Highly Luminescent Thermoresponsive Green Emitting Gold

Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Article

Highly Luminescent Thermoresponsive Green Emitting Gold Nanoclusters for Intracellular Nanothermometry and Cellular Imaging: A Dual Function Optical Probe Sangita Kundu, Devdeep Mukherjee, Tapas Kumar Maiti, and Nilmoni Sarkar ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00107 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Highly

Luminescent

Thermoresponsive

Green

Emitting

Gold

Nanoclusters

for

Intracellular Nanothermometry and Cellular Imaging: A Dual Function Optical Probe Sangita Kundu†, Devdeep Mukherjee ‡, Tapas Kumar Maiti ‡ and Nilmoni Sarkar†* †

Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India

‡

Department of Biotechnology, Indian Institute of Technology Kharagpur 721302, WB, India.

E-mail: [email protected] and [email protected] Fax: 91-3222-255303 Abstract In view of many promising applications of gold nanoclusters (AuNCs), nanothermometry is an important field of research in biology and medicine. Here, we demonstrate the temperature dependent photophysical properties of highly luminescent green emitting L-arginine/ 6-aza-2 thiothymine-stabilized Au nanosclusters (ATT/Arg Au NCs) by using steady state and timeresolved photoluminescence spectroscopy. Significantly, thermoresponsive properties of these highly photostable and biocompatible Au NCs are reversible, which endow the probe for further bioanalytical applications with great prospects. Additionally, protein-NC interaction mechanism has been elucidated in-vitro and in-vivo that dictates the complex behavior of the NCs with living organisms. These ultrasmall Au NCs are observed to accumulate in the cellular cytoplasm by translocating through the membrane as evidenced from the confocal laser scanning microscopy (CLSM). In vivo temperature sensing examined with human osteosarcoma cell line (MG-63 cell) by employing fluorescence lifetime imaging microscopy (FLIM) technique reveals the optimistic application of these lifetime based nanosensors in biomedicine and biotechnology. Keywords: gold nanoclusters, cellular imaging, nanothermometer, protein-corona, lifetime, fluorescence lifetime imaging microscopy 1. Introduction Temperature is the most fundamental and key parameter that governs almost all cellular responses within living organisms. Therefore, precise temperature measurement and its variation 1 ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

in vivo are very much essential for the deep understanding of the reactivity and dynamics of biomolecules inside cells responsible for all cellular events.1, 2 Intracellular temperature sensing represents a useful tool in the analysis of the behavior of cancer cells in correlation with the normal cells which further helps us to establish novel diagnostic and therapeutic methods in several diseases.3 Hyperthermia therapy used in the treatment of cancer is applied for the extraordinary heat production to kill the tumor related tissues and therefore it is very necessary to control the temperature distribution in different cellular components in order to make the therapeutic procedure more potential and effective.4 Even, in the seemingly homothermic cellular environment, slight variation in temperature affects the fundamental physiological processes like protein folding, calcium signaling, diffusion through the cell membrane, enzyme catalysis and so forth. Many approaches have been explored for carrying out thermometry having excellent thermal and spatial resolution such as 2D- Infrared (IR) thermography and infrared camera,5 spatially resolved NMR thermometry,6 raman thermography,7 atomic force microscope (AFM) based temperature mapping8 technique. But, the sensitive probing of temperature mapping at the molecular level is an outstanding challenge that encounters enormous difficulties in the interior of the living cell.9 In contrast to such modern technologies, fluorescent based nanothermometry is a powerful tool in recent area of research as these “non-contact” luminescent materials can act as dual function of imaging and sensing probes.10-15 Intracellular temperature sensing or mapping using nanoparticles16, proteins,19 fluorescent polymers20,

21

17

organic dyes,18 green fluorescent

have been investigated where the temperature dependent

emission of these luminescent materials has been exploited as the sensing mechanism for accurate determination of temperature in the field of biology. However, many of these existing methods can be limited by some technical and experimental drawbacks such as low quantum yield of these materials, low sensitivity, poor photostability, systematic errors as a consequence of fluctuations in the fluorescence rate and a general tendency of the fluorescence properties of these materials may vary with the local chemical environment and the optical properties of the surrounding medium. Satisfactorily, ultra bright luminescent nanoclusters (NCs) of the noble metals have attracted tremendous attention for their use in various biomedical applications in the field of biosensing, in vitro and in vivo bioimaging and cancer therapy owing to their ultrasmall size, high photo and colloidal stability, good biocompatibility and higher photoluminescence intensity. Shang et al.22 2 ACS Paragon Plus Environment

Page 3 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


have developed red luminescent Au NCs as potential thermometers because the thermal response of these nanoclusters reflected the thermal environment of living cells within the physiological temperature range. Biocompatible glutathione (GSH)-capped AuNCs have been revealed to sense temperature in the human and bacterial cells.23 Recently, Wu et al.24 have reported dual emissive ratiometric fluorescent probe consisting of bovine serum albumin (BSA) stabilized AuNCs and fluorescein-5-isothiocyanate (FITC) that exhibited temperature and pH responsive signals to determine temperature and pH simultaneously even in the living cells. Several fluorescent biosynthesized Cu NCs have also been explored which have shown intracellular temperature sensing and imaging applications because of their thermo-responsive properties over the physiological temperature range.25-27 While many red28,

29

or blue30,

31

luminescent

nanothermometry have been reported so far, similar nanocomposite emitting green luminescence is not common. So, there is a basic need to develop highly fluorescent green luminescent nanothermometer and bioimaging agent that can be used as an alternative of Alexa Fluor 488, common organic dye used in cell imaging in that wavelength region. However, while the applications of the nanoparticles for medical purposes are growing to increase, there are demanding concerns to characterize the effects after exposure to cells and to address their potential toxicity.32 It is now emerging to understand the biological identity of the nanomaterials which is inevitable for the determination of the physiological responses and to develop safe and effective nanomedicines.33 Upon encountering physiological environment, the nanoparticles are readily adsorbed proteins of different cellular compartments in living organisms forming “protein-corona” depending on the size, composition and charge of the nanomaterial surface.34 Moreover, due to the high surface to volume ratio of the metal NCs they are likely to interact with the proteins and thus the change in the surface properties due to the corona formation strongly affects their photophysical behaviors35 and biological responses like cellular uptake and toxicity. Therefore, from fundamental point of view, studying NC-protein interactions is of great benefit to apply them as therapeutic tools in nano-biotechnology. Herein, we have proposed luminescence and lifetime based nanothermometry with excellent temperature sensitivity and higher signal recognition ability of highly fluorescent robust Larginine/ 6-aza-2 thiothymine-stabilized Au NCs with supramolecular host-guest recognition employing the previously published protocol.36 Compared to the earlier reported Au NCs, our system is showing better temperature sensitivity (3.14% per °C) in the most relevant 3 ACS Paragon Plus Environment


physiological temperature range (36 °C to 43 °C). Interestingly, we have also observed complete reversible thermoresponsive optical properties of Au NCs. Due to the lack of molecular thermometer applicable for the live cell imaging, intracellular temperature imaging at the single molecular level was not well documented. With the help of fluorescence lifetime imaging microscopy (FLIM) technique, we have demonstrated in-vivo thermometric ability of our synthesized materials. The Au NCs are highly biocompatible, photostable, showing higher cellular uptake and exhibiting selective emission at the 530 nm wavelength range. Owing to their small size and high surface to volume ratio, Au NCs can permeate the cell membrane and accumulate in the cytoplasm as confirmed from the confocal laser scanning microscopy (CLSM) which indicates the tremendous potential of their subsequent application for staining the cytoplasm selectively in near future. In addition, internalization of the Au NCs inside cells has been supervised at the single molecular level by the fluorescence correlation spectroscopy (FCS) measurement. Moreover, detailed understanding of the interactions between proteins and preformed ligand protected small Au NCs have also been studied using fluorescence, time resolved spectroscopy and FCS. Additionally, the biological implications of the protein-corona formation have been quantitatively analyzed using cellular uptake and cytotoxicity study. So, our synthesized novel material with high temperature sensitivity in biological systems having simultaneous cellular imaging capability will pave us for devising practical development in the field of nanothermometry during the course of clinical treatment. 2. Experimental Section. 2.1. Materials and Sample Preparation. 6-aza-2-thiothymine (ATT) was bought from Alfa Aesar Chemicals Co. Ltd. HAuCl4.3H2O was received from Sigma-Aldrich. L-arginine (Arg) and NaOH were purchased from Sisco Research Chemical Laboratory (SRL), India. Bovine serum albumin (BSA) and human serum albumin (HSA) were bought from Sisco Research Chemical Laboratory (SRL) and TCI Chemicals (India) respectively. All aqueous solutions in the experiments were prepared in Milli-Q water. The chemical structures of capping ligands are shown in Scheme 1. 2.1.1. Synthesis of Water-Soluble ATT AuNCs. An aqueous solution of HAuCl4.3H2O (5 mL, 20 mM) was added to the ATT (63 mM) solution containing 0.2 M NaOH under vigorous stirring in the dark at room temperature for 6 h. The pH of the solution was maintained at 10 throughout the reaction. Then the resulting ATT Au NCs 4 ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were centrifuged at 13,000 rpm for 30 minutes to remove the extra capping ligands. The purified ATT capped Au NCs were dispersed in Milli-Q water and stored at 4 °C in dark prior to further experiments and characterizations. 2.1.2. Synthesis of Water-Soluble Arg/ATT AuNCs. L-Arg (555 µl, 32 mM) was added to the 5 mL as prepared ATT Au NC solution at pH10 and the reaction mixture was kept at 37 °C for 24 h. The as synthesized Arg/ATT Au NCs were further purified after centrifugation at 13,000 rpm for 30 minutes. The obtained purified solid Arg/ATT Au NCs were finally dispersed in Milli-Q water and stored at 4°C in dark for further use. Scheme 1. Chemical structures of ATT and L-Arg and structural model of Au NCs.

6-aza-2-thiothymine (ATT)

L-Arginine (L-Arg) O

O

H2N

O O

O

N O

N

N

O

N

N N

S

S

HN

N

O

N

N N

S

N O

N

S

N

N

S

S

S N

O

N

S N N

NH

H2N

N N

HN

O

O

H N

OH O O

N N

H2N

HN

O

NH2

NH

N

O

NH2

N

S

S

S NH2

HN

N N

NH

O

N

N

H2N

H2N

N H

O

NH2

N N N

H2N

O

H N

O O

N H

N NH

O

O

HN

N

N S

N O

O

N

S

S

N

N

N

O

H2N

S

H2N

NH

S

N

L-Arg

N

N

NH2

N H

O

NH

N N

N

H N

NH2

N N

N

NH2

O

HN

O

NH2

N H

O O NH2 O

ATT Au NCs

O

ATT/Arg Au NCs

2.2. Instrumentation: We have performed time resolved fluorescence measurement using time correlated single photon counting (TCSPC) method and Fluorescence Correlation Spectroscopy (FCS) technique. The 5 ACS Paragon Plus Environment


imaging techniques, we used: Fluorescence Lifetime Imaging Microscope (FLIM), Confocal Imaging microscope (CLSM), Transmission electron microscope (TEM). The detailed descriptions of the experimental techniques are discussed in the supporting information (See S2S5). 3. Results and discussion. 3.1. Spectroscopic and Microscopic Characterization of Au NCs. Au NCs were synthesized using one pot approach employing ATT as reducing cum capping ligand. Then, L-Arg was introduced into the capping layer of ATT where the guanidium group presented in the side chains of Arg forms supramolecular host-guest recognition due to the strong hydrogen bonding interaction with ATT molecules (Scheme 1).36 The optical properties of the Au NCs are studied using UV-vis absorption and fluorescence spectroscopy at room temperature. The absorption and emission spectra of ATT Au NCs are shown in Figure S1A. The emission, excitation and absorption spectra of Arg/ATT NCs are shown in Figure 1A and 1B. A broad excitation maximum appears at 375 nm with three small humps at 400, 475 and 496 nm that matches well with the absorption spectrum. The emission spectrum of yellow colored aqueous solution of ATT Au NCs shows maximum at 530 nm under excitation of 375 nm. Neither ATT nor HAuCl4 emits any fluorescence under the same conditions, thus the green fluorescence originates from ATT/Arg Au NCs only. It is very fascinating to observe that the fluorescence intensity of ATT Au NCs is strikingly increased by introducing Arg into the solution (shown in the inset of Figure 1A). The absorption spectrum and the color of the solution are also changed dramatically in the presence of Arg. It is may be due to the difference in the surface charge property of Au NCs indicating stronger interaction between ATT NCs and Arg molecules. The emission maxima of Arg/ATT Au NCs is independent of the excitation wavelength (data are not shown here), the emission profile is almost symmetrical and is relatively narrow (PL full width half maximum is nearly 32 nm) which correlates well with the narrow size distribution of the NCs. The ATT Au NCs shows characteristic tri-exponential decay 37, 38

with average time constant of 12.20± 0.91 ns (shown in the inset of Figure 1C). However,

upon binding with L-Arg, the average lifetime of Arg/ATT Au NCs is increased to 45.81± 3.44 ns (Figure 1C) that can be assigned to the suppressing the non-radiative relaxation pathways of the excited states.36 It is evident that the shorter lifetime components originate from the intraband d→sp transition of Au NCs38, 39 and the longest lifetime component (80.21±2.73 ns) may be due 6 ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to the singlet- triplet intraband transition.40 Several literature reports suggest that the longest lifetime component may be ascribed to the formation of complexes due to the metal (I)–metal (I) and ligand to metal (thiolate-Au (I)) charge transfer interactions40, 41 (Table S1). The morphology and size of ATT Au NCs and Arg/ATT Au NCs are confirmed from the transmission electron microscopy (TEM) and atomic force microscopy (AFM). The typical TEM images of Au NCs are shown in Figure S1B and 1D respectively. TEM image of more than 130 individual particles of Arg/ATT Au NCs shows an average diameter of 1.5± 0.15 nm. Likewise, for ATT Au NCs, there is no obvious change in the core size of Au NCs occurs after combination with Arg (shown in the inset of the Figure S1B and 1D). Furthermore, based on high resolution TEM (HRTEM) images (Figure 1E) we can also conclude that Au NCs are highly crystalline in nature where the lattice planes are well separated by 0.2 nm which matches well with the (111) plane lattice spacing of face centered cubic (fcc) Au.42 The AFM images (Figure S1C and 1F) reveal that both the NCs have typical heights in between 2.5 nm to 2.75 nm (shown in the inset of the Figure S1C and 1F). In the energy dispersive X-ray spectroscopy (EDAX) spectrum in Figure S2A and S2B, two distinct peaks of Au and S confirms the formation of Au NCs. Dynamic light scattering results also demonstrate that no further aggregates are formed after Arg addition (Figure S3).The small change in the hydrodynamic diameter may be attributed due to the thick layer of the Arg to the capping layer of the NCs. Furthermore, the zeta potentials of AuNCs have been determined to know the surface charge of the NCs. The zeta potentials of ATT Au NCs and ATT/Arg Au NCs are -35.2 mV and -28.5 mV which indicate good colloidal stability of the NCs. The small change in the value for ATT/Arg Au NCs can be ascribed due to the positive charge of the guanidium group of the Arg molecule in the ligand shell. Furthermore, X-ray photoelectron spectroscopy (XPS) has been employed to reveal the valence states of Au and S atoms in the Au NCs. It has been observed from the Au 4f XPS spectrum of Au NCs (Figure S4A), the binding energies of Au 4f7/2 and 4f5/2 are at 84.6± 0.1 eV and 88.5± 0.1 eV which indicates the presence of mixed valence states of Au(I) and Au(0) in the NCs. Furthermore, XPS spectrum of S 2p reveals the binding energies of S2p3/2 are at 161.3± 0.1 eV, 165.7± 0.1 eV(Figure S4B). Where, the dominate peak at 161.3 eV is attributed to the presence of Au-S bond in the NCs.36, 37 The other smaller peak is may be due to the unbound S present on the surface. The Au-S bonding in the Au NCs can be further confirmed from the Fourier Transform Infrared Spectroscopy (FTIR). In the FTIR spectra, the small peak at 2560 cm-1 is only found in the ATT whereas the peak is 7 ACS Paragon Plus Environment


absent in the ATT Au NCs and ATT/Arg Au NCs which suggests the direct covalent attachment of S to the Au NCs (Figure S5).The relatively strong peak in ATT Au NCs at 3411 cm-1 can be attributed to the presence of O-H stretching frequency. This peak becomes broad in ATT/Arg Au NCs due to the overlap of the O-H and N-H stretching frequency. Again, the peak at 1631 cm-1 in the NCs may be due to presence of carboxylate group of Arg or C=N stretching frequency of

0.4

b a

0.0

400 500 Wavelength (nm)

0.6 0.4 0.2

(B)

0.8 0.6 0.4 0.2 0.0 200

3

10

10

2

10

b a 0

200 400 600 Time (ns)

1

300 400 500 Wavelength (nm)

600

0

200 400 Time (ns)

(F)

10 0 0

3

10

(E)

20

(C)

Absorption Excitation

nm

(D)

Frequency (a.u.)

0.0 350 400 450 500 550 600 650 700 Wavelength (nm)

1.0

Counts (Log)

0.8

0.8

Counts (Log)

(A)

Normalized Intensity (a.u.)

1.0

PLInt. (a.u.)

ATT. Normalized PL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

3 2 1 0.0

1 2 Diameter (nm)

600

0.3 0.6 m

1μm

1 nm

Figure 1. Characterization of ATT/Arg Au NCs. (A) Fluorescence emission of ATT/Arg Au NCs (excitation at 375 nm). Inset shows emission profile of (a) ATT Au NCs and (b) ATT/Arg Au NCs. (B) Fluorescence excitation (red line, emission at 530 nm) and UV-vis absorption spectra (black line) of ATT/Arg Au NCs. (C) Time resolved emission spectrum of ATT/Arg Au NCs under excitation at 375 nm. Inset shows decay traces of (a) ATT Au NCs and (b) ATT/Arg Au NCs. (D) TEM image of ATT/Arg Au NCs. Inset shows the size distribution histogram of ATT/Arg Au NC. (E) HRTEM image of ATT/Arg Au NCs displaying the crystalline lattice planes of an individual NC. (F) AFM topography image of ATT/Arg Au NCs. The inset shows the height profile diagram along the marked line of the image. 3.2. Insights into the Interaction of Proteins with NCs.

8 ACS Paragon Plus Environment

Page 9 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Before proceeding for the in-vivo application of Au NCs, it is a primary concern to understand biological actions of NCs upon their exposure to the complex biological environment.43, 44 Here, we have studied the interaction of ATT Au NCs and ATT/Arg Au NCs in-vitro with two important serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA). It is observed that two proteins are bound to the ultrasmall Au NCs with micromolar affinities. We have studied the effects of proteins on the NCs with the help of steady state fluorescence, timeresolved spectroscopy and FCS analysis. First, we have investigated how the fluorescence profile of Au NCs is changing in presence of the proteins. It is observed from Figure S6A, the fluorescence of ATT Au NCs is 5 fold and 3 fold enhanced upon 10 µM BSA and HSA association respectively with a small 2-3 nm blue shift in emission maxima. Similarly, the fluorescence emission intensity of ATT/Arg Au NCs is increased almost 10 times and 5 times with respect to the blank buffer solution in presence of BSA and HSA respectively (Figure S6B). It is evident that protein molecules are non-fluorescent in this spectral window, so the increased fluorescence intensity of Au NCs is caused by the adsorption of the protein molecules onto the surface of Au NCs. To gain better insight into the mechanism of fluorescence enhancement, we have performed time-resolved fluorescence decay measurements. Here, for this protein adsorption experiments, the Au NCs are diluted in PBS buffer solution such that concentration becomes 5 nM to avoid the inner-filter effect and the samples are incubated at 37 °C for 6 h prior to the experiment. It has been found that diluted ATT Au NC solution possesses an average lifetime of 2.81± 0.18 ns that can be fitted by the tri exponential functions. Again, after addition of BSA and HSA the decay becomes slower (Figure S6C) and the average lifetime becomes 7.84± 0.54 ns and 6.46± 0.44 ns respectively (Table S2). Likewise, the average fluorescence lifetime of ATT/Arg Au NCs (12.18±0.84 ns) is prolonged to 23.08± 1.66 ns and 16.44± 1.18 ns upon 10 µM BSA and HSA adsorption (Figure S6D). All lifetime data of both NCs in buffer and in presence of two proteins are tabulated in Table S2. Detailed analysis reveals that the main contribution to the increment in the average lifetime data originates from the longest lifetime component. In the presence of BSA, the longest lifetime component is increased from 49.99 ns to 69.82 ns and the contribution of this lifetime component also increases from 8% to 17%. As a result, the average lifetime component is increased from 12.18± 0.84 ns to 23.08± 1.66 ns. So, the protein corona may have strong effect on the ligand to metal charge transfer and it can be proposed that the ligand related nonradiative relaxation pathways (intramolecular vibration and 9 ACS Paragon Plus Environment


Page 10 of 33

rotation) are getting suppressed because of the corona formation on the surface of the Au NCs which might be the origin of the strikingly prolonged lifetime of Au NCs induced by the proteins.35, 45 Therefore, our results imply that the protein molecules adsorb immediately onto the nanomaterial surfaces and form “protein-corona” that alters the luminescence properties of the NCs. Furthermore, Protein binding onto the surface of small Au NCs has been studied quantitatively by employing in situ FCS spectroscopy.46 We have first monitored. diffusional motion of ATT Au NCs and ATT/Arg Au NCs in water (Figure 2). For ATT Au NCs, the FCS trace is fitted well with normal 3D diffusion model

47

(details are described in the supporting

information) leading to the diffusion coefficient (Dt) 273 µm2s-1. From the value of Dt, we have determined hydrodynamic diameter employing Stokes−Einstein equation (equation 1). 𝐷ℎ =

𝑘𝐵𝑇

(1)

3𝜋ƞ𝑇

Where kB is the Boltzman constant, T is the absolute temperature; ƞ is the viscosity of the solution. In our case absolute temperature is 25 °C and viscosities of the protein solutions are measured. The hydrodynamic diameter of ATT Au NCs is 1.80 nm. It is obvious that in case of ATT/Arg Au NCs, the diffusion becomes slower due to the extra ligand shell of Arg. The diffusion coefficient of ATT/Arg NCs is obtained 201 µm2s-1 and the corresponding hydrodynamic diameter increases to 2.97 nm. Here the NCs are immersed in concentrated protein solution to ensure the complete surface coverage with the protein molecules. The diffusion of both the NCs become slower with increasing concentration of both the proteins in PBS buffer as a consequence of the increase in the hydrodynamic diameter of Au NCs. The normalized FCS traces of ATT Au NCs and ATT/Arg Au NCs in presence of increasing concentrations of BSA and HSA proteins are shown in Figure 2. Our observation indicates that both the serum proteins are adsorbed onto the surface of Au NCs with micromolar affinities and form the protein monolayer so called “protein-corona”. Consequently, the hydrodynamic diameter of the Au NCs increases due to the corona formation. The diffusion coefficient and hydrodynamic diameter of ATT Au NCs and ATT/Arg Au NCs are tabulated in Table 1. However, for two different proteins, a decrease in diffusion coefficient is observed with different extent and much slower diffusion is observed in presence of HSA compared to BSA. Moreover, ATT/Arg Au NCs exhibit stronger interaction with HSA in comparison to ATT Au NCs which is confirmed from the extent of increase in hydrodynamic diameter of ATT/Arg Au NCs in 10 ACS Paragon Plus Environment

Page 11 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence of HSA. These results are corroborated by size distribution obtained from the DLS measurements. DLS measurements showed that the bare ATT Au NCs possess an average diameter of 2.77 nm. But when incubated with the BSA and HSA protein solution the size distribution of NCs become broad with an average diameter of 12.46 nm and 14.02 nm respectively (Figure S7A). The change is more prominent in case of ATT/Arg Au NCs where after protein adsorption the hydrodynamic diameter of bare NC (5.36 nm) is increased to 19.60 nm and 18.93 nm in presence of HSA and BSA respectively (Figure S7B). To gain more insight into the formation of protein-corona, a protein layer surrounding the Au NCs has been visualized by TEM after 24 h incubation with the HSA protein at 37 °C (Figure 3).48 The results suggest that the size of ATT/Arg NC is increased after corona formation due to the adsorption of protein on the surface of NCs. Therefore, we can expect a change in the binding energy of the Au core levels of Au NCs due to the interaction with the proteins. Furthermore, we have employed XPS technique to investigate the interaction mechanism of ATT/Arg Au NCs with proteins. It has been observed that the binding energies of Au 4f7/2 and 4f5/2 of NCs are shifted to the 84.3± 0.1 eV and 87.9± 0.1 eV from 84.6± 0.1 eV and 88.5± 0.1 eV upon HSA adsorption indicating the change in the oxidation state (electronic configuration) of the Au atoms (Figure S8).35, 45 The XPS data strongly suggests the surrounding physical/chemical environment of core Au atoms is changed due to the protein adsorption. However, FTIR spectroscopy can be implied to investigate the interaction mechanism of Au NCs with proteins (Figure S9A). It is a well established technique to validate the changes in the secondary structure of proteins. According to the literature, the amide I band of the protein is mostly dependent on the secondary structure of protein.49, 50 The absorption maxima at 1655-1650 cm-1 have been attributed to the α helices, 1685-1663 cm-1 to β sheet or β turn structures, 1648-1644 cm-1 to random chains, 1639-1635 cm1

to extended chains and 1632-1621 cm-1 to extended chains or β sheet. We have compared the

relative amounts of the different structural components of protein by investigating the intensities of the deconvoluted amide I peak. After absorbing the protein molecules on the NC surface, the intensities of α helix peak (1655 cm-1) is decreased whereas the intensities of the random coil bands (1644 cm-1) and β sheets (1678 cm-1) are increased which indicates α helix structure of protein is somewhat reduced whereas the β sheet and open chain structure of protein is increased (Figure S9B).51,

52

Another evidence for the conformational changes of the protein can be

obtained by observing the changes in the amide III band between the region of 1200-1350 cm-1 11 ACS Paragon Plus Environment


(Figure S9C). For HSA, 1350 cm-1 peak corresponds to the α helix structure and 1250 cm-1 peak is attributed to the β sheet structure. It has been observed that compared to the HSA, the intensities of these two peaks have been altered in the HSA/Au NCs confirming the change in the conformation of protein upon absorption on the NC surfaces.

1.0 0.5 0.0 1.0 0.5 0.0

(A) (a) (b) (c)

0.6 0.0 0.6 0.0 10

(d) 100

(C)

1000 10000 Time (s)

100000

(c) (d) 100

(D)

(c) (d) 10000

(b)

0.6 0.0 10

(b)

1000 Time (s)

(a)

0.6 0.0

(a)

100

(B)

0.6 0.0

G()

1.0 0.5 0.0 1.2 0.6 0.0 1.0 0.5 0.0 1.0 0.5 0.0 10

1.0 0.5 0.0

G()

G()

.

G()

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

100000

1.0 0.5 0.0 1.2 0.6 0.0 1.0 0.5 0.0 1.0 0.5 0.0 10

1000 10000 Time (s)

100000

(a) (b) (c) (d) 100

1000 Time (s)

10000

100000

Figure 2. Fitted FCS traces of (A), (B) ATT Au NCs in presence of (a) 0 µM, (b) 37.5 µM, (c) 75 µM and (d) 112.5 µM BSA (A) and (a) 0 µM, (b) 37.5 µM, (c) 75 µM and (d) 112.5 µM HSA (B); (C), (D) ATT/Arg Au NCs in presence of (a) 0 µM, (b) 37.5 µM, (c) 75 µM and (d) 112.5 µM BSA (C) and (a) 0 µM, (b) 37.5 µM, (c) 75 µM and (d) 112.5 µM HSA (D).


Page 13 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Variation in Diffusion Time, Diffusion coefficient and Hydrodynamic Diameter of ATT Au NCs and ATT/Arg Au NCs in presence of different concentrations of BSA and HSA. Sample

τD (µs)

Dt (µm2s-1)

Rh (nm)

0 µM BSA

122.4±2.3

272.1

1.80

37.5 µM BSA

133.2±1.6

250.1

1.96

75 µM BSA

160.0±2.4

208.1

2.36

112.5 µM BSA

199.4±1.7

167.0

3.68

0 µM HSA

122.4±2.3

272.1

1.80

37.5 µM HSA

154.2±1.8

216

2.26

75 µM HSA

241.3±3.1

138

3.55

112.5 µM HSA

339.8±2.2

98

5

0 µM BSA

201.8±3.2

165

2.97

37.5 µM BSA

528.7±1.7

63

7.77

75 µM BSA

666.1±2.5

50

10

ATT/Arg Au 112.5 µM BSA

693.9±2.1

48

10.20

NCs

0 µM HSA

201.8±3.2

165

2.97

37.5 µM HSA

755.1±1.8

44.1

8.99

75 µM HSA

1443.1±3.3

23.1

16.47

112.5 µM HSA

1496.24±2.5 22.26

ATT Au NCs


17.08


(A)

Page 14 of 33

(B)

10 nm Figure 3. HRTEM image of (A) bare ATT/Arg Au NCs and (B) ATT/Arg Au NCs after 24 h incubation with HSA protein. Visualization of protein cloud surrounding the Au NCs are shown in the inset of the Figure (B). 3.3. Effect of protein adsorption on the cellular responses of Au NCs. It is now emerging how the presence of corona affects the biological consequences of these ultrasmall Au NCs inside living cells.53-55 To achieve this goal, NCs are treated to MG-63 cell for facile monitoring the internalization of the NCs. MG-63 cell line is derived from human osteosarcoma, a malignant bone tumor cells. Before that we have checked the stability of Au NCs in presence of reactive oxygen species (ROS) like H2O2. The fluorescence spectra and stability of ATT/Arg Au NCs exposed to the different concentrations of H2O2 (upto 10 mM concentration of H2O2) are shown in Figure S10. Due to the formation of host-guest supramolecular assembly between the ATT and guanidium group of Arg surrounding the Au core, the stability of the NCs is significantly improved. So, our synthesized ATT/Arg Au NCs is robust and exhibits excellent oxidation stability which can efficiently inhibit the diffusion of the small molecules like H2O2 on the Au core.56 In addition, we have also verified the interference of several biomarkers of cancer such as several amino acids (L-Tyrptophan, L-Hisidine, LMethionine) and biogenic amines (dopamine, histamine, trimethyl amine N-oxide, Gammaaminobutyric acid) and the results demonstrate (Figure S11) that these analytes are rarely interfering the fluorescence property of Au NCs. However, the efficient intracellular localization of these highly fluorescent Au NCs inside MG-63 cells has been corroborated from the FLIM 14 ACS Paragon Plus Environment

Page 15 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


images. With the advancement of newly developed techniques the lifetime resolved fluorescence imaging microscopy technique (FLIM) has been adopted as a powerful technique apart from the conventional confocal imaging microscopy for live cell imaging. In this technique the imaging agents are needed to emit the fluorescence signals that are quite distinguishable from the cellular autofluorescence backgrounds to detect the cell images and this method drastically reduces the interferences from the power of laser excitation source.37, 57 The lifetime images provide better contrast and sensitivity due to the spatial variations of the lifetime of the fluorophore and it is advantageous because the lifetime is dependent on the local environment of the imaging agent but is independent of the local probe concentration or fluorescence intensity. For FLIM, we monitor pixelwise single MG-63 cells confocally and record the lifetime in each pixel. The lifetime maps are constructed from the lifetime decay curves obtained from each pixel after fitting each curve with tri exponential function. The bright green fluorescence was observed from the cells under the excitation of 488 nm laser light which suggests the translocation of Au NCs through the cell membrane because of their small size (Figure S12A and S12B). The fluorescence lifetime distribution histogram obtained from the FLIM images is found quite broad which may be because of the localization of the NCs in different cellular compartments. From our experimental results, it has been observed that the ATT Au NCs in cells show lifetime peak centered at 1865± 4 ps, whereas ATT/Arg Au NCs show lifetime value around 2088± 5 ps. The increased lifetime value is due to the extra capping of Arg on the ATT capped gold core (Figure S12C). In addition, the confocal laser scanning microscope (CLSM) images of the MG-63 cells after incubation with ATT Au NCs and ATT/Arg Au NCs for 2 h in serum supplemented medium are shown in Figure S13 and 4 respectively. The images clearly show the bright green fluorescing NCs in the cytoplasm around the nucleus which confirm that the internalization of the NCs inside cytoplasm. However, we have not observed any colocalization of AuNCs with nuclear staining dye (Hoechst) after 2 h incubation. Thus, the confocal images further corroborate the good permeability of the green fluorescent Au NCs towards MG-63 cells which is the merit of these green emitting bioimaging probes. Furthermore, FCS measurements of Au NCs confirm the internalization inside living cells. Now, we have investigated the diffusion of ATT Au NCs and ATT/Arg Au NCs in MG-63 cell where the FCS traces fitted by the following equation.



𝐺(𝜋) =

1 𝑁

( ( ))( 1

𝜏 1+ 𝜏𝐷

𝛼

1

( ))

1 𝜏 1+ 2 𝜔 𝜏𝐷

Page 16 of 33

12

(2)

𝛼

Here, the value of α denotes the extent of deviation from normal diffusion (α =1). The representative autocorrelation traces in cells are shown in Figure 5A and B. In contrast to the diffusion behavior of NCs in water; both the NCs show anomalous subdiffusive motion in the interior of cell having anomality parameter α

Highly Luminescent Thermoresponsive Green Emitting Gold

Recommend Documents