Research Article pubs.acs.org/journal/ascecg
A Theragnosis Probe Based on BSA/HSA-Conjugated Biocompatible Fluorescent Silicon Nanomaterials for Simultaneous in Vitro Cholesterol Effluxing and Cellular Imaging of Macrophage Cells Shanka Walia,†,‡ Anika Guliani,†,‡ and Amitabha Acharya*,†,‡ †
Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur (H.P.) 176061, India Academy of Scientific & Innovative Research (AcSIR), New Delhi, India
‡
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S Supporting Information *
ABSTRACT: Fluorescent silicon NPs (Si-NPs) and 3-mercaptopropionic acid-coated CdS NPs (MPA-NPs) were prepared and conjugated with two different albumin proteins, viz., BSA (B) and HSA (H). The absorption, fluorescence, FTIR, circular dichroism, and gel electrophoresis studies confirmed the conjugation of proteins to NPs. DPPH assay confirmed that the conjugated proteins retained their functional activity even after chemical modifications. The sizes of Si-NPs by TEM were found to be ∼8.7 ± 2 nm, whereas MPA-NPs showed individual particle sizes of ∼4.6 ± 1 nm. The in vitro studies suggested that these NPs were highly biocompatible. The potential of these protein-conjugated NPs in cholesterol effluxing and fluorescence imaging was studied using two different macrophage cell lines, viz., human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). Results suggested that HSA-conjugated NPs showed better cholesterol effluxing ability and superior penetration toward these treated cells. Intracellular presence of Si-NPs was confirmed by confocal microscopic studies. All these studies unravelled the potential of Si-NPs as a theragnosis probe for future biomedical applications. KEYWORDS: Silicon NPs, Albumin proteins, Cytocompatibility, Cholesterol effluxing, Confocal imaging
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INTRODUCTION Different molecular imaging modalities have been used for diagnosis of diseases, but unfortunately, all suffer from some drawbacks and need improvements.1 Nanotechnology considerably facilitates the development of molecular diagnostic probes.2 In the past few years, fluorescent nanoparticles (FNPs) with targeted biological applications have gained the utmost attention.3,4 Fluorescence imaging plays an important role in clinical diagnosis due to their added advantages, viz., ease of detection of events in real time, high sensitivity images with dynamic details, and quantitative information at cellular/ subcellular levels with high spatial resolution at nanometer range.5,6 Additionally, FNPs are widely used because these are brighter and more stable than organic dyes and fluorescent proteins.7 The commonly used FNPs include inorganic NPs especially quantum dots (QDs), viz., CdSe, CdTe, CdS, PbS, ZnS, Ag2S, ZnO, etc.,2,8,9 rare earth oxides (iridium, ruthenium), lanthanide complexes, dye-doped silica NPs, etc.10,11 In the © 2016 American Chemical Society
literature, Cd-based FNPs have been extensively studied for bioimaging, but strong toxicity concerns restrict their clinical applications.8,11−13 In this respect, there are some better substitutes as compared to Cd-based QDs, viz., carbon dots, metal nanoclusters, upconversion luminescent NPs, and Si-NPs.4,14 Silicon (Earth’s second most abundant material) NPs possess strong fluorescence emission and offer excellent biocompatibility (since Si degrades into silicic acid which is nontoxic in nature and thus is easily excreted out from the body), with no or minimal inflammatory response in vivo.14−16 However, smaller particles resulted from the fast biodegradation of Si-NPs are generally recognized and scavenged as foreign particles by the immune system, which is inevitably reflected in the decrease in fluorescence intensity of these NPs and restricts their use in Received: August 19, 2016 Revised: December 5, 2016 Published: December 19, 2016 1425
DOI: 10.1021/acssuschemeng.6b01998 ACS Sustainable Chem. Eng. 2017, 5, 1425−1435
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ACS Sustainable Chemistry & Engineering long-term bioimaging.14 Therefore, to increase their bioavailability and retention time, it is indeed necessary to chemically modify their surface via oxidation, silanization, hydrosilylation, chemical conjugation, etc.15,17 Albumin proteins, viz., BSA (B) and HSA (H), share structural as well as ∼75.52% similarity in their amino acid sequence.18 Both of these albumin proteins can express their binding moieties for many ligands like various metabolites, hormones, etc., though to a different extent due to their difference in structural rigidity.18 It has also been known that these albumin proteins can play a significant role in cholesterol effluxing in plasma.19 Further, surface coating of NPs with these albumin proteins may significantly increase the retention time of the corresponding NPs.14 Cholesterol, synthesized by liver or supplied in diet, is important in constituting cell membrane structure, production of hormones, and shielding of nerves. Deposition of excess of cholesterol in cells or serum causes blocking of the arteries and is a major cause for cardiovascular disorders.20 Thus, cholesterol content in the cells should be balanced, otherwise it may lead to necrosis or apoptosis. Normally most of the cells cannot degrade cholesterol; however, numbers of intracellular proteins are responsible for maintaining the required concentration of the same, inside and outside the cells.21 Here in this paper, we have reported a mild chemical synthetic procedure for the preparation of two different FNPs, viz., silicon NPs (Si-NPs) and 3-mercaptopropionic acid-coated CdS nanoparticles (MPA-NPs). Further, to increase their efficacy in biomedical applications, these NPs were conjugated with BSA and HSA. Spectroscopic, microscopic, and gel electrophoresis studies were done to characterize and confirm the conjugation of the respective proteins on the NP surface. In vitro studies carried out with two different macrophage cells, viz., human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC), suggested excellent biocompatibility and increased cholesterol effluxing ability for the protein conjugated NPs. The intracellular presence of the prepared Si-NPs was confirmed by confocal microscopic studies.
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(MPA-NPs) and corresponding conjugates of BSA and HSA (M-B and M-H), and APTES-coated Si NPs (Si-NPs) and corresponding conjugates of BSA and HSA (Si-B and Si-H) were synthesized and characterized. Synthesis of GSH-NPs, MPA-NPs, and Si-NPs. Glutathionecoated CdS nanoparticles (GSH-NPs) were synthesized and characterized based on the report published elsewhere.8 MPA-NPs were prepared according to the already published literature report with modifications.22 Initially, 400 μL MPA was added to 40 mL CdCl2 (5 mM) and left for stirring at 160 °C for 1 h under inert atmosphere. The pH of the solution was raised to 6.5 by dropwise addition of NaOH (1 mM). The reaction mixture was refluxed for the next 30 min, and then, 40 mL of Na2S (2 mM) solution was added dropwise and refluxed, while the color of the solution turned yellow. The reaction mixture was concentrated and then purified using dialysis membrane against Milli-Q water for 6 h. For Si-NPs preparation, 3 mL APTES was added to 12 mL preheated water, and the solution was further heated at 80 °C for 20 min.23 Next, 3.75 mL (+)-sodium L-ascorbate (0.1 M) solution was added to it, and the reaction mixture was refluxed at 90 °C for 2 h until the solution turned reddish brown. The cooled solution was preserved in an ambercolored glass vial. Conjugation of BSA and HSA to Prepared Fluorescent NPs. The conjugation of proteins, viz., BSA and HSA, to the NP surface was done by using the EDC/NHS coupling reaction with some modifications.24 The activation of the protein surface was done by dissolving 4 mg/mL of respective proteins in 1 mL of EDC (0.1 M)/ NHS (0.4 M), diluted in 10 mM NaOAc (pH 5) buffer in an ambercolored vial, and the solution was stirred for ∼4 h. To these solutions, 1.0 mL of freshly prepared Si-NP solution was added, and the reaction mixture was stirred in the dark overnight. The as-prepared protein conjugates, viz., Si-B and Si-H, were purified by using an Amicon filter of molecular weight cutoff ∼ 50,000 Da. A similar procedure was also followed for the preparation of M-B/M-H and G-B/G-H though the activation using EDC/NHS was done on MPA-NPs and GSH-NPs surfaces, respectively. Characterization. The absorption and fluorescence spectra of the prepared samples were recorded in a NanoDrop 2000 UV−vis spectrophotometer and Varian Cary Eclipse fluorescence spectrophotometer (ex/em slit width was 10/10, respectively). Fourier transform infrared spectroscopy (FTIR) studies were carried out in Agilent Cary 600 Series FTIR spectrometer. The hydrodynamic diameter and zeta potential values were measured using a Zetasizer Nano ZS (Malvern Instruments). TEM and HRTEM images were obtained by Tecnai T20 twin, TEM 200 kV (FEI Netherlands). The confocal microscopy was done using a confocal Carl Zeiss LSM 510 META. For UV−vis and fluorescence spectroscopy, 3 mL of each of the samples were used, whereas for DLS/Zeta studies 1 mL of each of the samples was used. For CD spectroscopy studies, 100 μL of each sample diluted in 1 mL of water was used. Both TEM and HRTEM studies were performed by directly placing a drop of different NPs on carbon-coated copper grids. Bradford and DPPH Assay. Protein estimation was done using the Bradford assay, whereas the DPPH assay was performed to ascertain the functional activity of the conjugated proteins. SDS-PAGE. The protein-conjugated fluorescent NP complexes were run for gel electrophoresis studies (SDS-PAGE) to check the binding of proteins on the NP surface. The running time for gel electrophoresis was set for 6 h at 70 V and 50 mA. For gel studies, 10 μL of an aliquot of the individual samples were mixed with 10 μL of the loading buffer (50% glycerol, 10% SDS, 0.1% bromophenol blue, and tris-HCl at pH 6.8), and the mixture was cast into the gel wells and run along with the protein marker (12% resolving gel, 4% stacking gel). Coomassie brilliant blue was used for staining the gel for 1 h and then destained overnight. Circular Dichroism (CD) Spectroscopy. CD spectra of native BSA, HSA, and NP-conjugated BSA/HSA were recorded to observe the changes in the protein secondary and tertiary structures after chemical conjugation. The spectra were recorded between 190 and 340 nm. The helical content of free and bound BSA and HSA was calculated from the mean residue ellipticity (MRE), in terms of deg cm2 dmol−1.
EXPERIMENTAL SECTION
Materials. (3-Aminopropyl)triethoxysilane (APTES), (+)-sodium L-ascorbate, bovine serum albumin (BSA), human serum albumin (HSA), 3-mercaptopropionic acid (MPA), N-hydroxysuccinimide (NHS), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), ammonium persulfate, tetramethylethylenediamine, acrylamide, N,N′-methylenebis(acrylamide), coomassie brilliant blue, sodium dodecyl sulfate, glycerol, bromophenol blue, cholesterol efflux assay kit (MAK192), sulforhodamine B (SRB), poly-L-lysine, dialysis tubing cellulose membrane, Bradford reagent, 2,2-diphenyl-1-picryl hydrazyl (DPPH), and propidium iodide (PI) were purchased from Sigma-Aldrich. Amiconultra-15 centrifugal filter (cutoff ∼ 50,000 Da), trichloroacetic acid (TCA), and glacial acetic acid (GAA) were purchased from Merck. Cell lines, viz., human coronary artery endothelial cells (HCAEC), human umbilical vein endothelial cell (HUVEC), and their specific growth medias were purchased from Lonza, India. Sodium sulfide flakes, cadmium chloride monohydrate, glutathione (GSH), disodium hydrogen phosphate anhydrous, sodium dihydrogen phosphate monohydrate, NaCl, and NaOH were purchased from SRL, India. Trisodium citrate and tris HCl were procured from SDFCL, India. Cell line, namely, BALB/C monocyte macrophage (J774), was obtained from National Centre for Cell Science (NCCS), Pune, India, and its media RPMI 1640 was procured from Gibco, Invitrogen. Sterile chamber slides with covers were purchased from Thermo Scientific. Initially, GSH-coated CdS NPs (GSH-NPs) and corresponding conjugates of BSA and HSA (G-B and G-H), MPA-coated CdS NPs 1426
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ACS Sustainable Chemistry & Engineering Scheme 1. Chemical Structures of (a) APTES, (b) MPA, and (c) GSH
Figure 1. Absorption (A) and fluorescence (B) spectra of GSH-NP (black line), MPA-NP (red line), and Si-NP (blue line) respectively.
Figure 2. Photographic images of the prepared fluorescent NPs and their protein conjugates under visible (top row) and UV (bottom row, excitation 364 nm) light. Glass vials arranged from left to right correspond to Si-NP, Si-B, Si-H, MPA-NP, M-B, M-H, GSH-NP, G-B, and G-H, respectively. MRE =
θ 10rl(n)
Cell viability (%) =
Absorbance of sample × 100 Absorbance of control
Cholesterol Efflux Assay. The cholesterol efflux assay was performed with both HCAEC and HUVEC to check the efficacy of the prepared NPs. The assay was performed on a 96 well plate containing 1 × 105 cells/well. The adhered cells were first washed with RPMI media (without serum), and then, the cells were treated with cholesterol effluxing reagents as per the instructions given in the kit (100 μL/well) and incubated for 16 h. The labeling reagent was not added for the control samples. After 16 h, the cells were washed and treated with three different concentrations, viz., 32.5, 65, and 130 μg/ mL, of each sample, viz., Si-B, Si-H, M-B, M-H, and BSA and HSA as controls and incubated for 2 and 4 h. A similar concentration of Si-NPs and MPA-NPs were used by adjusting the dilution. At the end of incubation, the supernatants were separated, and their fluorescence was measured (ex/em = 482/515 nm). Finally, the cells were solubilized using 100 μL of cell lysis buffer, and the fluorescence was measured (ex/ em = 482/515 nm). The percentage of cholesterol efflux was calculated by the formula
where θ is the observed CD (in millidegrees), r = number of amino acid residues in proteins, l is the path length of the cell (in cm), and n is the molar concentration of albumin proteins.25,26 Cell Culture and Maintenance. Three different cell lines, viz., HCAEC, HUVEC, and J774, were maintained for in vitro studies. All the cells were incubated in a humidified incubator at 37 °C with 5% CO2, and the culture medium was changed as and when required. Cytocompatibility Studies Using SRB Assay. The cell viability was calculated using the SRB assay.27 The macrophage cell lines, viz., HCAEC and HUVEC, were dispersed in a 96 well plate with total cell density of 20,000 cells/well (100 μL/well) and maintained at 5% CO2 and 37 °C in a 95% air incubator for 12 h.28 Next, the cell media was removed, and the cells were treated with three different concentrations, viz., 32.5, 65, and 130 μg/mL (based on the concentrations of proteins attached on Si-B, Si-H, M-B, and M-H, as estimated from the Bradford assay). The control studies were carried out using similar concentration of native BSA and HSA. For Si-NPs and MPA-NPs, the concentration was adjusted by appropriate dilution. Cytocompatibilty studies were carried out at two different time intervals, viz., 24 and 48 h. After the desired time duration, the cells were fixed with 10% TCA and frozen at 4 °C for 30 min, and then, SRB was added to stain the fixed cells for the next 30 min. The excess dye was removed by washing the cells with 1% acetic acid 5−6 times, and then, the cells were air-dried. The protein bound dye was dissolved using 10 mM tris base. Finally, the plates were read using a microplate reader at optical density of 510 nm, and cell viability was calculated using the following formula:
Cholesterol efflux (%) =
Fm × 100 Fc
where Fm is fluorescence intensity of the supernatant and Fc is the fluorescence intensity of the cell lysate and supernatant. Confocal Microscopy. The imaging studies were done by seeding HCAEC, HUVEC, and J774 cells (6000 cells/well) in eight well chamber slides pretreated with poly-L-lysine and allowed to adhere for 24 h. After removing the media and subsequent washing with PBS, these 1427
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Figure 3. continued
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Figure 3. FTIR spectra of (A) MPA-NP and (D) Si-NP, whereas (B), (C), (E), and (F) represent corresponding spectra for M-B, M-H, Si-B, and Si-H, respectively. cells were incubated with 100 μL of each sample (65 μg/mL), viz., Si-B, Si-H, M-B, and M-H, for 2 h. BSA, HSA, Si-NPs, and MPA-NPs were used as controls. Then, each of the samples was incubated with 200 μL of 1.5 mM PI for ∼5 min followed by rinsing with 20 mM sodium chloride sodium citrate (SSC) buffer. Finally, the cells were imaged under a laser scanning confocal microscope. The band-pass for NPs was kept at 505− 530 nm, whereas for PI it was 560−615 nm.
respectively, suggesting none or minimal interference of protein conjugation on the individual NPs absorption profile (Supporting Information, Figure S1). The excitation (ex) wavelength vs fluorescence intensity plot corresponding to each fluorescence emission (em) wavelength for the individual NPs are summarized in Figure S2 of the Supporting Information. Fluorescence spectrophotometric results suggested that GSH-NPs showed maximum emission intensity of ∼52 at ∼527 nm when excited at 380 nm (Figure 1B, Supporting Information, Figure S2). Similarly, MPA-NPs showed a maximum fluorescence intensity of ∼754 at ∼558 nm when excited at 360 nm. Si-NPs showed a maximum fluorescence intensity of ∼490 when excited at 440 nm with emission at ∼528 nm. When BSA and HSA were excited at 280 nm, both showed emissions at ∼347 nm. The protein emission bands of conjugated NPs, viz., G-B/G-H, M-B/M-H, and Si-B/Si-H, were observed at ∼332/337, 343, and 341 nm, respectively (Supporting Information, Figure S3 and Table S1). Such a hypsochromic shift with low fluorescence emission intensity in G-B/G-H might have resulted from the poor conjugation of both the proteins on the GSH-NPs surface. When G-B/G-H, M-B/MH, and Si-B/Si-H were excited at their respective NP excitation wavelength, these showed emission peaks at ∼517, 558, and 512 nm, respectively. UV Irradiation Studies. When all the samples were irradiated with 364 nm light, Si-NP, Si-B, and Si-H showed blue fluorescence emissions, whereas MPA-NP showed orange emission, and M-B and M-H showed light yellow emissions
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RESULTS AND DISCUSSION The synthesis of GSH-NPs, MPA-NPs, and Si-NPs and their protein conjugates are described in the Experimental Section. The chemical structures of APTES, MPA, and GSH are depicted in Scheme 1. Spectroscopic Characterization of Prepared Nanomaterials. The absorption and fluorescence spectra of GSH-NPs, MPA-NPs, and Si-NPs were studied, and the data are compiled in Figure 1. The absorption peaks were observed at ∼440, 370, and 560 nm for GSH-NPs, MPA-NPs, and Si-NPs, respectively (Figure 1A). Further, attachment of BSA and HSA on GSH-NPs, MPA-NPs, and Si-NPs was also analyzed by spectrophotometric studies. Both BSA and HSA showed a characteristic protein absorption band at ∼277 and 275 nm, respectively, whereas protein-conjugated NPs showed a blue shift in the absorption band at ∼261, 260, and 259 nm for G-B/G-H, M-B/M-H, and SiB/Si-H, respectively (Supporting Information, Figure S1). Such a change in absorption band might have resulted due to the conjugation of proteins on the NP surface.29 Further, the characteristic absorption peaks of individual NPs were observed at ∼440, 370, and 560 nm for G-B/G-H, M-B/M-H, and Si-B/Si-H, 1429
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Figure 4. TEM micrographs of (A) MPA-NP, (B) M-B, (C) M-H, (G) Si-NP, (H) Si-B, and (I) Si-H. Corresponding HRTEM images are given in (D), (E), (F), (J), (K), and (L), respectively. Inset of (C) represents a closer look of M-H, with scale bar of 20 nm.
negative zeta potential value followed by GSH-NPs and Si-NPs, which can be correlated with the decreasing negative charge of individual ligands, viz., MPA, GSH, and APTES. The protein conjugated NPs, viz., G-B/G-H, M-B/M-H, and Si-B/Si-H, suggested the particle sizes were ∼6392/6483, 396 ± 27/436 ± 23, and 321 ± 10/422 ± 29 nm with corresponding zeta values in the range of ∼ −12.9 ± 15/−19.9 ± 14, −12.2 ± 10/−21.4 ± 12, and −6.5 ± 10/−9.2 ± 9 mV, respectively. The increase in the particle sizes as well as changes in the negative zeta potential values suggested protein conjugation on the NP surface. Further, the large particle sizes obtained for both G-B/G-H suggested
(Figure 2). It was further observed that neither GSH-NP nor G-B/G-H responded to UV irradiation. Size and Surface Charge Analysis. To illustrate further the effects of proteins on NPs size and charge, DLS and zeta potential studies were performed. DLS and zeta results suggested the sizes of GSH-NP, MPA-NP, and Si-NP were ∼12.9 ± 3, 93.8 ± 15 and 4.8 ± 1 nm and −36 ± 6, −37 ± 4 and −2.5 ± 2, respectively (Supporting Information, Figure S4 and Table S2). The higher size of MPA-NPs might have resulted from the aggregation of individual MPA-NPs, which was later confirmed by HRTEM studies. It can be noticed that MPA-NPs showed maximum 1430
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Figure 5. Plots for cholesterol efflux assay of (A) HCAEC and (B) HUVEC at 2 h (black bar) and 4 h (red bar). The numerical 1, 2, and 3 in the panels correspond to concentration of 32.5, 65, and 130 μg/mL, respectively.
the primary structure of the protein was retained even after chemical conjugation. The ability of the corresponding NPs to retain the secondary and tertiary structures of proteins was evaluated using CD spectroscopy (Supporting Information, Figure S8B). CD studies were carried out in the wavelength range of 190−340 nm, where 200−260 nm represents the far-UV range (for secondary conformational changes) and 250−340 nm signifies the near-UV range (for tertiary conformational changes). All the samples, viz., BSA, HSA, M-B/M-H, and Si-B/Si-H, showed the characteristic peak of α-helical conformation at ∼207 and 223 nm (originated from n → π* and π → π* transitions, respectively, of the amide groups in the peptide bond), indicating that protein secondary structures were retained after chemical conjugation.25,32 Further CD plots of the near-UV region resulted in no significant differences for native proteins and NP conjugated proteins, suggesting that the tertiary structure of the respective proteins were preserved even after chemical conjugation. Functional Status of Conjugated Proteins. To verify whether the conjugated proteins retain their functional activities on the NP surface, a DPPH assay was performed.33 Increased antioxidant activity was observed for all the NP−protein conjugates suggesting retention of functional activities for the conjugated proteins (Supporting Information, Figure S9). Morphological Characterization. All the NPs and their protein conjugated counterparts were characterized by TEM studies (Figure 4). The sizes of the individual Si-NPs were found to be ∼8.7 ± 2 nm, whereas MPA-NPs showed particle sizes of ∼4.6 ± 1 nm. It may be noticed that for Si-NPs mostly spherical and monodispersed particles were observed, though micrographs of MPA-NPs suggested aggregated and nonspherical structures. When both of these NPs were conjugated with BSA and HSA, larger sized polydispersed particles of sizes, ∼26.9 ± 5 (M-B), 49.5 ± 4 (M-H), 194 ± 25 (Si-B), and 141 ± 10 (Si-H) nm, were observed where metallic NPs appeared as dark spots, and the conjugated proteins can be visualized as a thin film present around the NP surface. Further, HRTEM studies revealed lattice fringes corresponding to Cd and Si for protein-conjugated NPs, thus suggesting the presence of both the metals in the respective protein counterparts. To evaluate the biological efficacy of the prepared nanomaterials, cytocompatibility and cholesterol efflux assay were performed on two different macrophage cell lines, viz., HCAEC and HUVEC, whereas suitability of these prepared nanomaterials as molecular imaging agents were studied by confocal microscopy additionally on J774 (BALB/C monocyte macrophage) cells. The selection of these cell lines were made on the basis of the
possible sedimentation of both of these samples, which in turn was supported by the precipitation observed for these samples (Supporting Information, Figure S5). Thus, for all future studies, only MPA-NPs and Si-NPs and their corresponding protein conjugates were used. Fluorescence Stability Studies. The stabilities of MPANPs/M-B/M-H and Si-NPs/Si-B/Si-H were analyzed by spectrophotometric studies over a period of 6 days (Supporting Information, Figure S6). Results suggested that MPA-NPs showed minimal change in fluorescence intensity as well as in peak position and symmetry, whereas Si-NPs indicated a decrease in fluorescence intensity. Protein conjugation on the NP surface is expected to decrease the fluorescence intensity, and this was observed for all the samples, viz., M-B/M-H and Si-B/Si-H. Further, it may be mentioned that the comparative decrease in the relative fluorescence intensity plot suggested BSA/HSA conjugation resulted in increased fluorescence stability for Si-NPs, which in turn is expected to enhance its utility in the biological milieu (Supporting Information, Figure S6). Anchoring of Proteins to NP Surface. FTIR analysis was carried out to confirm the conjugation of proteins on the individual NP surface (Figure 3, Supporting Information, Table S3). MPA-NPs showed the presence of a disulfide (−S−S−) peak at ∼507.15 cm−1, whereas the corresponding thiol (−SH) peak was absent at 2550−2600 cm−1, suggesting conjugation of MPA on the CdS NP surface. The IR spectra of M-B and M-H showed both amide I (∼1651.65 and 1650.76 cm−1, respectively, for CO stretching) and amide II (1555.16 and 1558.18 cm−1, respectively, for N−H bending) peaks, which correspond to protein conjugation on the MPA-NP surface. Similarly, Si-NPs showed strong peaks at ∼3487.68 and 1084.84 cm−1 corresponding to N−H (primary amine) and Si−OR stretching, respectively. Further, Si-B and Si-H showed amide I peaks at ∼1635.92 and 1644.64 cm−1 and amide II peaks at ∼1552.45 and 1552.67 cm−1, respectively. All these results suggested successful conjugation of proteins on the NP surface.30 Protein Structural Integrity on NP Surface. The concentration of proteins on the NP surface was estimated using the Bradford assay31 (Supporting Information, Figure S7) and suggested a ∼1.3 mg/mL of protein presence in the individual NP solution. The protein conjugation on the NP surface was further confirmed by SDS-PAGE studies. Both BSA and HSA showed a thick band at ∼66 kDa, which was also observed for M-B/M-H and Si-B/Si-H samples, suggesting the presence of the respective proteins on the NP surface (Supporting Information, Figure S8A). The gel studies further indicated that 1431
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Figure 6. Confocal microscopy images of different nanomaterial (65 μg/mL)-treated HCAEC (rows 1 to 3) and HUVEC (rows 4 to 6) after 2 h. The first, second, third, and fourth column corresponds to bright field, propidium iodide stained (PI, ex/em = 543/617 nm), nanoparticle treated (ex/em = 458/520 nm) and merged images, respectively.
Cytocompatibility Studies. Cytocompatibility experiments were performed to evaluate the cellular tolerance limit of these prepared nanomaterials (Supporting Information, Figure S10). To check the cell viability, both the cells were treated with three
fact that macrophages participate in cholesterol homeostasis. Further, the overdeposition of cholesterol causes atherosclerosis in foam cell macrophages in the arterial walls, and hence, these cells can act as cholesterol donors.34−36 1432
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Research Article
ACS Sustainable Chemistry & Engineering different concentrations of NPs, viz., 32.5, 65, and 130 μg/mL, in triplicate and were incubated for 24 and 48 h. Studies suggested that all the samples showed excellent cytocompatibility at both the time intervals and for both cell lines. Cholesterol Efflux Assay. Albumin proteins are known for their ability to act as reverse cholesterol transporters.19 The ability of the prepared nanomaterials to act as cholesterol efflux agents was tested against both the cells, viz., HCAEC and HUVEC (Figure 5). Appropriate control studies were performed to monitor the effect of protein attachment on the NP surface. From the graphs, it was evident that both BSA and HSA can act as cholesterol efflux agents, though at different extents for HCAEC (∼66%−85% and 92%−98% for BSA and HSA, respectively, at 2 to 4 h) and HUVEC (∼30%−50% and 30%−52% for BSA and HSA, respectively, at 2 to 4 h). Further, it was observed that for HCAEC, both Si- and MPA-conjugated protein NPs showed an increase in the cholesterol effluxing ability to a marginal extent (∼74%−98% for Si-B/Si-H and ∼84%−98% for M-B/M-H); however, no direct correlation could be established on the protein concentration/time duration and cholesterol effluxing ability of the NPs. Interestingly, when HUVEC cells were treated with Si-B/Si-H and M-B/M-H, an ∼2 times increase in cholesterol effluxing ability was observed, which was both concentration- and time-dependent; i.e., a higher cholesterol efflux was observed at higher concentration and longer time duration. Further, it was found that Si-B/Si-H showed better cholesterol effluxing ability compared to M-B/M-H. Cellular Imaging. To study the suitability of these NPs as bioimaging agents, three different cell lines, viz., HCAEC, HUVEC, and J774, were incubated with M-B/M-H and Si-B/SiH for 2 h and imaged under a confocal microscope along with the appropriate control NPs, viz., BSA, HSA, MPA-NP, and Si-NP (Figure 6 and Supporting Information, Figure S11). The green and red channels corresponds to NP and propidium iodide (PI) emissions, respectively, whereas the last column reflects the merged images. It can be noticed that the red fluorescence response was observed for all the samples though the green fluorescence response was observed only for Si-NP, Si-B, and Si-H, which was further supported by the corresponding merged images. Further, better fluorescence response was observed for HUVEC compared to HCAEC, and among the three samples, green fluorescence intensity followed an order of Si-NP < Si-B < Si-H, suggesting more intracellular presence of silicon nanoparticles in the case of Si-H. All these studies suggested that the prepared Si-based nanomaterials can be used as molecular imaging systems for HCAEC and HUVEC.
here that CdS is a direct band gap semiconductor with a band gap of 2.42 eV, whereas Si is an indirect band gap semiconductor with a band gap of 1.11 eV (at 320 K), and hence, Si enjoys a much longer excited state than the direct band gap semiconductor.38 The DLS and zeta studies suggested an increase in size as well as negative zeta potential of the individual NPs after protein attachment. Further, Si-NPs and Si-B/Si-H and MPA-NPs and M-B/M-H were found to be reasonably stable over a period of 6 days. The structural integrity and functional activity of the conjugated proteins on the NP surface was confirmed by SDSPAGE, CD, and DPPH studies. TEM studies suggested the size of Si-NP to be ∼8.7 ± 2 nm, whereas ∼4.6 ± 1 nm sizes were obtained for MPA-NP. Further, HRTEM and FTIR studies reflected that the metallic NPs are anchored on the large protein surface via chemical conjugation. Cytocompatibility studies suggested that MPA-NP showed reduced cell viability as compared to Si-NP for both the cell lines, though protein conjugation on both NP surfaces increases their cytocompatibility. The cholesterol efflux assay suggested both the cells behave differently in the presence of nanomaterials. In the case of HCAEC, no direct relation could be made between NPs or their concentration and cholesterol effluxing percentage. But for HUVEC, protein-conjugated nanomaterials showed better cholesterol effluxing with both a time- and a concentrationdependent manner. The differential cholesterol efflux ability of both cells can be attributed to the fact that HUVEC produces a lower expression level of ABCA1 compared to HCAEC.39 It has also been reported that under similar experimental conditions HCAEC produces a pattern of expression of chemokines distinct from that of HUVEC.40 The study suggested that NPs allow easy passage of protein inside the cellular environment. Further, it was also observed that both Si-B and Si-H showed better effluxing ability as compared to M-B and M-H, whereas Si-H showed maximum efficiency. The confocal studies suggested nonspecific distribution of Si-NP and Si-B/Si-H in all the cells, whereas more fluorescence response was observed in the case of Si-H with HUVEC. This is in accordance with the earlier cholesterol effluxing results. All these results suggested that HSA allowed more penetration of NPs than BSA in cells. Literature reports suggested that serum albumins with a number of binding sites acts as carriers for the transportation of different molecules/ ligands and through the bloodstream. The binding cavity of HSA is more open-type, whereas the comparatively rigid BSA structure restricts its availability for incoming ligands,41 and hence, these two proteins behave differently in a similar cellular environment. The present work reflects that with improved cholesterol effluxing ability and better fluorescence response in the visible range Si-NP as well as its protein conjugates might find their application as theragnosis probes for future biomedical applications.
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CONCLUSION Three different nanomaterials and their BSA and HSA conjugates were synthesized, viz., GSH-NP/G-B/G-H, MPA-NP/ M-B/M-H, and Si-NP/Si-B/Si-H. The absorption and fluorescence studies confirmed the formation of NP as well as protein attachment. The protein-attached nanomaterials showed a blue shift in the characteristic BSA/HSA peak, suggesting a change in the microenvironment of the tryptophan residue of the corresponding proteins.37 In the case of fluorescence studies, G-B/G-H did not respond strongly, reflecting poor conjugation of the proteins on the GSH-NP surface which was further proved by precipitation studies. Further, it was observed that protein conjugation has resulted in a blue shift of the emission of the NPs. When irradiated with 364 nm light, Si-NPs and Si-B/Si-H as well as MPA-NPs and M-B/M-H emitted strongly, whereas GSH-NPs/G-B/G-H did not respond. It should be mentioned
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01998. Absorption and fluorescence, DLS and zeta potential, stability analysis, FTIR, Bradford assay, DPPH assay, cytocompatibility studies, and confocal imaging. (PDF)
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AUTHOR INFORMATION
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DOI: 10.1021/acssuschemeng.6b01998 ACS Sustainable Chem. Eng. 2017, 5, 1425−1435
Research Article
ACS Sustainable Chemistry & Engineering ORCID
(16) Park, J. H.; Gu, L.; Von Maltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat. Mater. 2009, 8, 331−336. (17) Anglin, E. J.; Cheng, L.; Freeman, W. R.; Sailor, M. J. Porous silicon in drug delivery devices and materials. Adv. Drug Delivery Rev. 2008, 60, 1266−1277. (18) Akdogan, Y.; Reichenwallner, J.; Hinderberger, D. Evidence for water-tuned structural differences in proteins: An approach emphasizing variations in local hydrophilicity. PLoS One 2012, 7, e45681. (19) Zhao, Y.; Marcel, Y. L. Serum albumin is a significant intermediate in cholesterol transfer between cells and lipoproteins. Biochemistry 1996, 35, 7174−7180. (20) Luthi, A. J.; Zhang, H.; Kim, D.; Giljohann, D. A.; Mirkin, C. A.; Thaxton, C. S. Tailoring of biomimetic high-density lipoprotein nanostructures changes cholesterol binding and efflux. ACS Nano 2012, 6, 276−285. (21) Low, H.; Hoang, A.; Sviridov, D. Cholesterol efflux assay. J. Visualized Exp. 2012, 61, e3810−e3810. (22) Thakur, D.; Deng, S.; Baldet, T.; Winter, J. O pH sensitive CdS− iron oxide fluorescent−magnetic nanocomposites. Nanotechnology 2009, 20, 485601. (23) Wang, J.; Ye, D. X.; Liang, G. H.; Chang, J.; Kong, J. L.; Chen, J. Y. One-step synthesis of water-dispersible silicon nanoparticles and their use in fluorescence lifetime imaging of living cells. J. Mater. Chem. B 2014, 2, 4338−4345. (24) Orelma, H.; Morales, L. O.; Johansson, L. S.; Hoeger, I. C.; Filpponen, I.; Castro, C.; Rojas, O. J.; Laine, J. Affibody conjugation onto bacterial cellulose tubes and bioseparation of human serum albumin. RSC Adv. 2014, 4, 51440−51450. (25) Rogozea, A.; Matei, I.; Turcu, I. M.; Ionita, G.; Sahini, V. E.; Salifoglou, A. EPR and circular dichroism solution studies on the interactions of bovine serum albumin with ionic surfactants and βcyclodextrin. J. Phys. Chem. B 2012, 116, 14245−14253. (26) Matei, I.; Hillebrand, M. Interaction of kaempferol with human serum albumin: a fluorescence and circular dichroism study. J. Pharm. Biomed. Anal. 2010, 51, 768−773. (27) Pradhan, P.; Giri, J.; Banerjee, R.; Bellare, J.; Bahadur, D. Preparation and characterization of manganese ferrite-based magnetic liposomes for hyperthermia treatment of cancer. J. Magn. Magn. Mater. 2007, 311, 208−215. (28) Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 2006, 1, 1112−1116. (29) Singh, S.; Kaur, R.; Chahal, J.; Devi, P.; Jain, D. V. S.; Singla, M. L. Conjugation of nano and quantum materials with bovine serum albumin (BSA) to study their biological potential. J. Lumin. 2013, 141, 53−59. (30) Chatterjee, S.; Mukherjee, T. K. Spectroscopic investigation of interaction between bovine serum albumin and amine-functionalized silicon quantum dots. Phys. Chem. Chem. Phys. 2014, 16, 8400−8408. (31) Kumari, A.; Guliani, A.; Singla, R.; Yadav, R.; Yadav, S. K. Silver nanoparticles synthesised using plant extracts show strong antibacterial activity. IET Nanobiotechnol. 2015, 9, 142−152. (32) Zhu, L. Y.; Li, G. Q.; Zheng, F. Y. Interaction of bovine serum albumin with two alkylimidazolium-based ionic liquids investigated by microcalorimetry and circular dichroism. J. Biophys. Chem. 2011, 2, 147−152. (33) Gahlawat, G.; Shikha, S.; Chaddha, B. S.; Chaudhuri, S. R.; Mayilraj, S.; Choudhury, A. R. Microbial glycolipoprotein-capped silver nanoparticles as emerging antibacterial agents against cholera. Microb. Cell Fact. 2016, 15, 1−14. (34) Moore, K. J.; Sheedy, F. J.; Fisher, E. A. Macrophages in atherosclerosis: a dynamic balance. Nat. Rev. Immunol. 2013, 13, 709− 721. (35) Sontag, T. J.; Chellan, B.; Bhanvadia, C. V.; Getz, G. S.; Reardon, C. A. Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis. J. Lipid Res. 2015, 56, 470−483. (36) Sankaranarayanan, S.; de la Llera-Moya, M.; Drazul-Schrader, D.; Phillips, M. C.; Kellner-Weibel, G.; Rothblat, G. H. Serum albumin acts as a shuttle to enhance cholesterol efflux from cells. J. Lipid Res. 2013, 54, 671−676.
Amitabha Acharya: 0000-0002-9013-9027 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are thankful to the Director, CSIR-IHBT, for infrastructural facilities. A.A. acknowledges the financial support from CSIR, GOI in the form of BSC0213, BSC0112, and MLP0068. S.W. and A.G. acknowledge CSIR for a project fellowship in the form of BSC0213 and BSC0112, respectively. A.A. acknowledges Dr. Y. S. Padwad for cell culture facility and Mr. Sourabh Soni for his assistance in cell culture work. The CSIR-IHBT communication number of this manuscript is 4050.
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REFERENCES
(1) Jokerst, J. V.; Cole, A. J.; Van de Sompel, D.; Gambhir, S. S. Gold nanorods for ovarian cancer detection with photoacoustic imaging and resection guidance via Raman imaging in living mice. ACS Nano 2012, 6, 10366−10377. (2) Yi, X.; Wang, F.; Qin, W.; Yang, X.; Yuan, J. Near-infrared fluorescent probes in cancer imaging and therapy: An emerging field. Int. J. Nanomed. 2014, 9, 1347−1365. (3) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281, 2013−2016. (4) Zhang, Z. Y.; Xiong, H. M. Photoluminescent ZnO nanoparticles and their biological applications. Materials 2015, 8, 3101−3127. (5) Zhang, J.; Chen, R.; Zhu, Z.; Adachi, C.; Zhang, X.; Lee, C. S. Highly stable near-infrared fluorescent organic nanoparticles with a large stokes shift for noninvasive long-term cellular imaging. ACS Appl. Mater. Interfaces 2015, 7, 26266−26274. (6) Battistelli, G.; Cantelli, A.; Guidetti, G.; Manzi, J.; Montalti, M. Ultra-bright and stimuli-responsive fluorescent nanoparticles for bioimaging. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2016, 8, 139−150. (7) Chan, W. C.; Nie, S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281, 2016−2018. (8) Acharya, A.; Rawat, K.; Bhat, K. A.; Patial, V.; Padwad, Y. S. A multifunctional magneto-fluorescent nanocomposite for visual recognition of targeted cancer cells. Mater. Res. Express 2015, 2, 115401. (9) Alivisatos, A. P. Semiconductor clusters, nanocrystals, and quantum dots. Science 1996, 271, 933−937. (10) Walia, S.; Acharya, A. Silica micro/nanospheres for theranostics: From bimodal MRI and fluorescent imaging probes to cancer therapy. Beilstein J. Nanotechnol. 2015, 6, 546−558. (11) Reisch, A.; Klymchenko, A. S. Fluorescent polymer nanoparticles based on dyes: seeking brighter tools for bioimaging. Small 2016, 12, 1968−1992. (12) Hu, J.; Li, L. S.; Yang, W.; Manna, L.; Wang, L. W.; Alivisatos, A. P. Linearly polarized emission from colloidal semiconductor quantum rods. Science 2001, 292, 2060−2063. (13) Walia, S.; Sharma, S.; Kulurkar, P. M.; Patial, V.; Acharya, A. A bimodal molecular imaging probe based on chitosan encapsulated magneto-fluorescent nanocomposite offers biocompatibility, visualization of specific cancer cells in vitro and lung tissues in vivo. Int. J. Pharm. 2016, 498, 110−118. (14) Xia, B.; Zhang, W.; Shi, J.; Xiao, S. J. Engineered stealth porous silicon nanoparticles via surface encapsulation of bovine serum albumin for prolonging blood circulation in vivo. ACS Appl. Mater. ACS Appl. Mater. Interfaces 2013, 5, 11718−11724. (15) Kafshgari, M. H.; Delalat, B.; Tong, W. Y.; Harding, F. J.; Kaasalainen, M.; Salonen, J.; Voelcker, N. H. Oligonucleotide delivery by chitosan-functionalized porous silicon nanoparticles. Nano Res. 2015, 8, 2033−2046. 1434
DOI: 10.1021/acssuschemeng.6b01998 ACS Sustainable Chem. Eng. 2017, 5, 1425−1435
Research Article
ACS Sustainable Chemistry & Engineering (37) Gelamo, E. L.; Tabak, M. Spectroscopic studies on the interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants. Spectrochim. Acta, Part A 2000, 56, 2255−2271. (38) Gu, L.; Hall, D. J.; Qin, Z.; Anglin, E.; Joo, J.; Mooney, D. J.; Sailor, M. J.; Howell, S. B. In vivo time-gated fluorescence imaging with biodegradable luminescent porous silicon nanoparticles. Nat. Commun. 2013, 4, 1−7. (39) Sun, R. L.; Huang, C. X.; Bao, J. L.; Jiang, J. Y.; Zhang, B.; Zhou, S. X.; Cai, W. B.; Wang, H.; Wang, J. F.; Zhang, Y. L. CCL2 suppresses HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in human endothelial cells. J. Biol. Chem. 2016, 291, 19532−19544. (40) Briones, M. A.; Phillips, D. J.; Renshaw, M. A.; Hooper, W. C. 2001. Expression of chemokine by human coronary artery and umbilical vein endothelial cells and its regulation by inflammatory cytokines. Coron. Artery Dis. 2001, 12, 179−186. (41) Datta, S.; Halder, M. Detailed scrutiny of the anion receptor pocket in subdomain IIA of serum proteins toward individual response to specific ligands: HSA-pocket resembles flexible biological slidewrench unlike BSA. J. Phys. Chem. B 2014, 118, 6071−6085.
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