Photoluminescence Mechanisms of Dual-Emission Fluorescent

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Photoluminescence Mechanisms of Dual-Emission Fluorescent Silver Nanoclusters Fabricated by Human Hemoglobin Template: from Oxidation- and Aggregation-Induced Emission Enhancement to Targeted Drug Delivery and Cell Imaging Mojtaba Shamsipur, Fatemeh Molaabasi, Morteza Sarparast, Elahe Roshani, Zahra Vaezi, Mohsen Alipour, Karam Molaei, Hossein Naderi-Manesh, and Saman Hosseinkhani ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b02674 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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ACS Sustainable Chemistry & Engineering

Photoluminescence Mechanisms of Dual-Emission Fluorescent Silver Nanoclusters Fabricated by Human Hemoglobin Template: from Oxidationand Aggregation-Induced Emission Enhancement to Targeted Drug Delivery and Cell Imaging

Mojtaba Shamsipur,a*† Fatemeh Molaabasi,b,e*† Morteza Sarparast,f† Elahe Roshani,c Zahra Vaezi,c Mohsen Alipour,g Karam Molaei,b Hossein Naderi-Maneshc and Saman Hosseinkhanid a

Department of Chemistry, Razi University, Zakariya Razi Blvd, Kermanshah, Iran Department of Chemistry, cDepartment of Nanobiotechnology/Biophysics, dDepartment of

b

Biochemistry, Tarbiat Modares University, Al Ahmad Street, Tehran, Iran e

Department of Biomaterials and Tissue Engineering, Breast Cancer Research Center, Motamed

Cancer Institute, ACECR, Gandhi street, Tehran, Iran f

Department of Chemistry, Michigan State University, East Lansing, Michigan 48824-1322,

USA g

Department of Advanced Medical Sciences & Technologies, School of Medicine, Jahrom

University of Medical Sciences, Jahrom, Iran *Tel:

+98

83

34274515;

E-mail:

[email protected]

(M.

Shamsipur);

and

[email protected] (F. Molaabasi) †

These authors contributed equally to this work.

ABSTRACT: A novel fundamental understanding of the features of mechanism for the synthesis of luminescent silver nanoclusters (AgNCs) in human hemoglobin (Hb) as capping/reducing agent based upon simultaneously size transition and fluorescence enhancement phenomena is presented. The interesting features consist of both NC core oxidation and aggregation-induced emission (AIE) attributed to ligand-to-metal charge transfer (LMCT) or ligand-to-metal-metal charge transfer (LMMCT) from Ag(I)-Hb complexes (through oxygen, nitrogen, and sulfur atoms of Hb residues donation to the Ag(I) ions) forming Ag(0)@Ag(I)−Hb core−shell NCs, the origin and consequence of which being a dual emission/single excitation nanosystem with large stocks shift and high quantum yield obtained at even high temperature is a

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challenging subject, which is not reported until now. The bioconjugation of hyaluronic acid (HA) onto surfaces of Hb layer (HA/AgNCs) produced a biocompatible platform with doxorubicin drug (DOX) as DOX/HA/AgNCs for specific imaging and delivery of DOX via an efficient targeting of CD44-overexpressing cancer cells, which lead to an increased inhibition of tumor cell growth. Additionally, the cell viability analysis illustrated that the developed nanocarriers significantly enhanced the DOX uptake in HeLa cancer cells compared to HUVEC and HNCFPI 52 normal cells allowing a selective cytotoxicity to HeLa cells. The suggested LMCT/LMMCT mechanism for emission source combined with such attractive properties as simple one pot non-toxic synthesis rout, long lifetime and large Stocks shift, excellent aqueous stability and photostability and easy functionalization capability with good cell viability, provided the possibility to AgNCs nanoprobe for use to better understanding of the nucleation and growth mechanisms via computational modeling techniques (e.g. DFT study) and also for fabrication of new nanoprobes for developing multifunctional applications in the bio-based chemical and electrochemical fields and in in-vivo research.

Keywords: Dual-emission photoluminescence mechanisms, Aggregation-induced emission, Ag nanocluster, Targeted drug delivery, Cellular imaging

INTRODUCTION Protein-protected metal nanoclusters (NCs), in particular Au and AgNCs, with molecularlike properties have exhibited a considerable application in a variety of fundamental fields from chemistry and biology to biomaterials, due to such unique features as decent biocompatibility, ultra-small size, excellent stability and large stocks shift, to name but a few.1-5 The knowledge of luminescence origin is a key point to better design and synthesize multifunctional nanoprobes, which may improve and influence cluster size, synthesis kinetics, and such luminescence characteristics as quantum yield, lifetime, and stable emission ranging from UV to infrared.6-10 In general, it has been demonstrated that the photoemission of nanoclusters could be controlled not only by small size-quantization effects,11-12 but also by the type of surface anchoring ligands and the amount of surface charge on the cluster core,13-16 in both of which charge transfer mechanisms, so-called as ligand-to-metal charge transfer (LMCT) or ligand-to-metal-metal 2 ACS Paragon Plus Environment

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charge transfer (LMMCT), play an important role in enhancement of photoluminescence emission in the core-shell structure model.17-18 In this regard, it could be referred to LMMCT mechanism, due to coupling between surface plasmon and emitters in Ag-carboxylate NCs as found by Zhang et al.19 Aggregation-induced emission (AIE) phenomenon is another thoughtprovoking mechanism by which intramolecular rotation is restricted leading to an increase in QY upon aggregation and allowing variety of applications in biosensing, imaging, catalysis and optoelectronic devices.20 Such an attractive AIE effect was confirmed for Au(I)-,21-22 Au(I)/Ag(I)-,23 and Cu(I)-thiolate complexes,24 but not for protein as a scaffold-directed synthesis of NCs. Recently, our group has discovered the capability of human hemoglobin of α, β polypeptide chains as a scaffold to stabilize fluorescent AuNCs, while there was no detailed discussion about emission mechanisms occurring kinetically under alkaline condition.25 Surprisingly, in this work, kinetically synthesis was also occurred for AgNCs owing to both temperature and time dependent photoluminescence enhancement. Therefore, taking the abovementioned points into consideration, we decided to investigate and discover all possibilities of the emission mechanisms for as-prepared silver nanoclusters capped by hemoglobin as a surface protein ligand, thereby providing a new avenue in the development of state-of-the-art NC–based AIE molecules, and thus the synthesis of highly luminescent water soluble NCs possessing sizetunable energies and minimum matrix effect in in-vitro/in-vivo assays. It should be remembered that the studies of the surface protein ligand role for the fluorescence enhancement mechanism has only been limited to the BSA-protected luminescent gold nanoclusters under atmospheric pressure treatment.26 In this work, the key role of unfolding secondary structure of protein, the type of metal and protein as well as the key role of Ag14 compared to the Ag4 during oxidation/aggregation induced-emission were confirmed, which allowing the reversibility of size transition. This discovery is actually reported here for the first time in the case of macromolecules-capped clusters with two emission wavelengths. The protein-stabilized nanoclusters can readily act as biocompatible multifunctional nanoprobes because these interesting platforms not only have advanced characteristics associated with photoluminescent NCs, but also can be easily functionalized due to the existence of protein layer on NCs; besides, even sometimes the protein retains its catalytic activity and/or targeting function, so that there is no need for covalent attachment of NCs, as previously demonstrated for Hb-AuNCs,27 Tf-AuNCs28 and HRP-AuNCs29 as well. These properties convert NCs to 3 ACS Paragon Plus Environment


competitive particles towards any cytotoxic quantum dots (QDs) and photo-bleaching organic dyes, especially in biomedical applications13, 28, 30-31. Here, the importance of cancer diagnosis and therapy was evident in developing NCs-based nanocarriers and led to successful fabrication of hyaluronic acid (HA)-Hb/AgNCs as a targeted fluorescent probe with a negligible background for bioimaging and drug delivery to CD44-overexpressing cancer cells. In addition, notably, unlike many nano-conjugates in biolabeling studies, anchoring HA as targeting, biocompatible and biodegradable ligand was easily achieved due to a lot of attachment sites on the AgNCs surface. Moreover, the surface functionalization is relatively low cost and possess no negative effect on the NCs luminescence intensity, stability of non-toxic HA/AgNCs and drug uptake, while other targeting moieties have limitations, examples of which are folic acid (FA) with poorly water-solubility, peptides with limited targeting ability, and high-cost antibodies with the shortcomings of immunogenicity.32 The favorable properties of the prepared fluorescent nanoprobes were further demonstrated by DOX/HA/AgNCs based on increased specific cytotoxicity on HeLa cancer cells and minimal toxicity to normal cells. We believed that the outstanding findings obtained in this work and previous report33 can be utilized to growth of the Hb usage, having heme group as active center and high structural flexibility, in the emerging research field of nanobiotechnology and catalysis science.

EXPERIMENTAL SECTION Materials and Reagents. Doxorubicin drug (DOX) and sodium salt of hyaluronic acid (HA) (Mw =100-120 kDa) were obtained from the Iranian Red Crescent Society and Life core, respectively. EDC, NHS, AgNO3, ethanol, cysteine, H2O2, and NaOH were purchased from Sigma. All chemicals were used as received without further purification. Synthesis of Hb/AgNCs and HA/AgNCs Nanoplatform. In this study, Hb was chosen as the stabilizing/reducing agent, which

for the synthesis of fluorescent Ag nanoclusters

(AgNCs) which can be wieldy and easily prepared with high purity (> 95%) from human blood, according to the method of William and Tsay.34 Briefly, similar to previous reports,25-27 the AgNCs was synthesized by adding an aqueous AgNO3 (5 mL, 0.055–2.0 mM) solution to a Hb solution (5 mL, 0.11mM), followed by addition of NaOH (1.0 mL, 1000 mM) solution after 10


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min and then allowed to continue under vigorous stirring for 5 days at 37 ºC. Solutions were subsequently purified through centrifugation (12000g) to remove the large silver nanoparticles. The Hb/AgNCs were functionalized by conjugating of surface amines of Hb to HA via EDC-NHS crosslinking reaction. In brief, first, the carboxylic groups of HA (5 mg mL-1) were activated by EDC: NHS (1:1 mass ratio) for 3 h. Then, AgNCs (10 mg) was added to the mixture and the reaction was carried out for 24 h to produce the HA/AgNCs nanoplatform. The product was filtered (MWCO 100 kD) to eliminate the free AgNCs and next freeze dried. Characterization. Spectrofluorometric measurements were performed using a PerkinElmer LS-50B instrument (Perkin-Elmer, U.K.). The emission spectra for Hb/AgNCs were recorded over the wavelength ranges of 390-600 nm and 700-900 nm at 1500 nm min-1 scan rate. The absorption spectra were recorded on a Model Scinco UV S-2100 (Cinco, Korea), over the wavelength range from 250 to 600 nm. The TEM and HRTEM characterization were performed on a Tecnai G2 F20 (Philips, Holland) and JEM-2200FS (JEOL, Japan) operating at 200 kV, respectively. The hydrodynamic sizes and the surface charges of the nanoclusters in aqueous solution were measured using a Zetasizer nano ZS series dynamic light scattering (DLS) (Malvern Instruments Ltd., U.K.). All pH measurements were performed using a Metrohm 713 pH/ion-meter with a standard uncertainty of 0.1 mV (Metrohm, Switzerland). The X-ray photoelectron spectroscopy (XPS) measurements were carried out with an ESCALab220I-XL spectrometer (VG company, U.K.), and an Al Ka X-ray was used as the excitation source (hν = 1486.6 eV) operating at a vacuum < 10-7 Pa. All binding energy values were aligned by adjusting the carbon peaks at 285.0 eV. The FTIR spectra of freeze-dried samples were recorded using a Tensor 27 Bruker instrument (Bruker, Japan) having a resolution of 4 cm−1. In all cases, the data were averaged over 16 scans. The CD spectra of pure Hb and Hb/AgNCs in aqueous medium were recorded on a J-715 JASCO CD spectrometer (JASCO, Japan) with a cell of 1 mm path length. The average of three scans was adopted to increase the signal to noise ratio of the CD spectra. The MALDI MS analysis of Hb and Hb/AgNCs were conducted using a Kratos Axima CFRplus (Shimadzu Biotech, Manchester, U.K.). Lifetime values were measured by time correlated single photon counting (TCSPC) on a FLS920 spectrofluorometer (Edinburgh Instruments, UK) equipped with EPL375 pulsed laser diode (5 mW, 1 MHz). The instrument response function (IRF) was recorded with a Ludox solution (Sigma–Aldrich, USA). The exponential decays were fitted by using a nonlinear least 5 ACS Paragon Plus Environment


square curve fitting procedure to a function (( = ( ( − ′ comprising of / convolution of the IRF (E(t)) with a sum of exponential (R(t) = A + ∑ ) with pre

exponential factors (Bi), characteristic lifetimes (τi) and a back ground (A). The quality of the curve fitting was evaluated by reduced chi-square and residual data. It has to be noted that with time resolved instrument, we could resolve at least one fourth of the instrument response time constants after the de-convolution of the IRF. In Vitro Cytotoxicity. To investigate the in-vitro cytotoxicity of AgNCs and HA/AgNCs, and to evaluate the cell killing efficiency of DOX/HA/AgNCs, the MTT assay method for human cervical cancer HeLa (HA receptor-positive), normal cervical cell HNCF-PI 52 and normal human umbilical vein endothelial HUVEC cell lines was used; 200µL of cells, at a density of 2 × 104 cells/well, were seeded into 96-well plate, and incubated for 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. Then, different concentrations of AgNCs, HA/AgNCs (with the equivalent concentration of AgNCs), free DOX, and DOX/HA/AgNCs (with equivalent concentration of DOX) were added. The cells were then incubated for an additional 24 h at 37 °C. Then, the medium was removed, and 20 µL of MTT solution (15mg MTT in 3ml PBS) was added and incubated for 4 h. Finally, 150 µL of MTT solubilizing agents was added to the cell. Untreated cells were used as controls. The absorbance was measured at a wavelength of 570 nm. In Vitro Imaging and Evaluation of Cellular Uptake. The Hela cell was used to investigate the uptake of as-prepared AgNCs and HA/AgNCs. Cells were seeded on a sixchamber glass slide at 1 × 105 cells/well with 2 mL culture medium (PRMI medium with 10 % fetal bovine serum (FBS) and 1 wt. % of penciling-streptomycin) in a humidified 5% CO2 incubator atmosphere at 37 °C incubator, after 24 h, the culture medium was abandoned and cells were treated with Hb/AgNCs, and HA/AgNCs (with equivalent concentration of AgNCs), followed by incubation at 37 °C for 6 h. After that, the culture media were discarded, and the cells were washed with buffer (PBS 0.01 M, pH 7.4) three times to remove any unbound AgNCs or HA/AgNCs. The fluorescence images were captured by an Olympus IX-81 fluorescent microscope (Olympus Imaging System, Japan).

RESULTS AND DISCUSSION 6 ACS Paragon Plus Environment

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Synthesis, Characterization, and Stability of Hb/AgNCs. The recorded fluorescence (FL) spectra depicts that the emission intensity gradually enhanced and was shifted from 460 nm to 445 nm with increasing of AgNO3 concentration up to 0.5 mM (Figure 1a and Figure S1 a-d), then remained nearly constant up to 1 mM (Figure 1a and Figure S1e), and finally gradually decreased at concentration of 2.0 mM (Figure 1a and Figure S1f), probably due to the ineffective protection of AgNCs by the scaffold protein, which results in damaging the cluster formation.35 From the absorption spectra shown in Figure 1b and Figure S1aˊ-fˊ, no apparent SPR absorption band was observed over the wavelength of 400 to 500 nm, revealing the absence of nonfluorescent silver nanoparticles.36-38 Moreover, the hemoglobin Soret band was decreased with a blue-shift from 410 nm to 395 nm with an increase in concentration level, indicating that the AgNCs formation can affect the hydrophobic environment of the heme group.25 Because of incomplete reaction during 5 days, the time-dependent UV-Vis and PL measurements were performed and the results revealed that the highest FL intensity was obtained after 28 days (Figure 1c), without any measurable change over longer periods of time. As is obvious from Figure 1c, a solution of Hb and AgNO3 in the absence of NaOH shows no fluorescence emission at 450 nm. However, under optimized pH of 12.4 and relative Hb/AgNO3 concentration ratio of 7.0 mg mL-1/0.5 mM, the formation of the Hb/AgNCs begins immediately after adding NaOH, as confirmed by observation of a weak emission at 450 nm. However, no measurable shift in maximum emission at 450 nm was observed (Figure 1c) which suggests the presence of single fluorescent species in solution. Meanwhile, from the time-dependent UV-Vis spectra shown in Figure 1d, unfolding of the protein skeleton with cluster formation is obvious, as the bands belong to Hb located at 280 nm (due to the phenyl group of Trp and tyrosine residues), 349 nm (ε band), 540 and 575 nm (related to oxy-band or Q-band) disappeared after formation of AgNCs and/or addition of NaOH, and even the intensity of porphyrin–Soret band of Hb at 415 nm is decreased along with a shift toward the UV region (405 nm).25 After addition of NaOH, with elapse of time, a broad absorption band related to nanocluster was appeared at 330 nm, and increase gradually, while no obvious shift corresponding to emission of fluorescent NCs was observed, indicating that the fluorescence enhancement could not be due to the quantization effect of the metal core. In other words, it can be said that, after the coordination of Ag ions with various functional groups of Hb protein such as –SH, –NH, and –OH, the cluster nucleation is 7 ACS Paragon Plus Environment


started at a basic pH of ~12.4), which is higher than the pKa of Tyr (∼10) and Cys thiols (~ 7.49.1).1, 25 In this condition, Tyr residues can greatly reduce Ag(I) ions by their phenolic groups. Moreover, a simultaneous redistribution of silver ions between Hb proteins induces the conformational changes of the protein and allows the effective formation of AgNCs, as confirmed by UV spectra.39 To further study the formation mechanism of the AgNCs in the Hb aqueous solution, the FT-IR spectra of pure Hb and Hb/AgNCs solutions were recorded (Figure S9). The FTIR spectra are expected to provide information on the secondary structural change before and after metal nanocluster encapsulation, because the amide bands are highly sensitive to environmental change and characteristic hydrogen-bond patterns. In the recorded IR spectra show the following main bands: amide I C=O stretching (1600 –1680 cm−1), amide II band arising from N–H bending (60%) and C–N stretching (40%) (1500 –1620 cm−1) and saturated C–H stretching vibrations at ~2960 cm−1. Other bands include the stretching vibration of N–H of amide group (ca. amide A′) at ~ 3425 cm−1 and C–N stretching vibration of aromatic amine at ~1395 cm−1 (Figure S9).25 Compared to the IR spectra of native protein, the IR spectra of the Hb/AgNCs showed several distinct changes. After the formation of AgNCs, the shape and peak position of the amide I band (1658 cm−1) of Hb is nearly the same while decreased in intensity. But the amide II (1535 cm−1) and aromatic amine band (1395 cm−1) present in free Hb are disappeared in Hb/AgNCs in the expense of appearance of a new band located at 1442 cm−1 (Figure S9). It should be noted that tyrosine is a very strong IR absorber, which over dominated the amide II bands. Thus, the obvious changes observed for amide II band and the aromatic amine band are responsible for the binding of silver ions with protein via free amine groups and the decreasing in intensity of amide I and amide A′ bands suggest that there is a substantial change in the conformation of the Hb from the free state, i.e. fewer helical structures are present as a result of interaction with the AgNCs. In addition, the formation of Hb/AgNCs lead to the enhancement of the peak intensity centered at 1442 cm−1, which could correspond to the vibration of tryptophan (Trp) when nanoclusters are prepared at high pH (Figure S9). The XPS results in the literature also indicate that the thiol-containing amino acids were effective for the synthesis AgNCs resulting in controlled AIE phenomenon confirmed by the photoluminescence spectra (see Figures 1c and 1e), the UV-Vis absorbance (Figures 1b and 1f), DLS (Figures 2a and S8a) and TEM images (Figures 3a-d). Therefore, it can be concluded that 8 ACS Paragon Plus Environment

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both the extremely alkaline pH condition in the synthesis of Hb/AgNCs and the consequent binding interactions between silver ions and thiol and amine groups of Hb, as excellent nucleating agents, may cause energy level splitting, electron redistribution on the cluster surface and, thus, the variation in optical properties of NCs. In other words, these are most possibly responsible for change in secondary structure arrangement of Hb and promoting the formation of clusters and fluorescence enhancement of AgNCs.26 For a more thorough elucidation of the formation mechanism, the conformational behavior of free Hb and Hb/AgNCs solutions were investigated by time dependent circular dichroism (CD) measurements. As shown in Figure S2, the free Hb shows a positive CD at 193 nm and two negative CD bands at 210 nm and 221 nm, which are characteristics of a high αhelical content. The secondary structural elements were calculated using the J-700-Standard Analysis program and found that the addition of NaOH and consequent formation of AgNCs results in a ~65% decrease in α-helical (i.e., mean residual ellipticity at 221 nm), and 76% and 12% increase in β-sheet and random coil (i.e., mean residual ellipticity at 195 and 200 nm) structures, respectively, and after that a slight change in β-sheet structure occurred during 1 day to 28 days (Figure S2b), which was partly corroborated by the size transition described in the next section. Meanwhile, after the formation of AgNCs, the negative bands at 210 nm and 221 nm and the positive band at 193 nm present in CD spectrum of the free Hb are blue shifted to 203 nm and 214 nm, and red shifted to 197 nm, respectively (Figure S2). As is known, the conformation of protein should undergo a certain degree of changes to facilitate the effective formation of NCs.39 These results are partly different from the BSA ligand with more helical ordered structures for the synthesis of AuNCs and Au/AgNCs,2, 52 which indicates that the type of ligand protein and the consequent unfolding of its structure play an essential role in determining the mechanism of cluster formation.52 Notably, α-helix has 3.6 amino acids per turn of the helix and β-sheet structure can be seen as a kind of special α-helix only with two amino acid residues through stretching. α-Helix is formed when the carbonyl oxygen of the ith amino acid bonds to the amide H of the ith + 4 amino acids away. Therefore, H bonding is the main factor to maintain the α-helix structure of the protein. Thus, based on findings from UV-vis, FTIR and CD spectroscopies, it can be concluded that the strong binding of the surface of nanoclusters to the amino acid residues of the main polypeptide chains of Hb protein causes the unfolding of the protein skeleton, which is favorable 9 ACS Paragon Plus Environment


for cluster formation and a corresponding fluorescence enhancement. In fact, the loss of α-helix stability due to breaking of hydrogen bonds, leads to an α-β transition and, consequently, results in changing of the structure of the heme group and perturbation of a lot of microenvironments around the deprotonated aromatic amino acid residue.1,25 This finally changes the microenvironment of the silver core and affect the Ag(I)−Ag(I) interactions that can be due to the ionization of both the amino and carboxyl groups in Hb and, consequently, in an increased protein charge to the metal core.25-27 It should be noted that these results are the first reports in the case of Hb protected luminescent silver nanoclusters, which are opposite to the findings reported previously for the small molecule and BSA protected luminescent gold nanoclusters1, 26 and to a certain extent in agreement with the BSA protected luminescent copper nanoclusters.34 Although the fluorescent metal nanoclusters have drawn considerable research interest in the fields of biomaterials and biology, studies on the role of surface protein ligand in the fluorescence enhancement mechanism is still limited to the BSA-protected luminescent gold nanoclusters under atmospheric pressure treatment.26 In this way, density functional theory (DFT) calculations, which is under investigation, may help a better understanding of the response of amino acids towards silver ions and the role of conformation changes in promoting the cluster formation. It is interesting to note that there was a slight change in β-sheet structure of Hb/AgNCs corroborated by the size transition, as will be discussed in the next section, so that the β-sheet band is decreased in 0 and 9 days and after that increased in 12, 16, 21, and 28 days (Figure S2b). Therefore, this is a possibility for the existence of a correlation between cluster size transition and β-sheet structure of Hb protein in the course of oxidation/aggregation process. One remarkable observation was that, in addition to emission at ~450 nm, the Hb/AgNCs exhibited a near-infrared (NIR) fluorescence emission at λem of 760 nm (λex = 320 nm) attributed to a larger cluster size, which suggesting the producing two cluster sizes, i.e. blueemitting and red-emitting clusters using Hb protein (Figure S3).13, 40-41 In this way, we used the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), as a powerful analysis method, to determine the number of Ag atoms in the protein-capped NCs at pH 12. As shown in the Figure S4 and Table S1, the free Hb has distinct peaks at m/z 30 574 - 32 027 Da, 47 440 Da and 62 887 Da, which are probably associated with dimer, trimer and tetramer of Hb subunits, respectively. As a result of AgNCs formation and following the unfolding of the Hb template, the subunit peaks were significantly decreased, broadened and shifted into two mass 10 ACS Paragon Plus Environment

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peaks at 47 894 and 48 928 Da for trimeric species, and the 63 364 and 64 397 for tetrameric species. The differences in molecular weight due to lower mass peaks (i.e., ~ 454 Da for trimer and 477 Da for tetramer) and higher mass peaks (i.e., ~ 1488 Da for trimer and 1510 Da for tetramer) could be attributed to the blue emitting Ag4NCs and red emitting Ag14NCs, respectively, in accordance with the results obtained from fluorescence measurements (Figure S4).40-43 Since the dimer is rather complex, we did not assign the fragmented peaks of clusters. As Shown in Figure S5, the bioconjugated AgNCs on Hb template was also analyzed by native polyacrylamide gel electrophoresis (PAGE) (left, 15%) and agarose gels (right, 1%). Obviously, upon Coomassie staining, distinct bands became visible for Hb and as-synthesized AgNCs at 37 °C and as-synthesized AgNCs at 67 °C, respectively (Figure S5, upper left panel, lane a, d, e) whereas Hb bands in the presence of sodium hydroxide and AgNCs with low concentration (0 day) were smeared without any distinct band in the native PAGE analysis (Figure S5, upper left panel, lane b, c). The increased mobility of the Hb/AgNCs compared with Hb, Hb/NaOH and Hb in the presence of low concentration of NCs (0 day) can also be explained based on the surface charge (zeta potential) changes (Figure S5, upper left panel). In addition, relatively narrow luminescent bands were observed under UV light for as-synthesized AgNCs so that the nanoclusters synthesized at 67 °C showed stronger greenish-blue intensity than those synthesized at 37 °C (Figure S5, down left panel). This is consistent with obtained quantum yield, further confirming that the luminescence was generated by the AgNCs in our sample and not by dark Ag NCs. Moreover, as seen, there is no band for dark NCs or other species (Figure S5) which is well verified by MALDI mass analysis (Figure S4). Moreover, as shown in the luminescence feature at 440 nm and 760 nm (Figure 1e) belonging to the Ag4 and Ag14 NCs, demonstrating that the NCs were nearly pure in the as-synthesized form and this species seems to be the most abundant product, with a yield of 41.4% for Hb/AgNCs at 37 °C and 55.4 % for Hb/AgNCs at 67 °C, as compared with the amount of Coomassie stain in the Hb bands (lane a, d, e).44-45 Similar results were also obtained with agarose gels (Figure S5, right panels). Following the above observations, it can be suggested that the ligand Hb has several beneficial roles for stable silver nanocluster formation and fluorescence enhancement of AgNCs. Interestingly enough, in contrast to Hb, using such proteins as BSA,46 HSA,13, 47 Lys,48-49 and dBSA50 as capping agents need the addition of hazardous additive and/or NaBH4 as strong reducing agent, and results in formation of single silver cluster size. These phenomena may be 11 ACS Paragon Plus Environment


due to: (i) the extraordinary flexibility of Hb structure; (ii) the high amine/thiol ratio in Hb (94/6) compared to BSA (102/35) and Lys (18/8), which allow an effective coordination of Ag ions; and (iii) the reduction of Ag ions with 18 tyrosine/tryptophan residues in alkaline media according to its pKa (~10), resulting in the formation of stable luminescent Hb/AgNCs in aqueous solution.25,

51-52

The other interesting finding was to shorten the reaction time by

increasing of temperature from 27 to 67 ºC (Figure S6) so that the maximum emission intensity for both wavelengths (450 nm/760 nm) were recorded at 67 ºC until 10 days, with similar characterization regarding to low temperature synthesis (Figures 1e and 1f). Hb/AgNCs at the physiological temperature (37 °C) showed a quantum yield of 2.8 % which increased to 3.4% at 67 ºC, using quinine sulfate as the standard (QY = 54%, in 0.1 M H2SO4).25 A number of stability tests including photostability during UV-A irradiation for 120 min (λex/λem= 320 nm/440 nm, wide-range pH test (2-12) and the ionic strength of 1.0 M NaCl were carried out on as-prepared AgNCs (Figures 1g-1i). None of cases had significant effect on the luminescence intensity of AgNCs, implying that Hb with large size (574 AA) can effectively cover the AgNCs and inhibit a cluster aggregation so that the synthesized AgNCs can be stored up to 6 months at 4 ºC without any type of change in quality.51 Moreover, at room temperature, the powder of the Hb/AgNCs was stable for at least 3 months, as it was evident from the corresponding HRTEM image (Figure 3e) and zeta potential (-48.7 ± 1 mV).53 The as-prepared AgNCs retained 85-90% of the fluorescence intensity both at the room temperature (25 °C) and at physiological temperature (37 °C) during one month in solution (data not shown). The TEM image of the luminescent AgNCs synthesized after 10 days of reaction, showed an ultrasmall and monodisperse NCs with an average diameter of 2.2 ± 0.3 nm, revealing that the Hb protein is an interesting candidate to very efficiently production of AgNCs under one-pot, green and very simple method (Figure 1j). To clarify the oxidation state and binding properties of silver nanoclusters capped by Hb, the XPS measurements were performed (Figures 1k and 1l). As shown in Figure 1k, the binding energy of Ag 3d5/2 could be deconvoluted into Ag(I) and Ag(0) components centered at 369.8 (~54%) and 368.1 eV (~46%), respectively.37, 46 The results suggest the presence of Ag(I)-Hb complex shell on the AgNC core and thus proposes Ag(0)NCs@Ag(I)-Hb complex core−shell structural model, so that the luminescent can be related to the Ag(I)-Hb complex shell.18-19, 23,54-55 Moreover, the binding energy of S 2p3/2 can be deconstructed into three different components at 12 ACS Paragon Plus Environment

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161.2 (~29%), 163.5 (~60%), and 166.7 eV (~11%) which are assigned to Ag–S, S, and oxidized sulfur, respectively (Figure 1l).37, 46,56 The high contribution of the unbound sulfur as well as low contribution of oxidized sulfur indicates the partial binding of AgNCs to the sulfur groups accompanied with a slight sulfur oxidation during the NC growing process. It is worth commenting that considering the XPS spectra, the large Stoke’s shift of the NIR emission (440 nm), and a LMCT mechanism, the emission at NIR (760 nm) could be attributed to metalcentered triplet excited state and charge transfer from Ag(I)-Hb complexes to the Ag atoms.23,5758

Mechanism for Luminescence Behavior of AgNCs. In order to investigate the luminescent enhancement mechanism, some experiments can be performed to facilitate the discussion. Dynamic light scattering (DLS) measurements were first performed as a function of reaction time to provide a conclusive evidence of size transition (Figure 2a). As seen, the average diameter was found to be 6.3 nm after 2 days, which decreased to 5.3 nm and 2.2 nm after 5 and 9 days, respectively, which caused a substantial increase in the zeta potential and, thus, the stabilization of AgNCs. Surprisingly, the average diameter begins to increase to 2.8 nm after 12 days, followed by increased to 5.1 nm on 16 days, and then remained rather constant after 21 days (~5.3 nm) and 28 days (~5.4 nm). This pattern resulted in a decrease in zeta potential arising from aggregation of nanoclusters (Figure 2b). Notably, it is not expected to consider any hydrodynamic diameter for Hb/AgNCs, because according to the CD analysis results, a complete conformational transition of α to β chains of Hb occurred in the course of cluster encapsulation in the protein, which results in exposing more hydrophobic residues of Hb on the surface; such conformational changes will limit the solvation of AgNCs and their hydrodynamic diameters.59-60 Furthermore, TEM images obtained in three time intervals of the reaction (i.e., first, middle, and last days) and surprisingly showed a size transition in the course of AgNCs formation. As shown in Figure 3a-d, the average diameter of NCs at first day of reaction was 5.71 ± 0.65 nm which decreased to 2.96 ± 1.0 nm at the middle day and after that increased to 5.16 ± 0.78 nm at the last day. The observed mean diameter of the clusters well matched the corresponding average diameters obtained from DLS and thus the size transition event was well demonstrated.53 Besides, as it is seen from Figure 3a, in the first day, the number of clusters is low in accordance with the weak luminescence intensity observed and at the middle day the number of clusters is increased corresponding to evolution of the PL spectra of AgNCs with the elapse of 13 ACS Paragon Plus Environment


time (Figure 3b) and at the last day the number of clusters has largely increased (Figure 3c, d) resulting in the maximum fluorescence intensity (Figure 1c). A high-resolution TEM image with Fourier transform (FT) pattern was recorded and is shown in the inset of Figures 3e. The HRTEM of the AgNCs, after 3 months storage at RT, revealed a clear lattice fringes with interplanar spacing distances of 0.205 and 0.239 nm, which could be attributed to two {200} and {111} main crystal planes of the face-centered cubic (fcc) Ag, as confirmed by the FT pattern (Figure 3e). This could point to the absence of such by-products as AgCl and Ag2O and, thus, supported the high purity of the AgNCs.53,61 The chemical composition of Hb/AgNCs was further proved by energy dispersive X-ray (EDX) spectrum (Figure S7) and identified the existence of Ag, Fe, and S, revealing that a single molecule of Hb protein contains 6 cysteines and 4 heme groups.41 It seems that a two-step mechanism implies to the observations which could lead to the observed size transition: a first oxidation process followed by an aggregation-induced emission (AIE), so that the presence of the two processes can inhibit a drastic size change during the reaction (Figure 3a-d). It is well known that the AEI process and the consequent cluster size changes are dependent to the nature of metal and capping agent and synthesis condition. In this way, we used hemoglobin as capping agent for the synthesis of AuNCs,25 PtNCs and compared them to AgNCs (Figure 3). Surprisingly, it was found that the size of AuNCs remained without changes during the synthesis (Figure 3f), while a drastic size change occurred for PtNCs, so that the PtNCs with size of 2-3 nm converted to porous Pt nanotetrahedra with a size of ~ 15 nm without any reversibility in size transition (Figure 3g). More investigation showed that the only effective mechanism for PtNCs size variation is AEI; thus, the key role of oxidation in size transition for AgNCs was well demonstrated. In order to investigate the effect of capping agent on the cluster size, BSA was also used for the synthesis of PtNCs and no drastic size or no emission enhancement with time was observed (Figure 3h). It was previously reported that a drastic size change for nanoclusters usually occurs in the presence of an external reagent,62-63 while in the case of our AgNCs biotemplate, a size transition was occurred during the reaction time without using any external reagent. On the other hand, from the next discussion on lifetime, it will be demonstrated that the occurrence of oxidation/AEI mechanism results in more effect on the size of Ag14NCs with near-IR emission compared to that on blue emitting Ag4NCs with respect to their PL intensity. Notably, it was hard to get clearer TEM images on these samples, 14 ACS Paragon Plus Environment

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since the distribution of nanoclusters was significantly influenced by increasing voltage or magnification of the instrument so that, in some cases, the agglomeration is occurred. This is because of the fact that the nanoclusters are capped into a bio-polymer (protein), and the high intensity electron beams can destroy it. To further elucidate these possibilities according to the previous literature, several control experiments were designed by separately introducing H2O2, cysteine, and ethanol to the Hb/AgNCs aqueous solution and investigating its fluorescence and size variation, partly supported by DLS and zeta analysis. In the case of H2O2, fluorescence response enhanced by addition of H2O2 even at different temperatures, together with a size reduction from 5.78 nm to 5.28 nm (-∆zeta = 1.5) with 10 mM H2O2 and 2.96 nm (-∆zeta = 3.9) with 100 mM H2O2 (Figure 2c-e), which could be due to an increase in electropositivity (or oxidation state) of the Ag core, similar to GSH/Au25NCs reported by Wu et al.17,19 The presence of cysteine also enhanced the fluorescence intensity with good linearity with its increased concentration (i.e. by a factor of 1.7 at 100.0 mM) that was based on the charge transfer from S atom to the Ag center,64 leading to the aggregation of Ag atoms (Ag clusters) to a larger extent, and hence bigger NCs formation from 6.35 nm to 14.18 nm (∆ zeta = 14.4) (Figure 2f-h). This finding is remarkably different from the luminescent GSH/AgNCs54 and GSH/Au@AgNCs23 which decomposed by cysteine thorough interacting with Ag(I) ions, to form Ag(I)-thiolate complexes and, thus, allowing the fluorescence quenching. Meanwhile, in the case of ethanol, an increase in fe (fe = volethanol/vol(ethanol+water)) to 40% induced a strong fluorescence enhancement with only slightly decrease in emission from 40% to 60% (Figure 2i) along with a surprisingly large increase in size (Figure 2j); however, the gradual neutralization of negative charge of AgNCs with increased ethanol concentration (Figure 2k) caused the Ag(I)−Hb complexes to get closer together and, consequently, potentially facilitated the Ag(I)…Ag(I) argentophilic interactions and finally bring about an increase in the aggregation degree of oligomeric Ag(I)−Hb complexes and consequent dense aggregate formation.24, 57-58, 6566

Besides, the addition of high volume fractions of ethanol (above 60%) disrupted the hydration

shell of Ag(I)−Hb complexes and hence might lead to a decrease in fluorescence intensity owning to the formation of smaller NC aggregates (Figure 2i).58 It follows from all these observations that the surface complex Ag(I) ions in Ag(0)NCs@Ag(I)-Hb core−shell structure play an absolutely pivotal role in exploring the causes for the time-dependent fluorescence 15 ACS Paragon Plus Environment


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enhancement phenomenon accompanying with size transition during long-term AgNCs synthesis.21 This may be explained by the combination of the two mechanisms shown in Figure 2l that: (i) the oxidation of NCs core and/or oxygen etching effect using O2 (in air) as an oxidant can induce a slower reaction rate together with promoting the charge transfer from the electronrich surface functional groups (e.g., carboxylic and amino groups) present at Hb layer to the Ag core (LMCT/LMMCT) via oxygen, nitrogen, and sulfur atoms,19 similar to the AuNCs, as described by Jin et al.;17 (ii) the occurrence of AIE mechanism in which the degree of NCs aggregation could be controlled using the intra- and inter-complex argentophilic Ag(I)···Ag(I) interactions and, as a consequence, the restriction of intramolecular motions of the Ag(I)-Hb complexes linked to the core.24, 54-55 In this sense, the LMCT/LMMCT can also contribute to the emission from the NCs aggregates in which nonradiative relaxation of excited states may be blocked and hence radiative activation occurs.20,

57-58

Bringing this in mind, these two

mechanisms in general dictated the origin of luminescent enhancement of silver nanoclusters in alkaline media (pH ∼12) in which the Ag (I) precursor was first converted into oligomeric Ag(I)X complexes (X: thiol, carboxyl and amine groups), and then reduced to Ag(0) nuclei by the reductive residues, i.e., tryptophan, tyrosine and phenylalanine in human hemoglobin;25, 67 after that, the sequestration of Ag(0) cores by Ag(I)-X complex shell and, thus, production of Ag(0)on-Ag(I)-X intermediates is followed by Ag core oxidation and/or slow aggregation into Ag(0)NCs@Ag(I)Hb complex core-shell nanostructure.6,

18-20, 58, 67

This process is facilitated

with the rise in temperature (from 27 to 67 ºC) without any other change in the experimental parameters, so that highly luminescent AgNCs can be obtained in shorter synthesis time at the elevated temperature of 67 °C (Figures 1e and S6a).58 These results indicates that high temperatures accelerate the reduction of Ag(I)−X complexes to Ag(0) with a rather broad size distribution (Figure S8a) compared to prepared AgNCs at a relatively low temperature (Figure 2a).52 In recent years, we discovered that there is an effective electron transfer between NCs and the heme group of Hb as active center in Hb/AuNCs platform. 27 According to this finding and also by considering iron-mediated polyol process,68-69 there is a possibility for the heme group containing a trace amount of iron species to slow down the reduction reaction of the Ag(I)X complexes, during which the Ag nuclei will oxidize to Ag(I) by Fe(III) ions and after that the resulting Fe(II) can be readily converted to Fe(III) by oxygen. However, by an increase in 16 ACS Paragon Plus Environment

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temperature to 67 °C, the heme degradation and/or iron release from Hb takes place (after 2 days) and causes a faster reduction of Ag(I)−X complexes to Ag (0) and favoring the formation of Ag NC aggregates. The heme degradation was indicated by the change in solution color from red to brownish-yellow, and the absence of Soret band in near-UV (400-436 nm) (Figure 1f). It should be noticed that, the heme group was partly intact after 48 h, which is potentially useful in photo- and electrocatalytic applications as well as in photovoltaic processes (Figure S6b). To further understanding of the possible reasons for the occurrence of luminescence enhancement associated with size transition, the excited-state dynamics were investigated by a time-correlated single photon counting (TCSPC) at two wavelengths of 440 and 760 nm for Hb/AgNCs prepared at different reaction times (Table 1, Figure 4). All luminescence decay spectra could be fitted by a biexponential function with χ2 values ≤ 1.3 (Figure 4c-f). As shown, the average lifetime of emission at 440 nm increased from 2.481 to 2.656 ns corroborated with the fluorescence enhancement during 28 days of synthesis, which seems to be independent of the NCs size (Table 1, Figure 4a). However, the average lifetime of emission at 760 nm decreased from 0.983 µs in 0 day to 0.948 µs and 0.885 µs after 5 days and 9 days, respectively, along with an oxidation feature (Table 1). Surprisingly, after that, the average lifetime increased to 0.903 µs after 12 days and 1.043 µs after 28 days (with AIE feature) (Table 1, Figure 4b). Therefore, in contrast to nanosecond results, the obtained microsecond results showed the size-dependent behavior and interestingly it is almost in agreement with the size transition as demonstrated by DLS and TEM measurements. As mentioned above, this can be due to the fact that the NIR emission in silver clusters originates from the charge-transfer triplet excited state of the surface complex Ag(I) ions through Hb layer (LMCT/LMMCT) whereas the blue emission is mainly attributed to the singlet excited states which could be attributed to Ag(0) core transitions.20,24,57-58 In addition, as seen in Table 1, the size-dependent triplet excited state behavior of Hb/AgNCs was confirmed in the presence of additives including ethanol, Cys, and H2O2 as follows. The microsecond time was reduced in the presence of H2O2 as the cluster size decreased due to oxidation Ag (0) core, while the microsecond lifetime was increased with the addition of Cys and ethanol because the AEI phenomena strengthened via the charge-transfer triplet excited state (Table 1). In this connection, DLS analysis and time-resolved measurements, as other control experiment, were also performed for AgNCs synthesized at 67 °C over 9 days. The results 17 ACS Paragon Plus Environment


showed a decrease in size from 7.1 nm in 0 day to 2.6 nm in 1 day and after that an increase in size to 4.1 nm, 5.2 nm and 6.1 nm were observed after 4, 7, and 9 days, respectively (Figure S8a). Meanwhile, the microsecond lifetime showed a decrease from 2.616 µs in 0 day to 1.172 µs in 1 day and then increased to 1.277 µs and 1.912 µs after 4 and 9 days, respectively (Table 1). Therefore, it can be concluded that the presence of both oxidation and AIE mechanisms simultaneously or sequentially affect the lifetime- and size-transition process during fluorescence enhancement of Hb/AgNCs. The nanosecond and microsecond lifetimes in luminescent Hb/AgNCs were similar to the those of AgNCs reported previously, such as DPA/AgNCs51 and GSH/[email protected] The data well indicate that the large stocks shift and the long lifetime belong to the red emitting AgNCs could be assigned to the oxidation state of metal center, phosphorescence emission, and also size transition event via metal-metal and metal-ligand charge transfer with triplet multiplicity.70-71 Considering the observed advantages of photo and chemical stability, biocompatibility, long lifetime and larg stocks shift, make the AgNCs suitable for in vitro and in vivo bioimaging as well as multimodal imaging for efficient and accurate diagnosis of various diseases.4,18 The present work is the first document for the observation of the size transition during fluorescent enhancement phenomenon that, according to the corresponding mechanisms, can create a novel proposal to stimulate experimental and theoretical design of metal nanoclusters and their use in developing biosensors, cancer cell imaging, targeted drug delivery nanosystems, and optoelectronic nanodevices. In vitro Cytotoxicity and Live Cell-Specific Targeting. Thanks to the unique properties of the as-prepared bionanomaterials, we designed novel imaging and targeted drug delivery nanocarriers for cancer treatment based on applying hyaluronic acid (HA) binding Hb-stabilized AgNCs.4,72 In this sense, EDC/NHS activation was used to link the primary amines of Hb/AgNCs to carboxyl group of HA at pH ~7 as confirmed by the related FTIR spectra in which the reduced intensity of carboxyl (1618 cm-1) and the appearance of ether groups (1038 cm-1) in HA/AgNCs well established the successful carboimide crosslinking reaction (Figure S9 with more detail in SI).73 Two cell lines with the above-mentioned interesting aims, i.e., HUVEC and HeLa cell lines, were chosen and incubated with various concentrations of the as-prepared AgNCs and HA/AgNCs (0.5−5 mg mL-1) to investigate their cell viability using MTT assay. As shown in Figure 5a,b, both Hb/AgNCs and HA/Hb/AgNCs displayed high biocompatibility and low cytotoxic activity for both cancer cells and normal cells at even high concentrations, as the 18 ACS Paragon Plus Environment

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viability remained above ca. 85% after 24 h incubation time,31 revealing that both NCs are of high biocompatibility and promising potential to use in tumor targeting, which is definitely due to the presence of Hb protein and HA, because the surface properties of nanoparticles play an absolutely crucial role in their nanotoxicity to cells. Furthermore, it has been proved that the proteins, as scaffold, can reverse the cytotoxicity to the cells by the positive nutrition and growth-promoting effect; additionally, their conjugation with HA, possessing targeting and biocompatible behavior, will substantially improve the noncytotoxity of the as-prepared nanoclusters.72,74-75 It is worth mentioning that the slight differences between cytotoxicity of Hb/AgNCs and HA/Hb/AgNCs on HUVEC and Hela cells could be attributed to: i) different uptake potential of the cell lines; ii) different growth rate of the used cell lines, which is directly related to the time required for the toxicity of nanoplatform on cells to emerge.76 The inhibitory effect of DOX released from DOX/HA/ AgNCs nanohybrid on the growth of cancer HeLa cells at a series of DOX concentrations (0.2-5.0 µg mL-1) loaded was investigated (see Figure 5c). The cytotoxicity of free Dox, as control experiment, was also tested and added to Figure 5c. The results depict that DOX/HA/AgNCs present a much higher cytotoxicity to HeLa cells compared to the normal HUVEC cells (Figure 5c, d). Moreover, compared to free Dox, the cytotoxicity of DOX/HA/ AgNCs was more to HeLa cells, and less to HUVEC cells. (Figure 5c,d). This phenomenon can be attributed to more uptake of DOX by HeLa cells, which results from CD44 receptor-mediated specific endocytosis and allowing growth inhibition and, thus, the cell death (Figure 5c, d). Consequently, the DOX released from DOX-loaded HA/AgNCs holds a potent anticancer and tumor-targeting activity.32,78 The biocompatibility of HA with CD44 less-expressed normal cells was also demonstrated by nonspecific permeation of the free DOX, leading to a similar (with 2 and 5 µg mL-1 DOX) and/or even lower cell viability (with 0.2-1 µg mL-1 DOX) in normal HUVEC cells compared to the CD44 over-expressed HeLa cells (Figure 5c, d). Notably, since the HeLa cell was known as a cancer cervical cell line, all the above-mentioned cytotoxicity experiments were also performed on HNCF-PI 52 cells, as a normal cervical cell line, to provide a more accurate comparison measurement of cytotoxicity (Figure S10). However, almost similar results were observed for both HNCF-52 PI cell and HUVEC cell compared to the HeLa cell (Figure S10). Human cervical cancer HeLa cell lines were selected in order to study the cellular uptake of AgNCs and HA/AgNCs, and capability of cancer-targeting yet sustained drug delivery 19 ACS Paragon Plus Environment


potential of the nanohybrid. CD44 over-expressing HeLa cells were incubated with both Hb/AgNCs and HA/AgNCs (1 mg mL-1) and the related images were recorded at 37 ºC after 2 h. As shown in Figure 6b,c, a strong blue fluorescence, originating from Hb/AgNCs was observed inside the HeLa cancer cells incubated with HA/AgNCs, which may be due to the specific interaction of HA with CD44 receptor, which results in an efficient uptake of HA-functionalized NCs (Figure 6a-c).75 In contrast, the uptake of un-functionalized AgNCs, by which weaker fluorescence signals was obtained in CD44 over-expressing HeLa cells, was rare even after 4 h of incubation, indicating a decreased amount of silver nano clusters internalized by the cancer cells (Figure 6d-f).72,78 The reason behind the above mentioned observations may be due to the fact that the HA/AgNCs utilized both the HA receptor-mediated specific endocytosis and the enhanced permeability and retention (EPR) mechanisms, while the Hb/AgNCs can only utilize the EPR route to accumulate in the cancer cells.15,74,76-77 Interestingly, the results well demonstrated the excellent protective effects of Hb layer against proteolysis to maintain the fluorescent property of Hb/AgNCs during the endocytosis. The selective cellular uptake of the drug was well clarified by comparison of the DOX release between free DOX and DOX/HA/AgNCs with different concentrations of drug (0.2-5 µg mL-1) against HeLa cancer cells under green excitation fluorescence microscope (Figure S11). As seen, after 2 h incubation at 37 °C, the DOX signal increased in a dose-dependent manner; the DOX/HA/AgNCs showed an efficient inhibitory effect on cellular uptake at lower DOX concentrations compared to the free DOX; however, good uptake was only occurred only at high concentration (5 µg mL-1) for free DOX.32 As a result, less DOX regime is needed by utilizing DOX/HA/AgNCs, which absolutely decrease the side effects of a high dose drug regime, such as improper efficacy and toxicity. The other interesting finding was discovered from the red fluorescence intensity inside cells at the same DOX concentration (1 µg mL-1 DOX) for three incubation time points (i.e., 0.5, 1, and 4 h) and surprisingly significant uptake was observed after 0.5 h of incubation for DOX/HA/AgNCs, which enhanced with increased incubation time, indicating a great amount of nanocarriers internalized into the cancer cells by a short time contact with the cells, while free DOX exhibited scanty uptake even at the 4 h time point in HeLa cancer cell lines indicating that specific cancer cell release could be achieved with the DOX/HA/AgNCs system triggered by HA binding to CD44 overexpressed on HeLa cells in


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receptor-mediated endocytosis event leading to faster drug release at lower DOX levels (Figure 7).32, 78-79

CONCLUSIONS

In summary, human hemoglobin as a stabilizer, reducer, and linker was found to play a key role in developing new concept for the synthesis mechanism of ultrasmall Hb capped silver nanoclusters (Hb/AgNCs) with single excitation/dual maximum emission fabricated by a facile one-pot green approach under mild condition, without any toxic additive (e.g. NaBH4 and organic solvent). The successful characterization of the as-prepared AgNCs offers Ag(0)@Ag(I)−Hb core−shell structure formation controlled by combination of two interesting growth mechanism features oxidation of NCs core and aggregation-induced emission phenomena, owing to the aggregation of oligomeric Ag(I)-Hb intermediates causing luminescent enhancement as well as well-defined size transition event occurring during NCs synthesis. These NCs aggregates served as both selective nanocarrier and imaging probe in targeted cancer cell therapy by easily conjugation to hyaluronic acid (HA) with high biocompatibility, specificity and affinity toward CD44 receptor. Importantly, compared to free DOX, it was found that DOX/HA/AgNCs nanohybrid could cause an efficient uptake by HA reporter-mediated endocytosis at lower DOX concentrations for a short period time, allowing effective inhibition of HeLa cancer cell growth without any obvious side effects to normal cells. The reasonable cell viability, long lifetime, large Stocks shift, and excellent stability results such as good photostability, long-term colloidal stability for at least 6 months and also easy functionalization due to existence of Hb protein located at the surface of AgNCs enabled the proposed nanoprobe to construct novel biocompatible and stable multimodal nanosystems/nanoconjugates using a simple, low-cost, mild and non-toxic route, making it a promising candidate for imaging in vivo, gene delivery, biosensing, photocatalysts, and even for electrochemical applications.

Supporting Information: Detailed description on characterizations and optimization of the hemoglobin-stabilized Ag nanoclusters together with additional microscopic images are presented in supporting information. 21 ACS Paragon Plus Environment


Notes The authors declare no competing financial interest.

AUTHOR INFORMATION Corresponding Authors * Mojtaba Shamsipur, E-mail: [email protected]. Tel: +98 83 34274515. * Fatemeh Molaabasi, E-mail: [email protected]

Author Contributions †

M. Shamsipur, F. Molaabasi and M. Sarparast contributed equally to this work.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the support of this work by Research councils of Razi and Tarbiat Modares Universities, Iran National Science Foundation, and National Elite Foundation of I.R. Iran. The kind assistance of Fatemeh Rabani, Abouzar Ravandi and Behnam Hajipour from Department of Biology of Tarbiat Modares University is also acknowledged.

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Figures and Legends

Figure 1. Photophysical characterization and optimization. (a, b) fluorescence and UV−vis spectra for as prepared AgNCs using different concentrations of AgNO3 with λex = 360 nm, respectively. (c, d) fluorescence and UV−vis spectra of Hb-stabilized AgNCs upon different times of synthesis (λex = 360 nm, λem = 450 nm). (e, f) UV−vis and fluorescence spectra of AgNCs upon different times of synthesis at 67 ºC with λex = 320 nm, λem = 440 / 760 nm. (g) Photostability of Hb/AgNCs after 2 h with λex/λem= (320 nm)/ (440 nm). (h) The pH dependence fluorescence intensity of Hb/Ag NCs in 10 mM of PBS solution. (i) Fluorescence spectra of Hb/AgNCs in the absence and presence of 1 M NaCl. (j) TEM image of Hb/AgNCs. (k, l) X-ray photoelectron spectrum of Ag (3d) and S (2p) for the Hb/AgNCs obtained after 28 days at 37 ºC. [Hb] = 0.11 mM, [AgNO3] = 0.5 mM, [NaOH] = 1.0 cc of 1 M, Vtotal = 10 cc.


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Figure 2. Synthesis mechanism identification. (a) Size distribution and (b) zeta potential of AgNCs obtained from monitoring reaction times with 0.11 mM Hb and 0.5 mM AgNO3 at 37ᵒC. (c, d) Fluorescence enhancing effect of Hb/AgNCs at 450 nm (λex = 360 nm) in the presence of 12.1 mM H2O2 (c) after 5 min and (d) at different temperatures. (h) Size distributions of AgNCs in the absence and presence of H2O2. (f) Fluorescence spectra of AgNCs representing the enhancing effect upon addition of different concentrations of Cys at 450 nm (λex = 365 nm). (g) Plot of fluorescence ratio (F/F0) of the Hb/AgNCs at 450 nm for different concentrations of Cys. (k) Size distributions of AgNCs in the absence and presence of Cys. (i) Fluorescence spectra of Hb/AgNCs in mixed solvents with different fe (ethanol/water) at λex = (360 nm)/(450 nm). (j) Size changes, and (k) zeta potential of Hb/AgNCs versus f e.

Figure 3. TEM analysis. TEM images for as-prepared Hb/AgNCs at three interval times of reaction: (a) first day; (b) middle day; (c, d) last day. (e) indicates the HRTEM of Hb/AgNCs with the corresponding FFT pattern after being exposed to the electron beam. TEM images for (f) Hb/AuNCs; (g) Hb/PtNCs; (h) BSA/PtNCs. Scale bars: 20 nm for (a-d, h) images, 5 nm for (e); 10 nm for (f); 100 nm for (g). Figure 4. Lifetime investigation. Luminescence decay curves for AgNCs obtained at different reaction times at (a) λem = 440 nm and (b) λem = 760 nm. The exponential fit curves of the experimental data and corresponding residuals of fitting for (c, e) λem = 440 nm and (d, f) λem = 760 nm. The inset in (a) represents the Y-axis in log scale to depict the differences at long time of as-prepared AgNCs during reaction. Figure 5. MTT test. (a, b) In vitro cytotoxicity of the blank AgNCs and HA/AgNCs using MTT assay after 24 h of treatment against Hela cells and HUVEC cells, respectively. (c, d) Cytotoxicity of the DOXloaded AgNCs and free DOX at the different concentration against Hela cells and HUVEC cells, respectively.

Figure 6. Cellular imaging. Fluorescence images of HeLa cells incubated with (a-c) HA/AgNCs and (d-f) AgNCs solutions for 2 h under a concentration of 1 mg/mL AgNCs. Scale bar, 20 µm

Figure 7. Effect of incubation time of AgNCs on the HeLa cell uptake. Fluorescence microscope images of HeLa cells incubated with the DOX-loaded AgNCs and free DOX (DOX equivalent = 1 mg/mL) for 0.5, 1, and 4 h at 37 °C followed by Dox fluorescence (red color). Scale bar is 20 µm.



Page 34 of 43

Table 1. Lifetimes together with the normalized fractional populations for the assynthesized luminescent Ag NCs Luminescent AgNCs synthesized at 37 °C

τ1 (ns)

τ2 (ns)

0 day

0.56 (28.3%) 0.52 (26.4%) 0.54 (27.0%) 0.48 (24.6%) 0.55 (27.5%)

λem = 440 nm 3.24 2.481 (71.7%) 3.31 2.573 (73.6%) 3.34 2.584 (73.0%) 3.36 2.651 (75.4%) 3.40 2.616 (72.5%)

5 days 9 days 12 days 16 days

‹τ › (ns)

χ2

τ1 (µs)

τ2 (µs)

1.08

0.58 (90.0%) 0.64 (91.6%) 0.59 (86.2%) 0.59 (85.4%) 0.55 (83.3%)

λem = 760 nm 4.6 0.983 (10.0%) 4.32 0.948 (8.36%) 2.73 0.885 (13.8%) 2.74 0.903 (14.6%) 3.83 1.097 (16.7%)

1.50 1.10 1.19 1.20


‹τ › (µs)

χ2 1.02 1.20 1.12 1.12 1.13

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28 days 28 days/Ethanol (20% vol) 28 days/Cys 28 days/H2O2 Luminescent AgNCs synthesized at 67 °C 0 day 1 day 4 days 9 days

0.53 (26.7%) 0.48 (24.4%) 0.58 (28.3%) 0.53 (27.0%)

3.43 (73.3%) 3.32 (75.6%) 3.36 (71.7%) 3.32 (73.0%)

0.55 (26.3%) 0.58 (26.1%) 0.55 (23.4%) 0.54 (24.2%)

3.30 (73.7%) 3.34 (73.9%) 3.42 (76.6%) 3.39 (75.8%)

2.656

1.30

2.627

1.20

2.573

1.16

2.565

1.20

2.577

1.16

2.619

1.50

2.748

1.20

2.701

1.20

0.61 (85.8%) 0.74 (73.5%) 0.53 (78.0%) 0.4

3.67 (14.2%) 4.73 (26.5%) 3.63 (22.0%) -

0.22 (60.7%) 0.38 (76%) 0.62 (86.5%) 1.10 (79%)

6.32 (39.3%) 3.68 (245) 5.50 (13.5%) 4.97 (21%)


1.043

1.03

1.796

1.07

1.211

1.02

0.400

1.20

2.616

1.13

1.172

1.01

1.277

1.35

1.912

1.30


Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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Figure 6.



Figure 7.


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TOC

A dual emission silver nanocluster was synthesized in human hemoglobin and the all possible mechanisms of emission including “Aggregation-Induced Emission” and “Oxidation” was investigated. The as-synthesized nanoclusters exhibited a promising potential in targeted drug delivery and cell imaging via hyaluronic acid networks.


Photoluminescence Mechanisms of Dual-Emission Fluorescent

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