinetic rate constants and fluorescence quenching rate constants for the interaction.
Protein
|
CdTe QDs
|
CdTe/ZnS QDs
|
|
Ka (103 /M·s)
|
Kdis (10−4/s)
|
Kq (1013 L/M·s)
|
Ka (103 /M·s)
|
Kdis (10−4/s)
|
Kq (1013 L/M·s)
|
FIB
|
0.9420
|
4.414
|
0.8318
|
3.547
|
2.190
|
1.0960
|
PLG
|
1.703
|
<0.001
|
0.9971
|
12.33
|
<0.001
|
1.4560
|
PTM
|
1.440
|
<0.001
|
1.6190
|
6.274
|
<0.001
|
4.3970
|
As a thermodynamic technique for directly measuring the heat released or absorbed during a biomolecular binding process, ITC assays could not only directly acquire the information of numbers of binding sites (n) and affinity constant (KD), but also simultaneously determine the enthalpy change (ΔH), gibbs free energy change (ΔG) and entropy change (ΔS) during the molecular titrating reaction process.[29] Fig. 5 showed the raw data collected at each injection (top panel) and the fitting curve of a plot of the heat flow per injection of QDs versus the molar ratio of protein (bottom panel). As the thermodynamic parameters of the titration process showed in Fig. 6, the binding processes between QDs and three proteins were endothermic and spontaneous because of an enthalpy gain (ΔH > 0) and negative Gibbs free energy change (ΔG < 0). For large proteins or protein conjugates which may behave as colloids, entropy-driven adsorption may be common.[30] In the presence of entropy-enthalpy compensation, an unfavorable enthalpy contribution (ΔH > 0) was partially offset by a large favorable entropy change (ΔS > 0), affording negative free energy (ΔG < 0).[31]
The interaction of biomacromolecules with ligands is a complex process that can be accompanied by various weak noncovalent interaction, including hydrophobic interaction, van der waals forces, multiple hydrogen bonds and electrostatic forces.[32, 33] The positive ΔH and ΔS suggested that the interaction between the QDs and three proteins were mainly driven by a hydrophobic interaction.[34] Hence, it was necessary to correlate the binding affinity with the hydrophobicity of protein molecules. The amino acid compositions were taken from the protein database and the hydrophobic index (Hindex) were calculated (Table 2) for all the proteins under consideration as detailed in reference.[35] These results yield that KD for interaction of proteins and QDs followed the order of PTM>PLG>FIB, indicating the more positive is the Hindex, the higher is the protein-QDs binding. Besides, the numbers of binding sites for the interaction of proteins and CdTe QDs followed the order of FIB>PTM>PLG, while for the interaction of proteins and CdTe/ZnS QDs were similar. It was speculated that the smaller surface of CdTe QDs may reorganize upon the binding of the first protein molecule to expose more sites for interaction with the subsequent protein molecule. In comparison, the larger surface area of CdTe /ZnS QDs for binding may lead to only local reorganization of the surface groups and, thus, less impact on the subsequent binding.[26]
Table 2
Binding affinity constants and numbers of binding sites for the interaction.
Protein (Hindex)
|
CdTe QDs
|
CdTe/ZnS QDs
|
KD (10−6 M)
|
n
|
KD (10−6 M)
|
n
|
FIB (-0.746)
|
28. 74
|
2.643
|
2.809
|
1.411
|
PLG (-0.688)
|
3.290
|
1.977
|
1.816
|
1.495
|
PTM (-0.539)
|
1.336
|
2.511
|
0.2982
|
1.472
|
In the thermodynamic and kinetic processes of the interaction between proteins and QDs, Ka and KD were larger for interaction with CdTe/ZnS QDs than CdTe QDs, that also explained why CE-ICP-MS found CdTe/ZnS QDs-protein conjugates formed faster than CdTe QDs-protein conjugates. These results were also in agreement with Bohidar et al in that a 3-fold increment in KD for BSA, HSA and β-Lg interacting with ZnSe@ZnS core-shell structure as compared to that of ZnSe core only QDs.[36] We speculated that it may due to a coordinated binding with proteins by the Zn atoms at the surface of ZnS shell imperfections.[37, 38] Besides, the larger contact area between proteins and core-shell QDs compared with core only QDs may also strengthen the protein-nanoparticle interaction.[39]
Fluorescence quenching of proteins by QDs
For understanding the binding mechanism of FIB, PLG and PTM with QDs, the fluorescence spectra of the proteins in the presence of different concentrations of CdTe QDs and CdTe/ZnS QDs were recorded at 280 nm excitation. From Fig. 7, it can be observed that the fluorescence quenching of three kinds of proteins occured remarkably upon addition of CdTe QDs and CdTe/ZnS QDs. The fluorescence quenching of molecules can proceed via two major mechanisms, usually classified as static quenching and dynamic quenching. Dynamic quenching is due to the collision between the fluorophore and the quencher, whereas static quenching refers to the formation of the ground-state conjugates between them.[40] In order to obtain a clear insight into the quenching mechanism, the fluorescence quenching data were analyzed using the Stern-Volmer equation[41]
F 0/F=1+KSV[QDs]= 1+ Kqτ0 [QDs]
where F0 is the total fluorescence intensity of protein (in the absence of QDs), and F is the fluorescence intensity of protein at a specific QDs concentration. [QDs] represents the molar concentration of QDs, and Ksv refers to the Stern−Volmer quenching constant, a measure of the quenching efficiency. Kq accounts for the quenching rate constant, and τ0 (~10−8 s) is the average lifetime of protein in the absence of the quencher.[42] The inset in Fig. 7 demonstrated the linear dependence of (F0-F)/F of FIB, PLG and PTM as function of QDs concentration. As mentioned in Table 1, the obtained values of Kq are greater in comparison to the maximum scatter collision quenching constant of various quenchers (2.0×1010 L/M·s).[43, 44] Therefore, the nature of quenching was not arising from dynamic collision, but was due to the formation of ground-state conjugates between QDs and the proteins subsequently causing static quenching.[45] Meanwhile, the Kq for interaction of both CdTe QDs and CdTe/ZnS QDs with proteins followed the order of PTM>PLG>FIB, which is also identical to the order of KD. For same protein, the Kq of CdTe/ZnS QDs was larger than CdTe QDs.
QDs-induced proteins conformational changes
In solution, proteins fluctuate between many different conformations due to structural flexibility and optimized interaction with the surface of metallic NPs by adapting their structures.[46] To examine the potential structural changes of proteins upon interaction with QDs, CD spectra experiments were performed. In the ultraviolet region, two negative bands characteristic of the typical α-helix structure of protein were observed in the CD spectra (208 and 222 nm). The negative peak at 208 nm is contributed from the π-π* transition, and 222 nm corresponds to the n→π* transition due to the peptide bond of α-helix.[47] As can be seen in Fig. 8 and Fig. 9, these proteins underwent conformational changes upon interaction with CdTe QDs and CdTe/ZnS QDs. For FIB, in the presence of CdTe QDs with increasing concentrations, the α-helix content increased more significantly but β-sheet content decreased less remarkably than with addition of CdTe/ZnS QDs. For PTM, the α-helix content increased after addition of both CdTe QDs and CdTe/ZnS QDs, the β-sheets decreased after addition of CdTe QDs and the β-turns decreased after addition of CdTe/ZnS QDs. For PLG, the α-helix content and random coils content decreased, and β-turns content increased with increasing concentration of both CdTe QDs and CdTe/ZnS QDs. β-sheets increased after the addition of CdTe QDs, but were not notably influenced after the addition of CdTe/ZnS QDs. For the secondary structure of proteins, the α-helix structure shows the order of protein molecules, while β-sheets, β-turns and random coils reflect the looseness of protein molecules. These results indicated that the refolding and conformational changes of FIB and PTM occurred in the presence of QDs. However, the hydrogen-bonding networks of PLG were destroyed, causing unfolding of PLG and the exposure of amino acid residues folded inside the protein to the solution. In addition, the different proportions of secondary structures of three proteins induced by CdTe QDs and CdTe/ZnS QDs were inconsistent and irregular. The conformational changes of proteins upon QDs-protein interaction may depend on both the physicochemical characteristics of QDs and the properties of specific protein, such as isoelectric point, molecular weight and hydrophobicity. It seemed that little can be generally said about how NP adsorption induces the extent of protein conformational rearrangement, even under conditions where the protein binding constants are rather similar.[21]
Molecular docking analysis of L-GSH and L-Cys with proteins
In order to further identify the specific binding sites and elucidate the binding forces between proteins and QDs, molecular docking analysis of the modified groups L-GSH and L-Cys of QDs to FIB, PLG and PTM receptors were performed. Among the 10 retrieved possible docking poses for each ligand, the sulfhydryl facing outward poses with highest scores were selected for further analysis, that can also be compatible when they linked with QDs. FIB is a soluble glycoprotein composed by disulfide-linked dimer of three nonidentical polypeptide chains, Aα, Bβ, and γ.[48] Previous results showed that multiple cavities more densely located in the N-terminal central nodule E-region of FIB corresponded to the thrombin binding-domain, which have critical importance for the blood coagulation process.[49] Therefore, the molecular docking simulations focused on the potential docking interaction between L-GSH and L-Cys with E-region of FIB. As shown in Fig. 10A, the binding occurred at polar uncharged threonine (Thr), glycine (Gly), Cys, serine (Ser) sites and nonpolar proline (Pro) site, and the binding formation involved hydrogen bonding and hydrophobic forces. Under the presence of ligands (L-GSH and L-Cys), some residues can be perturbed by modifications in the symmetry architecture of the Bβ-γ/Bβ-γ dimeric domain of E-region and could induce potential hematotoxicity effects-mediated like fibrinolysis.[50]
PLG consists of seven domains: A N-terminal plasminogen-apple-nematode (PAN) domain, followed by five kringle domains (K1−K5) and the C-terminal catalytic trypsin-like serine protease (SP) domain.[51] Due to possessing lysine binding sites and the permanent exposure, K1 domain plays a key role in initial binding to lysine (Lys)-rich target surfaces like C-terminal fibrin monomers.[52] As shown in Fig. 10B, the ligands L-GSH and L-Cys were predicted to bind to the active pocket of K1 domain via hydrogen bonding and hydrophobic forces, and the binding sites included nonpolar leucine (Leu), negatively charged polar glutamic (Glu) and aspartic (Asp), positively charged polar argnine (Arg) and Lys, as well as polar uncharged Ser, asparagine (Asn) and Cys. The binding with active pocket of K1 domain may trigger the conformation transition of PLG into an open form, promoting the initial binding to Lys residues on target sites like fibrin.[53]
PTM is a modular protein composed of the Gla domain, two kringle domains (K1 and K2), and the serine protease domain connected by three intervening linkers.[54] The intramolecular collapse of tryptophane93 (Tyr93) in kringle-1 onto Trp547 in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin.[55] As shown in Fig. 10C, the ligands L-GSH and L-Cys were predicted to bind to nonpolar Pro, Trp, as well as polar uncharged tyrosine (Tyr), Asn, Cys via hydrogen bonding, hydrophobic forces and pi-sulfur bonding. It is worth noting that the interaction of L-GSH and L-Cys with Tyr93 may perturb the closed-open conformational equilibrium of PTM and promote the conversion of prothrombin to thrombin.[55]