Surface group mediates the adsorption of in situ generated thrombin and its interaction with anti-thrombin in the protein corona of SBA-15

Abstract

The surface groups of mesoporous silica (MPS) play an important role in mediating the hard protein corona (HPC) formation on its surface, leading to bio-identities with completely different procoagulant activities.

---

Mesoporous silica (MPS) has been considered as a prominent candidate for biomedical applications (e.g. drug delivery, enzyme immobilization, hemostatic response) due to its high surface area, large pore volume, controllable pore size, a large variety of surface function groups and low toxicity combined with biodegradability over time in the body.^1–4 It is inevitable that the surface of MPS will be coated by a hard protein corona (HPC) with high affinity when it enters the human body. It has been generally accepted that the protein corona largely defines the biological impacts of nanomaterials.⁵ In this regard, the effects of biological fluid,⁶ particle size⁷ and plasma concentration⁸ on the protein corona composition on nanomaterials have been extensively studied. However, there are few research studies focusing on the bio-impacts of protein corona formation on MPS.^9,10 The understanding of the relationship between the biological functions of these functional corona proteins and the structural parameters of the MPS is even more challenging. On the other hand, we notice that anti-coagulant plasma has been commonly used in HPC analysis, in which calcium ions have been chelated by sodium citrate to block the coagulation pathway.^6–8 The use of anti-coagulant plasma cannot fully replicate the pathway activation process in the human body, such as the complement pathway or coagulation pathway,¹¹ as zymogen activation will affect the protein composition in plasma and thereby lead to a change in the protein corona.

Herein, we studied the effect of modified surface groups of MPS on the protein corona formation and the procoagulant activity of the HPC formed in natural plasma. It is shown that the adsorption of in situ generated functional protein (thrombin) and its interaction with anti-thrombin in the protein corona of MPS is mediated by the surface group. The –OH and –SH modified MPS adsorb the in situ generated thrombin and protect it from complete inhibition by anti-thrombin, leading to a high procoagulant activity after HPC dressing. On the contrary, the in situ generated thrombin will be quickly deactivated by anti-thrombin in the protein corona of –COOH modified MPS owing to the competing adsorption of other non-functional corona proteins that weakens the interaction between MPS and thrombin.

The MPS SBA-15 with one-dimensional, hexagonally packed mesochannels was used in this study since it was one of the most recognized MPS materials.¹² The SBA-15 was synthesized by a hydrothermal method.¹³ Mesoporous SBA-15 is obtained by calcination at 350 °C (denote as SBA-15-OH). Further surface group modification is based on reported methods,¹⁴ and denoted as SBA-15-SG (SG = –SH, –NH₂ and –COOH). The detailed material synthesis and characterization are shown in the ESI (Fig. S1 and Table S1).† All SBA-15-SG samples show typical MPS structural characteristics, such uniform pore size distribution (∼5.0 nm), high surface area (190–300 m² g⁻¹), and large pore volume (0.23–0.37 m³ g⁻¹). The presence of the functional groups on the SBA-15 surface is confirmed by FT-IR and elemental analysis (Fig. S2 and Table S2†).

We first investigated the procoagulant activities of the SBA-15-SG by an in vitro clotting assay. A negatively charged surface had been confirmed to facilitate the contact activation pathway of the blood coagulation cascade.¹⁵ The clotting time of natural plasma activated by SBA-15-OH is 4.93 ± 0.35 min (Fig. 1, blue column), which shows a great decrease compared to the natural clotting time (11.83 ± 0.51 min) without the addition of any solid hemostatic agent. The –COOH modification increases the negative charge of MPS (from −19.3 ± 5.2 mV to −29.4 ± 0.3 mV), accompanied by a decrease of the clotting time from 4.9 min to 4.1 min. On the other hand, the MPS SBA-15 becomes positively charged with –NH₂ modification. It has been reported that the positively charged surface can decrease thrombin formation due to binding of factors VII and IX to the surface, which results in depletion of the respective protein in solution.¹⁶ In our case, SBA-15-NH₂ displays an anti-coagulant property with a clotting time much longer than the natural clotting time.

Fig. 1 The clotting time of the HPC/SBA-15-SG composites as determined using in vitro clotting assays. SBA-15-SG (50 mg) was separately incubated with different volumes of natural plasma to form a HPC/SBA-15-SG composite.

After contact with natural plasma, the surface of SBA-15-SG is immediately dressed by the HPC. The procoagulant properties of the HPC/SBA-15-SG composites are compared with the plasma clot. The plasma clot shows a moderate procoagulant activity (the clotting time of 200 μl plasma clot is ∼132 s), since the in situ generated thrombin in the plasma clot binds to the fibrin clot and partially retains its catalytic activity.¹⁷ The incubation of the natural plasma (100–400 μl) with SBA-15-COOH shows similar procoagulant properties as the plasma clot. However, the HPC dressing of SBA-15-OH and SBA-15-SH significantly changes their procoagulant activities. The clotting time of HPC/SBA-15-OH (-SH) is as low as ∼26 s when incubated with large volume (≥200 μl) of natural plasma, a five-fold reduction as compared to the plasma clot (132 s) (Fig. 1 and Table 1). The HPC/SBA-15-NH₂ displays the anti-coagulant property in spite of the change in the incubation plasma volume. The results indicate that the surface group of MPS plays a critical role in determining the procoagulant activity of the HPC/SBA-15-SG composites, which has not been reported so far.

Table 1 The zeta potential of SBA-15-SG and its procoagulant/thrombin activities with/without HPC dressing

-SG	Zeta potential/mV (±SD)	Clotting time^a/s	Thrombin activity (NIH U/50 mg)
a The number in parentheses is the clotting time of SBA-15-SG without HPC dressing.
–OH	−19.3 ± 5.2	27 (296)	3.68
–SH	−15.6 ± 3.5	26 (283)	4.00
–COOH	−29.4 ± 0.3	168 (246)	0.39
–NH2	26.4 ± 4.3	>900 (>900)	—
Plasma clot		132	1.31

---

The procoagulant activities of HPC/SBA-15-SG composites directly relate to their thrombin activities. Thrombin acts as a serine protease, converting soluble fibrinogen into insoluble strands of fibrin to form the final clots. The thrombin activity of HPC/SBA-15 (200 μl natural plasma incubated with 50 mg SBA-15-SG) was then evaluated by thrombin chromogenic substrate S2238. The composites of HPC/SBA-15-OH show much higher thrombin activity than HPC/SBA-15-COOH (3.68 NIH U vs. 0.39 NIH U, Table 1). The low thrombin activity of HPC/SBA-15-COOH is responsible for its low procoagulant activity.

The direct identification of active thrombin in HPC/SBA-15-SG is analyzed by immunoblot using rabbit polyclonal to thrombin. HPC/SBA-15-OH and HPC/SBA-15-SH show two discrete protein bands at 31 kDa and ∼95 kDa, corresponding to free thrombin and inactive thrombin–antithrombin complex (Fig. 2a).^18,19 Only one protein band at 95 kDa is observed for HPC/SBA-15-COOH, which is much lighter than that for HPC/SBA-15-OH. It reveals that the thrombin content in HPC/SBA-15-COOH is much less than that for HPC/SBA-15-OH (-SH), and most thrombin molecules in HPC/SBA-15-COOH are inhibited by anti-thrombin. Comparing the HPC/SBA-15-SG with high and low procoagulant activities and their corresponding thrombin content in HPC, we can conclude that thrombin is the key functional corona protein contributing to their procoagulant activities.

Fig. 2 (a) The western blot analysis of proteins retrieved from HPC/SBA-15-SG formed in natural plasma after extensive washing. The SDS-PAGE profiles of (b) HPC/SBA-15-SG and (c) BSA/SBA-15-SG.

To understand how the surface functional group affects the thrombin generation and its interaction with anti-thrombin, we compared the protein profiles of HPC/SBA-15-OH and HPC/SBA-15-COOH by SDS-PAGE experiment (Fig. 2b and S3†). The results reveal that the corona protein content on the SBA-15-COOH surface is higher than that on SBA-15-OH on the whole. We also use BSA to mimic the role of other corona proteins in HPC/SBA-15 composites. It is found that SBA-15-COOH does show a strong adsorption capability for BSA molecules, while a weak adsorption is confirmed for SBA-15-OH (Fig. 2c). Plasma proteins compete for the SBA-15 surface and lead to the protein corona formation. The strong adsorption of other non-functional proteins on the SBA-15-COOH surface weakens the interaction between thrombin and the MPS. This can explain why a low content of thrombin is observed for SBA-15-COOH. At the same time, the weak interaction between thrombin and the SBA-15-COOH cannot prevent the binding of thrombin by anti-thrombin, which leads to the deactivation of thrombin and results in a very low thrombin activity as shown in Table 1.

Based on the above results, we proposed a model for how the surface group mediates the adsorption of in situ generated thrombin and its interaction with anti-thrombin on the SBA-15 surface (Scheme 1). In natural plasma, the coagulation cascade can be activated on the negatively charged surface (–OH, –SH, and –COOH) and inhibited on a positively charged surface (–NH₂) due to its low affinity to corona proteins. The more negatively charged –COOH groups show strong adsorption to non-functional corona proteins that compete with in situ generated thrombin for the silica surface. This results in a weak adsorption of thrombin on SBA-15-COOH, leading to its deactivation by anti-thrombin binding. –OH and –SH surface groups show intermediate negative charge and interaction with non-functional corona proteins. The in situ generated thrombin can strongly bind to its surface, partially retaining its catalytic activity for blood coagulation.

Scheme 1 The surface group mediates the activation of blood coagulation and the adsorption of in situ generated thrombin in the protein corona of SBA-15.

The use of natural plasma is critical for the observed surface group dependent in situ thrombin generation and its corresponding procoagulant activities. If we replace the natural plasma with anti-coagulant plasma, which has been commonly used in other HPC studies,^6–8 no thrombin is detected by the immunoblot experiment (Fig. 2a), corresponding to the limited procoagulant activity of HPC/SBA-15-OH formed in anti-coagulant plasma (Fig. S4†). The use of anti-coagulant plasma cannot fully replicate the pathway activation process in the human body, such as the complement pathway or coagulation pathway.¹¹ This fact has been overlooked all along.

The HPC mainly formed outside the mesopores of SBA-15. The pore size of SBA-15 is ∼5 nm. Only the proteins that are smaller than the pore opening of SBA-15 can access the large internal surface. The molecular weight of most HPC proteins is larger than 50 kDa as shown in the SDS-PAGE analysis (Fig. S3†) (the dimension of ovalbumin, 45 kDa, is 4.0 nm × 5.0 nm × 7.0 nm), making them very difficult to enter the mesopores of SBA-15. On the other hand, the functional protein thrombin is generated by proteolytic cleavage of prothrombin (72 kDa), which is catalyzed by the prothrombinase complex. The prothrombinase complex consists of the serine protease, Factor Xa (56 kDa), and the protein cofactor, Factor V (300 kDa). The large molecular size of the proteins involved in the thrombin activation suggests that thrombin could only be generated outside the mesopores.

We also synthesized conventional SBA-15 with a rope-like morphology according the reported method.¹² We compared the HPC formation on its surface and the corresponding procoagulant activity with rod-like SBA-15-OH. As shown in the SDS-PAGE of the protein profiles, there is no obvious difference in the HPC formation on rod-like or rope-like SBA-15-OH (Fig. S5†). Although the procoagulant activity of naked rope-like SBA-15-OH is slightly higher than that of rod SBA-15-OH, there is no significant difference in the procoagulant activity of HPC/SBA-15-OH formed in natural plasma (Fig. S6†).

In summary, we show that the surface groups of MPS play a critical role in the formation of functional corona proteins (e.g. thrombin) in the HPC. There is an increasing need for understanding the fundamental interaction of nanomaterials within biological environments to further nanomedicine development. Our discovery of the surface group-mediated thrombin activity reveals a new strategy for regulating enzyme activity and specificity, and suggests that the HPC/SBA-15-OH (-SH) composites with high thrombin activity and procoagulant activity could serve as a new target for hemostatic development that has been dominated by organic-based molecules or materials.²⁰

Notes and references

1. I. I. Slowing, J. L. Vivero-Escoto, C. W. Wu and V. S. Y. Lin, Adv. Drug Delivery Rev., 2008, 60, 1278–1288.
2. C. L. Dai, Y. Yuan, C. S. Liu, J. Wei, H. Hong, X. S. Li and X. H. Pan, Biomaterials, 2009, 30, 5364–5375.
3. S. P. Hudson, R. F. Padera, R. Langer and D. S. Kohane, Biomaterials, 2008, 29, 4045–4055.
4. C. H. Lee, T. S. Lin and C. Y. Mou, Nano Today, 2009, 4, 165–179.
5. M. P. Monopoli, C. Aberg, A. Salvati and K. A. Dawson, Nat. Nanotechnol., 2012, 7, 779–786.
6. M. Lundqvist, J. Stigler, T. Cedervall, T. Berggard, M. B. Flanagan, I. Lynch, G. Elia and K. Dawson, ACS Nano, 2011, 5, 7503–7509.
7. S. Tenzer, D. Docter, S. Rosfa, A. Wlodarski, J. Kuharev, A. Rekik, S. K. Knauer, C. Bantz, T. Nawroth, C. Bier, J. Sirirattanapan, W. Mann, L. Treuel, R. Zellner, M. Maskos, H. Schild and R. H. Stauber, ACS Nano, 2011, 5, 7155–7167.
8. M. P. Monopoli, D. Walczyk, A. Campbell, G. Elia, I. Lynch, F. B. Bombelli and K. A. Dawson, J. Am. Chem. Soc., 2011, 133, 2525–2534.
9. A. J. Paula, R. T. Araujo, D. S. T. Martinez, E. J. Paredes-Gamero, H. B. Nader, N. Duran, G. Z. Justo and O. L. Alves, ACS Appl. Mater. Interfaces, 2013, 5, 8387–8393.
10. A. Yildirim, E. Ozgur and M. Bayindir, J. Mater. Chem. B, 2013, 1, 1909–1920.
11. B. Dahlback, Lancet, 2000, 355, 1627–1632.
12. D. Y. Zhao, J. L. Feng, Q. S. Huo, N. Melosh, G. H. Fredrickson, B. F. Chmelka and G. D. Stucky, Science, 1998, 279, 548–552.
13. C. Z. Yu, J. Fan, B. Z. Tian, D. Y. Zhao and G. D. Stucky, Adv. Mater., 2002, 14, 1742–1745.
14. X. Feng, G. E. Fryxell, L. Q. Wang, A. Y. Kim, J. Liu and K. M. Kemner, Science, 1997, 276, 923–926.
15. R. L. Heimark, K. Kurachi, K. Fujikawa and E. W. Davie, Nature, 1980, 286, 456–460.
16. C. Oslakovic, T. Cedervall, S. Linse and B. Dahlbäck, J. Nanomed. Nanotechnol., 2012, 8, 981–986.
17. J. I. Weitz, M. Hudoba, D. Massel, J. Maraganore and J. Hirsh, J. Clin. Invest., 1990, 86, 385–391.
18. D. Sommeijer, R. van Oerle, P. Reitsma, J. Timmerman, J. Meijers, H. Spronk and H. ten Cate, Thromb. J., 2005, 3, 12–21.
19. D. A. Lane and R. Caso, Baillieres. Clin. Haematol., 1989, 2, 961–998.
20. H. E. Achneck, B. Sileshi, R. M. Jamiolkowski, D. M. Albala, M. L. Shapiro and J. H. Lawson, Ann. Surg., 2010, 251, 217–228.

---

Footnote

† Electronic supplementary information (ESI) available: Experimental and characterization. See DOI: 10.1039/c4ra02106a

---