In vitro hemocompatability evaluation of gold nanoparticles capped with Lactobacillus plantarum derived lipase 1

Abstract

BACKGROUND:

Gold nanoparticles (GNPs) are key diagnostic and therapeutic agents in biomedical sciences. Several studies have been carried out in different therapeutic areas such as in cancer treatment, antibacterial topical agents, imaging agents etc. There is a necessity to evaluate the gold nanoparticles cytotoxicity at all fronts. Since blood is the first point of contact in any therapy, it is required to have a thorough in vitro investigation of gold nanoparticles to avoid any adverse effects.

OBJECTIVE:

The objective of the current study is to evaluate the effect of gold nanoparticles capped with lipase on blood clotting factors, platelets, coagulation time and blood clotting strength.

METHODS:

Whole blood samples were drawn from healthy volunteers. Plasma and plasma with platelets were isolated from the blood and all the samples were treated with lipase capped gold nanoparticles, except control. Plasma fibrinogen formed in the blood coagulation process after contacting with nanoparticles was quantitatively evaluated. In addition, platelet aggregation, blood clotting kinetics, strength of the blood clot and time were evaluated post nanoparticle treatment.

RESULTS:

The work primarily explores the effect of GNPs on blood with changing concentrations of lipase capping. Plasma fibrinogen levels of plasma samples were found to be moderately elevated, however, there is no significant effect on blood clotting kinetics, strength, and platelet aggregation. Also, the study showed that lipase capped GNPs did not result in aggregation upon interaction with plasma components and remained stable for 1 hour after incubation.

CONCLUSIONS:

Our study revealed that lipase capped GNPs synthesized using NaBH₄ approach were stable and hemocompatible. There is an increase in fibrinogen levels after the exposure to nanoparticles, an observation which is consistent with other studies. However, the functional consequences of such increase are unknown. The results of no significant platelet aggregation, change in blood clotting time, kinetics, and clot strength revealed the non-toxic effect of lipase capped GNPs towards blood components, which is essential for any in vivo applications.

Keywords

Hemocompatibility of Au nanoparticles platelet aggregation plasma fibrinogen test blood clotting

1 Introduction

Gold nanoparticles (GNPs) are emerging as potential agents for treatment in biomedical field. GNPs are unique due to their physiochemical properties such as smaller size, intense surface plasmon resonance in visible light wavelength region, excellent biocompatibility, and chemical stability. In this regard, the studies are currently being carried out with attention in many areas starting from targeted drug delivery in cancer treatment, antibacterial agents to tissue imaging agents [1 –3]. Synthesis of GNPs involves reduction of Au (III) to Au (0) ions using different reducing solutions such as sodium citrate, NaBH₄, alcohols etc, which result in the size range of 3 to 200 nm depending on the synthetic route [4]. The synthesized GNPs are usually stabilized by the capping agents that prevent particle aggregation and provide interfacial properties [5]. The surface properties can be tailored by the judicial choice of capping agent. The use of biomolecules (proteins, carbohydrates) as capping agents will provide interaction via free end groups present in them. Till date much research has been focused on using GNPs for various applications and their clinical evaluation. However, relatively little is known about the potential biological risks associated with GNPs and several barriers such as chronic cytotoxicity, tumor targeting efficacy, ability to avoid generating an immune response for in vivo applications [6 –8]. Thus recently several groups focused on risk assessment on clinical evaluation of medically applied nanoparticles and there is a shift in research focus towards probing the effect of GNPs on biological processes that are critical for the cell functions. Many studies have found that polymeric nanoparticles, dendrimers, quartz particles, carbon nanotubes are cytotoxic and induce blood clotting [9 –14]. Extensive in vivo hemocompatibility analyses have been carried out only for a few nanoparticles; however, the data interpretation is complicated due to the lack of adequate nanoparticles’ characterization in terms of surface charge, size and variability of the experimental medium [15]. Recently, there is an upsurge in study on hematocompatibility of GNPs with emphasis on blood coagulation factors [16]. It is known that the size, shape, surface charge, and capping agents influence the blood compatibility of GNPs [17 –20]. Studies determining plasma binding profile of citrate-stabilized GNPs indicate that major blood proteins such as albumin, γ-globulin, fibrinogen, interact strongly with GNPs and also results in hydrodynamic size doubling of nanoparticles [21]. Studies with chitosan, and pyrimidine functionalized GNPs show that platelets do not aggregate in the presence of nanoparticles thereby affecting thrombin and fibrin components of blood clotting factors and prolong the blood clotting time [17 –20]. Since all the above studies were based on measuring prothrombin time, they avoided major cell components present in the whole blood. In this study, we carried out lipase capped GNPs’ in vitro blood interaction studies using citrated whole blood (CWB) through monitoring the viscoelastic changes, platelets aggrometry tests, and plasma fibrinogen changes [16, 22]. The results are significant in determining the effect of protein capped GNPs interaction with human blood components.

2 Materials and methods

2.1 Whole blood collection and platelets isolation

All experiments were done with prior approval from ethical committee, Institutional Review Board (IRB:2016/11/009) of Deccan College of Medical Sciences, Hyderabad, Telangana, India and the written informed consent forms were collected from the healthy individuals participated in the study. Healthy volunteers were defined according to criteria of the Nordkem-Workshop. Blood was taken from healthy volunteers of age 18–30 who were free from platelet function affecting medication for a month. Donors suffering from any metabolic disorder or ailments were not included. The data of the donors such as (sex, age, height, weight, body mass index, blood pressure, heart rate, haemoglobin percentage, WBC count) was collected and only the volunteers within the reference range for healthy humans were included in the study [23]. Whole blood samples were drawn from healthy volunteers using BD Vacutainer (3.8% sodium citrate). To isolate platelet rich plasma (PRP), the collected whole blood was diluted using Lymphoprep™ physiological saline at 1 : 1 ratio in a centrifuge tube and performed a soft spin at 130 g, at 20°C, 15 min. The obtained supernatant after centrifugation was collected into another sterile tube followed by hemocytometer count. The collected blood was again centrifuged at higher speed to obtain platelet poor plasma. Two fractions were obtained after centrifugation, the upper 2/3rd fraction is platelet-poor plasma (PPP) and the lower fraction is 1/3rd platelet rich plasma (PRP) [24, 25]. The PRP was dissolved in appropriate amount of PPP. The concentration of the platelets within the PRP was assessed, and standardized to 1200×10³ platelets/mL by adding the appropriate amount of PPP and used for the study [26].

2.2 Synthesis and characterization of gold nanoparticles (GNPs)

The lipase used in this study was isolated from Lactobacillus plantarum (MTCC 4461) [27 –29]. Hydrogen tetrachloroaurate (III) hydrate (HAuCl₄), and NaBH₄ were purchased from Sigma Aldrich.

Lipase capped GNPs were prepared by adding1 mL of 1 mM HAuCl₄ solution to a 500μL solution of lipase in different concentrations (0.1, 0.5, 1, 1.5, and 2 mg/mL) in Tris-HCl buffer, followed by addition of 300μL of 25 mM NaBH₄ solution. The successful formation of GNPs was confirmed using UV-visible spectrophotometer (Shimadzu UV-3600 plus), and dynamic light scattering (DLS) (Zetasizer Nano-ZS, Malvern). The size of the GNPs was also confirmed using high-resolution transmission electron microscopy (HR-TEM, FEI, Tecnai G2, F30). DLS experiments were done to determine changes in size of GNPs after incubation in PRP, and PPP. Citrate-capped GNPs were synthesized using standard Turkevich method for comparison [30].

2.3 Platelet aggregation studies

Two different concentrations of GNPs namely 1.0 nM and 5 nM with respect to gold concentration was used to evaluate the blood coagulation properties at 37°C. Aggregation studies were performed in glass cuvettes coated with silicon using the instrument platelet aggregometer (Chrono-Log 490 model, Chrono-Log corp). This spectrophotometer mimics the blood flow conditions, creates a shear stress using magnetic stirrers and measures the aggregation as function of difference between transmission wavelength of PPP and PRP with aggregation agonists (here L-GNPs, ADP) expressed in percentage aggregation. The recordings were carried out until ten minutes after adding lipase capped GNPs and 25 uL of 2μM Adenosine Diphosphate (ADP) to PRP.

2.4 Plasma fibrinogen tests

Plasma fibrinogen tests were determined before and after incubation with lipase capped GNPs. Plasma fibrinogen test was performed based on sandwich Enzyme-Linked Immunosorbent Assay (AbFrontier, Catalog # LF-EK0153). This test was used to quantitatively determine the in vitro fibrinogen in human plasma. To make the standard curve, appropriate volume of standard solution with dilution buffers was added to microtiter wells and then human plasma was added to the sample wells, which was diluted by at least 4000 folds. Then the solutions were incubated for two hours, after which the incubation the solutions were discarded and the wells were washed adequately. About 100μL of working secondary antibody solution was pipetted into each well and incubated for 1 hour at room temperature. After incubation, the secondary antibody solutions from the wells were decanted, followed by washing of the wells. Then the working Avidin-Horseradish Peroxidase solution was added to each well and incubated in dark for 1 h. After incubation, the wells were washed, the substrate solution was added and the plate was incubated at room temperature, followed by O.D. determination at 450 nm by the microtiter plate reader.

2.5 Blood clotting tests

Blood coagulation kinetics of lipase capped GNPs was studied employing Thromboelastography TEG^®. The clotting time (R) was observed as a function of increase in clot strength upon Ca^2 + activation of coagulation in the presence of GNPs until an increase in elasticity corresponding to 2 graphical mm was observed. A shorter R value indicates hypercoagulability. Clot strength (MA) is the measure of maximum clot strength (in mm) developed with function of time, which mainly depends on the activity of blood clotting cascade that is usually expressed in dynes/cm². The clot kinetics is measured in terms of α-angle° (clot angle).

3 Results and discussion

The work primarily explores the effect of GNPs on blood with changing concentrations of lipase capping. Therefore, we have chosen 0.1 to 2 mg/mL lipase capping solution, while keeping the gold solution and reducing agent concentrations constant. The samples are coded based on the amount of lipase capping agent used are as follows: 0.1 mg/mL – (i); 0.5 mg/mL – (ii); 1 mg/mL – (iii); 1.5 mg/mL – (iv); 2 mg/mL – (v); and citrate-capped GNPs as reference – Citrate. Scheme 1 depicts the strategy of the present hemocompatibility study on human blood using lipase-capped GNPs. Figure 1 shows the representative UV-visible spectrum of 1 mg/mL lipase capped GNPs. A gentle, but prominent plasmonic peak at ∼521 nm reveals the successful formation of GNPs. The hydrodynamic size of the synthesized nanoparticles was evaluated before and after incubation with blood plasma by DLS measurements. Table 1 lists the mean particle diameter of the different amounts of lipase-capped GNPs employed in this study ranging from 7 to 10 nm. The hydrodynamic size of lipase capped GNPs after incubation with blood plasma (PPP) was also evaluated using DLS. It was found that the particle size remain unchanged even after 1 h of incubation in the blood plasma, suggesting that there was no aggregation of nanoparticles in blood plasma due to the efficient lipase capping which had stabilized the nanoparticles and prevented the aggregation in plasma solution. Representative HR-TEM images of 1 mg/mL lipase capped GNPs are shown in Fig. 2. It can be seen that the synthesized lipase capped GNPs were found to be in the sub-10 nm in size and the particle morphology was nearly spherical.

Scheme 1

Study of hemocompatibility of lipase capped GNPs on human blood.

Fig.1

UV-VIS Spectra of lipase capped GNPs.

Table 1

Particle size analysis using DLS for the lipase-capped and citrate-capped GNPs

Sample	(i)	(ii)	(iii)	(iv)	(v)	Citrate-capped
Particle diameter (nm)	9.4	9.2	8.8	7.5	10.1	9.7

Note: (i) 0.1 mg/mL (ii) 0.5 mg/mL (iii) 1.0 mg/mL (iv) 1.5 mg/mL (v) 2.0 mg/mL lipase capped and citrate capped GNPs.

Fig.2

HR-TEM images of lipase capped GNPs.

Plasma fibrinogen levels of plasma samples were evaluated after treatment with lipase capped GNPs and the results are presented in Fig. 3. It was found that there was no significant difference among the GNPs at the two different concentrations used. However, compared to the control, a two fold increase in fibrinogen levels was observed post incubation with lipase capped GNPs. Several other studies also reported that fibrinogen is one of the major components of blood that interacts with colloidal nanoparticles [31]. There may be several reasons; one of the differences could be attributed to the slight variation in protein charge in presence of nanoparticles.

Fig.3

Plasma fibrinogen test (A) 1 nM lipase capped GNPs treatment of whole blood (B) 5 nM lipase capped GNPs treatment of whole blood. i) 0.1 mg/mL ii) 0.5 mg/mL iii) 1.0 mg/mL iv) 1.5 mg/mL v) 2.0 mg/mL lipase capped GNPs, citrate capped GNPs and control.

Therefore, it is imperative to study the effect of the increase in fibrinogen content towards strength and kinetics of blood coagulation. Thus, we further studied blood coagulation using Thromboelastography TEG^® analysis in the presence of lipase GNPs (Figs.4–6). In this experiment, in vitro blood coagulation experiments with gold concentrations of 1 nM and 5 nM with varying lipase (0.1, 0.5, 1.0, 1.5, 2.0 mg/mL) capping agent was performed. We studied the effect of blood clotting on clot strength (MA), clotting time (R) and clot kinetics (α-angle°) in the presence of GNPs along with CaCl₂ (Ca^2 +) to activate blood coagulation since blood was collected using citrated vials. TEG^® analysis of blood with 1 nM and 5 nM GNPs with varying lipase capping indicated that despite two fold increase in the fibrinogen levels, there was no significant effect on clot formation kinetics (α-angle), clot strength and blood clotting time when compared with control [16]. Furthermore, there was no clear trend in relation to the difference in lipase capping of GNPs and also the use of different concentrations of gold. This could be due to the smaller size of the nanoparticles which are usually in the range of sub-10 nm and their inability to hyperactivate the clotting cascade. Even after increasing the amount of nanoparticle concentration to 5 nM, we could not detect any significant effect on the blood coagulation process. Several studies reported that treatment with GNPs results in increase in fibrinogen, but there is no evidence that the GNPs are thrombogenic in nature. The increased fibrinogen binds to gold nanoparticle surfaces, which is usually size dependent and increases with decrease in particle size, but such a binding does not usually cause coagulation [32]. Studies have shown that citrate stabilized GNPs potentiate ADP-induced platelet activation due to rapid internalization of smaller sized GNPs and subsequent fibrinogenesis [33]. In our study we used GNPs of size ∼10 nm, which could have resulted in internalization by platelets and thereby increasing the fibrinogen content. The specific GNPs-protein corona has not been studied in detail so far and also the components and the parameters involved in blood clotting cascade that determine the clotting time and formation are not clearly understood [34 –36].

Fig.4

Estimation of clot strength (A) 1 nM lipase capped GNPs treatment of whole blood (B) 5 nM lipase capped GNPs treatment of whole blood. i) 0.1 mg/mL ii) 0.5 mg/mL iii) 1.0 mg/mL iv) 1.5 mg/mL v) 2.0 mg/mL lipase capped GNPs, citrate capped GNPs and control.

Fig.5

Estimation of clot time test (A) 1 nM lipase capped GNPs treatment of whole blood (B) 5 nM lipase capped GNPs treatment of whole blood. i) 0.1 mg/mL ii) 0.5 mg/mL iii) 1.0 mg/mL iv) 1.5 mg/mL v) 2.0 mg/mL lipase capped GNPs, citrate capped GNPs and control.

Fig.6

Estimation of clot formation (A) 1 nM lipase capped GNPs treatment of whole blood (B) 5 nM lipase capped GNPs treatment of whole blood. i) 0.1 mg/mL ii) 0.5 mg/mL iii) 1.0 mg/mL iv) 1.5 mg/mL v) 2.0 mg/mL lipase capped GNPs, citrate capped GNPs and control.

The platelet aggregation in the presence of GNPs was evaluated with a positive control ADP. The percentage of platelet aggregation (%) in the presence of lipase capped GNPs was found to be significantly lower in the different concentrations tested in this study. The percentage aggregation of platelets after 10 minutes of incubation for different concentrations of lipase capped GNPs is presented in Fig. 7. The results suggested that blood platelet aggregation was not activated in presence of GNPs. Earlier reports have shown that the nanoparticles of 20 nm size and above were actively involved in platelet aggregation [37]. The lack of aggregation can be attributed to inactivation of platelets by the smaller size of the nanoparticles used for testing.

Fig.7

Estimation of platelet aggregation in presence of lipase capped GNPs. i) 0.1 mg/mL lipase capped GNP’s ii) 0.5 mg/mL iii) 1.0 mg/mL iv) 1.5 mg/mL v) 2.0 mg/mL, citrate capped GNPs, control.

4 Conclusion

The present study revealed that lipase capped GNPs synthesized using NaBH₄ approach are stable and hemocompatible. It was found that there was two-fold increase in fibrinogen levels after the exposure to nanoparticles, which is consistent with other studies. However, the increase in fibrinogen levels was found not to have any adverse effect on the blood coagulation parameters such as clotting time, clot strength and clot formation kinetics. Furthermore, our study also revealed that there was no significant change in the platelet aggregation behavior. The hemocompatibility is attributed to the very small size regime (sub-10 nm) and the eco-friendly behavior of the probiotic lipase-capped GNPs. Future studies can be directed to determine the coagulation effects of GNPs as a function of size as well as at elevated concentrations.

References

Yeh

Y-C

, Creran

, Rotello

. Gold nanoparticles: Preparation, properties, and applications in bionanotechnology. Nanoscale. 2012;4(6):1871–80.

Yadava

, Prakash Singh

, Rengan

, Shanavas

, Srivastava

. Gold laced bio-macromolecules for theranostic application. Int J Biol Macromol. 2017. doi:10.1016/j.ijbiomac.2017.10.124.

Ghosh

, Han

, De

, Kim

, Rotello

. Gold nanoparticles in delivery applications. Adv Drug Deliv Rev. 2008;60(11):1307–15.

, Murphy

. Room temperature, high-yield synthesis of multiple shapes of gold nanoparticles in aqueous solution. J Am Chem Soc. 2004;126(28):8648–9.

Rastogi

, Kora

. Highly stable, protein capped gold nanoparticles as effective drug delivery vehicles for amino-glycosidic antibiotics. Mater Sci Eng C. 2012;32(6):1571–7.

Cai

, Gao

, Hong

, Sun

. Applications of gold nanoparticles in cancer nanotechnology. Nanotechnol Sci Appl. 2008;19(1):17–32.

Yigit

, Medarova

. In vivo and ex vivo applications of gold nanoparticles for biomedical SERS imagingi. Am J Nucl Med Mol Imaging. 2012;2(2):232–41.

Rengan

, Bukhari

, Pradhan

, Malhotra

, Banerjee

, Srivastava

, et al. In vivo analysis of biodegradable liposome gold nanoparticles as efficient agents for photothermal therapy of cancer. Nano Lett. 2015;15(2):842–8.

Dobrovolskaia

, Patri

, Zheng

, Clogston

, Ayub

, Aggarwal

, et al. Interaction of colloidal gold nanoparticles with human blood: Effects on particle size and analysis of plasma protein binding profiles. Nanomedicine Nanotechnology Biol Med. 2009;5(2):106–17.

10.

Casals

, Pfaller

, Duschl

, Oostingh

, Puntes

. Time evolution of the nanoparticle protein corona. ACS Nano. 2010;4(7):3623–32.

11.

Benetti

, Fedel

, Minati

, Speranza

, Migliaresi

. Gold nanoparticles: Role of size and surface chemistry on blood protein adsorption. J Nanoparticle Res. 2013;15(6):1694–6.

12.

Goy-López

, Juárez

, Alatorre-Meda

, Casals

, Puntes

, Taboada

, et al. Physicochemical characteristics of protein– NP bioconjugates: The role of particle curvature and solution conditions on human serum albumin conformation and fibrillogenesis inhibition. Langmuir. 2012;28(24):9113–26.

13.

Khan

, Gupta

, Verma

, Nandi

. Kinetics of protein adsorption on gold nanoparticle with variable protein structure and nanoparticle size. J Chem Phys. 2015;143(16):164709.

14.

Walkey

, Olsen

, Song

, Liu

, Guo

, Olsen

DWH

, et al. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano. 2014;8(3):2439–55.

15.

Dobrovolskaia

, Neun

, Man

, Ye

, Hansen

, Patri

, et al. Protein corona composition does not accurately predict hematocompatibility of colloidal goldnanoparticles. Nanomedicine Nanotechnology, Biol Med. 2014;10(7):1453–63.

16.

Ajdari

, Vyas

, Bogan

, Lwaleed

, Cousins

. Gold nanoparticle interactions in human blood: A model evaluation. Nanomedicine Nanotechnology, Biol Med. 2017;13(4):1531–42.

17.

Szymusiak

, Donovan

, Smith

, Ransom

, Shen

, Kalkowski

, et al. Colloidal confinement of polyphosphate on gold nanoparticles robustly activates the contact pathway of blood coagulation. Bioconjug Chem. 2016;27(1):102–9.

18.

Zhao

, Tian

, Cui

, Liu

, Ma

, Jiang

. Small molecule-capped gold nanoparticles as potent antibacterial agents that target gram-negative bacteria. J Am Chem Soc. 2010;132(35):12349–56.

19.

Ehmann

HMA

, Breitwieser

, Winter

, Gspan

, Koraimann

, Maver

, et al. Gold nanoparticles in the engineering of antibacterial and anticoagulant surfaces. Carbohydr Polym. 2015;117:34–42.

20.

Sanfins

, Augustsson

, Dahlbäck

, Linse

, Cedervall

. Size-dependent effects of nanoparticles on enzymes in the blood coagulation cascade. Nano Lett. 2014;14(8):4736–44.

21.

Aggarwal

, Hall

, McLeland

, Dobrovolskaia

, McNeil

. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv Drug Deliv Rev. 2009;61(6):428–37.

22.

Park

, Martini

, Dubick

, Salinas

, Butenas

, Kheirabadi

, et al. Thromboelastography as a better indicator of hypercoagulable state after injury than prothrombin time or activated partial thromboplastin time. J Trauma Inj Infect Crit Care. 2009;67(2):266–76.

23.

Braune

, Sperling

, Maitz

, Steinseifer

, Clauser

, Hiebl

, Krajewski

, Wendel

, Jung

. Evaluation of platelet adhesion and activation on polymers: Round-robin study to assess inter-center variability. Colloids Surf B Biointerfaces. 2017;158:416–22.

24.

Dhurat

, Sukesh

. Principles and methods of preparation of platelet-rich plasma: A review and author’s perspective. J Cutan Aesthet Surg. 2014;7(4):189–97.

25.

Szponder

, Wessely-Szponder

, Smolira

. Evaluation of platelet-rich plasma and neutrophil antimicrobial extract as two autologous blood-derived agents. Tissue Eng Regen Med. 2017;14(3):287–96.

26.

Braune

, Basu

, Kratz

, Johansson

, Reinthaler

, Lendlein

, Jung

. Strategy for the hemocompatibility testing of microparticles. Clin Hemorheol Microcirc. 2016;64(3):345–53.

27.

Khan

, Ray Dutta

, Ganesan

. Lactobacillus sps. lipase mediated poly (ɛ-caprolactone) degradation. Int J Biol Macromol. 2017;95:126–31.

28.

Ramyasree

, Dutta

. The effect of process parameters in enhancement of lipase production by co-culture of lactic acid bacteria and their mutagenesis study. Biocatal Agric Biotechnol. 2013;4(2):393–8.

29.

Khan

, Dutta

, Ganesan

. Enzymes’ action on materials: Recent trends. J Cell Biotechnol. 2016;1(2):131–44.

30.

Kimling

, Maier

, Okenve

, Kotaidis

, Ballot

, Plech

. Turkevich method for gold nanoparticle synthesis revisited. J Phys Chem B. 2006 110 15700–7.

31.

Radomski

, Jurasz

, Alonso-Escolano

, Drews

, Morandi

, Malinski

, et al. Nanoparticle-induced platelet aggregation and vascular thrombosis. Br J Pharmacol. 2005;146(6):882–93.

32.

Chen

, Ni

, Zhou

, et al. Fibrinogen clot induced by gold-nanoparticle in vitro. J Nanosci Nanotechnol. 2011;11(1):74–81.

33.

Dobrovolskaia

, Patri

, Zheng

, et al. Interaction of colloidal gold nanoparticles with human blood: Effects on particle size and analysis of plasma protein binding profiles. Nanomedicine. 2009;5(2):106–17.

34.

Deb

, Patra

, Lahiri

, Dasgupta

, Chakrabarti

, Chaudhuri

. Multistability in platelets and their response to gold nanoparticles. Nanomedicine. 2011;7(4):376–84.

35.

Nel

, Madler

, Velegol

, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater. 2009;8(7):543–57.

36.

Sperling

, Fischer

, Maitz

, Werner

. Blood coagulation on biomaterials requires the combination of distinct activation processes. Biomaterials. 2009;30(27):4447–56.

37.

Lacerda

SHDP

, Park

, Meuse

, Pristinski

, Becker

, Karim

, et al. Interaction of gold nanoparticles with common human blood proteins. ACS Nano. 2010;4(1): 365–79.