Silver nanoparticle-human hemoglobin interface: time evolution of the corona formation and interaction phenomenon
© The Author(s) 2017
Received: 17 August 2017
Accepted: 13 October 2017
Published: 30 October 2017
In this paper, we have used spectroscopic and electron microscopic analysis to monitor the time evolution of the silver nanoparticles (Ag NP)–human hemoglobin (Hb) corona formation and to characterize the interaction of the Ag NPs with Hb. The time constants for surface plasmon resonance binding and reorganization are found to be 9.51 and 118.48 min, respectively. The drop of surface charge and the increase of the hydrodynamic diameter indicated the corona of Hb on the Ag NP surface. The auto correlation function is found to broaden with the increasing time of the corona formation. Surface zeta potential revealed that positively charged Hb interact electrostatically with negatively charged Ag NP surfaces. The change in α helix and β sheet depends on the corona formation time. The visualization of the Hb corona from HRTEM showed large number of Hb domains aggregate containing essentially Ag NPs and without Ag NPs. Emission study showed the tertiary deformation, energy transfer, nature of interaction and quenching under three different temperatures.
Recently metal nanoparticle has shown its potential in diagnosis and therapy, particularly drug delivery, gene therapy, biosensor, bio imaging [1–5]. There are few successful applications of nanomaterials in biomedical domain include bacteria detection, alzheimer disease, early detection of cancer, protein fibrillation and so on. The silver nanoparticle (Ag NP) is used widely in environment, food and cosmetics industry [6–9]. Ag NP related products are used in technology, including biomedical and pharmacological applications [10, 11]. Small size silver nanoparticles (1–50 nm) exhibits larger surface area compared to its volume. This relatively large surface area increases their free energy and reactivity, which in many instances also increases toxicity . The toxicity can limit the use of Ag NP in biology unless the nano-bio interaction is fully understood. Thus the fundamental question related its safety issue to health science, remains numerous challenges. Within this field, an area that has been largely unexplored is the interaction of metal nanoparticles with proteins [13–16]. Here, we used human hemoglobin (Hb) as model protein for study the interaction with Ag NPs. Hemoglobin is a tetrameric, globular and oxygen carrier protein. It composed of four subunit among which two are identical α-chains (α1, α2 contain 141 amino acid residues each) and two identical β-chains (β1, β2 each contain 146 amino acid residues) with each subunit has one oxygen binding heme-pocket [17–19]. Hb is rich of α-helix in native state . It has various spectral signatures in electronic as well as vibrational spectroscopy . When nanosize particles contact with Hb, Hb coronas are formed on NPs. Hb corona changes the surface properties of nanoparticles and governs the interaction between nanoparticles and Hb [14–16]. It causes Hb misfolding, which is intimately related to protein-mediated diseases . In 1962, Leo Vroman showed that adsorption (which leads to corona) of blood proteins to a nanoparticle surface is time dependent .
In this paper the corona formation and structural deformation of human hemoglobin (Hb) induced by the assembly on silver nanoparticle (AgNP) surface were studied in time dependent manner by various spectroscopic techniques and electron microscopic method. The kinetic of the corona of Hb to the metalic Ag nanoparticle surface showed by the kinetic change in the LSPR of Ag nanoparticles and surface Zeta potential study along with dynamic light scattering (DLS) measurement. The circular dichroism (CD) spectra of the Hb–AgNPs bioconjugate system showed that AgNPs could dynamically induce the conversion of α-helix to β-sheet structures and deformation of the hemoglobin structure. The corona formation was studied by using DLS and zeta potential measurement.
2.1 Synthesis of silver (Ag) nanoparticles
Here, we used a simple wet chemical method for Ag NPs synthesis . Silver nitrate (AgNO3) and sodium borohydride (NaBH4) is used. Concisely, 40 mL, 20 mM AgNO3 solution was included drop wise into 60 mL, 100 mM of NaBH4 solution that had been chilled in an ice bath. Here, NaBH4 is a reducing agent which reduces the silver from AgNO3. The solution turned brighter yellow after all of the AgNO3 was added. The mixture was stirred by magnetic stirrer. The entire addition took ~ 30 min, after which the stirring was stopped. The clear yellow colloidal silver particle is stable at room temperature and stored in a transparent vial.
3 Results and discussion
3.1 Physicochemical characterization of silver nanoparticles
3.2 Effect of silver nanoparticles (Ag NPs) concentration on the hemoglobin (Hb) absorption
3.3 Kinetic study of the adsorption of Hb to the AgNPs
We have found, t 1 = 9.51 min for the binding of Hb with surface of Ag NPs for the formation of corona and t 2 = 118.48 min revels to reorganization of Hb due to binding with Ag NPs. The higher value of A2 (0.035) than A1 (0.006) implies that the Hb corona formation is more prominent than unfolding, which well agreement with other results (CD measurement, Zeta potential, DLS). The exponential association confirms that the formation of ‘Ag NPs–Hb’ corona starts immediately after incorporation of Ag NPs into Hb. The hemoglobin (Hb) needs a relatively long time to shield the original surface of Ag NPs and reorganization of Hb occurs [51, 52].
3.4 Kinetics of Ag–Hb corona by autocorrelation function and dynamic light scattering (DLS) study
3.4.1 Time dependent zeta potential of Ag–Hb corona
Changes of the surfaces charges with the increase of the time of the corona
Time of the corona (min)
Average value of the surface zeta potential in mV
3.5 Time dependent circular dichroism (CD) spectra
The change in α-helical content of Hb is not significant after a certain time of the corona, which implies that the protein could retain most of its helical structure after conjugation with AgNPs. Therefore, loss of secondary structure of the protein was expected for the basic form of Hb . This change in the original structure of the protein may result in loss of its biological activity or the activation of immune response .
3.6 Visualization of the hemoglobin corona by high-resolution transmission electron microscopy (HRTEM)
Some of these Hb aggregates have well-defined profiles while others exhibit a random, amorphous morphology. The SAED pattern of the Hb conjugated Ag NPs shows amorphous in nature due to Hb and few clear dark spot due to Ag NPs inside the Hb matrix (Fig. 9a).
3.7 Emission of Hb–Ag NPs corona under three different temperatures: fluorescence quenching study and energy transfer efficiency
The thermodynamic parameters and binding parameters
− 99.19 kJ/mol
The resultant values of the thermodynamic parameters are summarized in Table 2 to account for the main forces contributing to the stability of Hb and binding mechanism. The negative value of ΔG indicates the spontaneity of the binding of Hb to Ag NPs. The negative value of ΔH represents that binding is an exothermic interaction process. The positive value of ∆S represents that electrostatic interaction between Trp of Hb and surface of Ag NP. This electrostatic force is the foremost force for binding of Ag NPs–Hb. After electrostatic interaction, the hydrophobic interaction takes place as evident from red shift of fluorescence peak of Hb in presence of Ag NPs.
Here, I and I0 are the relative emission intensity of the Hb, in the absence and the presence of Ag NPs respectively. The energy transfer efficiency increases as the increase of concentration of the Ag NPs. The maximum calculated efficiency is around 48% corresponding to 600 µM of the Ag NPs (almost same for all three experimental temperatures). The variation of energy transfer efficiency with concentration of different temperature of the corona is shown in Fig. 11B.
To the best of our Knowledge, this is the important report to study the time dependent interaction as well as the corona formation of Hb–Ag NPs bioconjugate. The red shift of SPR peak of Ag, in Ag NPs–Hb system, confirms the complex formation of Ag NPs with Hb. The results from the time dependent SPR absorption confirm the corona formation of Hb with the surface of Ag NPs through the formation of stable corona. The time constants for surface binding and reorganization are found 9.51 and 118.48 min, respectively. The hydrodynamic size of the corona along with zeta potential and SPR absorption of the Ag NP stops increasing at some point (after 120 min), support the idea of complete corona formation needs few hours. This Hb absorption process gives the idea about ultra high molecular sensitivity method. Thin shell of Hb corona formed over the AgNP and Ag nanoparticle aggregates are distributed throughout the Hb network leads to corona and deformation of Hb structure in time dependent manner as observed from HRTEM image and CD spectroscopy. The amount of α-helix decreases 63.8%, and β-sheet increase 8.42% along with increasing the time (200 min) of the corona resultant structural deformation of Hb is also found in presence of Ag NPs. The change of intensity of the fluorescence emission peak of the Hb in the Ag NPs–Hb conjugated system confirms the occurrence of fluorescence quenching and energy transfer. The quenching process occurs via interaction of the Trp residues accessible to the metallic surface of the Ag NPs. We have found that the Trp moieties are the most favorable binding sites of Hb with Ag NPs. The positive cooperative binding at lower temperature and negative cooperative binding at body temperature of Trp with Ag NPs induces the Hb molecules to organize at the surface boundaries of Ag NPs. Our Hb conjugate Ag NPs could cover the manner for scheming new optical based materials for the application in chemical sensing or biosensing. The Ag NPs–Hb interaction represents the fundamental of bioreactivity of metal nanoparticles. A significant increase in biomedical applications of Ag nanoparticles and their potential toxicity requires several studies by different way to determine Ag NPs-protein interactions. Our results are the awareness of the health effect of Ag nanoparticles.
The experimental details and characterization methods is included in the supporting section (Additional file 1).
AKB carried out the measurement and manuscript writing. TK assisted the problem of the research. SS assisted the measurement. TK discussed and helped draft the manuscript. All authors read and approved the final manuscript.
The authors are grateful to UGC and DST for their constant financial assistance through SAP and FIST program for Department of Physics and Technophysics of Vidyasagar University. The authors are also thankful to CRF, IIT Kharagpur and IACS Kolkata for measurement facilities.
The authors declare that they have no competing interests.
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