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31 July 2015 Optical properties of plasmon-resonant bare and silica-coated nanostars used for cell imaging
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Abstract
We synthesized and characterized gold nanostars and their silica-coated derivatives with 7- to 50-nm shell thicknesses as contrast agents for optical imaging. The scattering and absorption coefficients of the nanoparticles (NPs) were estimated by means of collimated transmittance and diffuse reflectance/transmittance analyses. The contrasting properties of the nanostructures were studied in optical coherence tomography glass capillary imaging. The silica-coated nanostars with the thickest shell have higher scattering ability in comparison with bare nanostars. Viability assays confirmed weak in vitro toxicity of nanostructures at up to ∼200-μg/mL concentrations. We showed real-time visualization of nanostars in both agarose and cultured cells by analyzing the backscattering signal using a conventional laser confocal microscope. The signal intensity detected from the silica-coated NPs was almost 1.5 times higher in comparison with bare nanostars. To the best of our knowledge, this is the first time that conventional laser confocal microscopy was applied in combined scattering and transmitted light modes to detect the backscattered signal of gold nanostars, which is useful for direct monitoring of the uptake, translocation, and accumulation of NPs in living cells.

1.

Introduction

Theranostics (simultaneous diagnosis and therapy within a single protocol) of cancer is an emerging trend in nanobiotechnology, necessitating multifunctional nanoparticles (NPs) that combine therapeutic, diagnostic, and sensing abilities in a single nanostructure.13 These nanostructures are often composed of core–shell type composites, where the plasmon-resonant core simultaneously functions as an optical label and a therapeutic agent. The silica shell covering the NP also has a number of roles. It can enhance cargo volume, surface area, and colloidal stability, and it can also improve size- and shape-dependent optical properties.4,5 Branched and star-shaped gold NPs become perspective candidates for use as a plasmon-resonant core of nanocomposites (NCs) due to their strong absorption in the near-infrared (NIR) wavelength range, optimal for the “diagnostic/therapeutic window” with enhanced light penetration into biotissue. In addition, the strong electric field concentrated at their branches6,7 enables applications such as surface-enhanced Raman spectroscopy,8 photodynamic therapy,9 targeted delivery optical imaging,10,11 and photothermolysis.12

Integration of plasmon-resonant nanoparticles (PRNPs) into biomedical applications requires the ability to change their absorption and scattering properties and tune the position of the resonance peak and associated scattering and absorption for optimal use. Although some experimental data on the relationship between the absorption and scattering properties of nanospheres and nanoshells,13,14 nanorods,15,16 and nanocages17 are available, the majority of studies are devoted to the absorption properties of plasmonic NPs. Despite their importance as contrast agents in biophotonics, scattering characteristics attract much less attention from researchers. Here, we focused on the scattering component of the spectrum, crucial for imaging applications such as dark-field and confocal microscopy or optical coherence tomography (OCT). The latter is a noninvasive technique based on low-coherence interferometry utilizing backscattered light for in vivo imaging of layered tissues, allowing for distinguishing between normal and pathological tissue and for monitoring changes in optical properties.18,19 The excellent contrast properties of gold nanoshells with a silica core,20 functionalized gold nanospheres,21 nanorods,22 and nanocages23 for OCT skin imaging have been exploited and also modeled with Monte Carlo simulations.24 The measured absorption cross section of nanocages appeared to be about fivefold larger compared with conventional dyes.25 On the other hand, branched gold NPs were shown to amplify the signal in water and agarose tissue-mimicking phantoms even under excitation not matching the resonance peak of the particles.26

Due to the high scattering, NPs are attractive agents for visualizing living cells and the dynamic processes occurring in them by using optical imaging techniques such as confocal,27 differential interference contrast,28 and dark-field29 microscopic techniques. It has recently been demonstrated how different microscopic techniques, including laser confocal microscopy, can provide useful information about particle localization and properties.30 Along with the integration of plasmonic nanostructures into the biological field, the uptake and toxicity of gold NPs and their derivatives is an emerging topic of modern nanomedicine and nanobiotechnology.31,32 The effect of NPs on living cells and interactions between living and inorganic matter are important factors that may limit their use in biomedical applications.33 Although no interaction between gold NPs and hemoglobin was found,34 PRNPs can bind to a variety of plasma proteins, leading to an adsorbed protein layer or “corona” that largely defines the biological identity of these particles.35 However, to study the effect of NPs on living cells, standard cell lines, such as A-549, HeLa, and 3T3 cells, are usually applied.36,37 A recent study showed that the morphological features and surface chemistry of NPs define their cellular translocation dynamics.38 Since both intracellular translocation after uptake and accumulation of NPs in cells are of high importance, here we summarize and extend our recent results39,40 on the synthesis, optical, and contrasting properties of nanostars (NSts) and their silica-shell-coated NCs as well as their toxicity based on colorimetric and fluorescent viability assay analyses. For the first time, the scattering and absorption coefficients of bare and silica-coated nanostars were separated by means of collimated transmittance and diffuse reflectance/transmittance analyses and compared with the scattering and absorption properties of bare (NSps) and silica-coated (NCSps) nanosphere suspensions. In addition, we applied conventional laser confocal microscopy in combined scattering and transmitted light modes to detect the backscattered signal of gold nanostars for real-time visualization of intracellular localization of nanostars on living cells.

2.

Materials and Methods

2.1.

Chemicals

Gold(III) chloride trihydrate (HAuCl4×3H2O), trisodium citrate dihydrate (C6H5O7Na3×2H2O), 1 N HCl, L(+)-ascorbic acid (AA), tetraethyl orthosilicate (TEOS), sodium borohydride (NaBH4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly(vinyl pyrrolidone) (PVP; Mw=55,000), and silver nitrate (AgNO3) were purchased from Sigma-Aldrich with the highest purity grade available. Absolute isopropyl alcohol (IPA; 99.99%), ammonium hydroxide NH4OH (29.5%), Dulbecco’s modified Eagle’s medium (DMEM), agarose, and penicillin-streptomycin solution were purchased from Sigma-Aldrich. DMSO (ACS grade) was purchased from Amresco; Milli-Q water (18MΩ-cm; Millipore) was used in all preparations.

2.2.

Nanoparticle Synthesis

Nanostars were synthesized with a slightly modified seed-mediated growth method, as described by Yuan et al.41 Gold nanospheres were used as seeds. The nanosphere solution was prepared by reducing HAuCl4 with sodium citrate using Grabar’s method.42 To prepare 15- and 50-nm nanospheres, 4.5 and 2.1 mL of 1% sodium citrate, respectively, were added quickly to boiling 0.03% HAuCl4 (50 mL), resulting in a change in solution color from pale yellow to deep orange-red (15-nm NSps) or pink-red (50-nm NSps). For nanostar synthesis, 10μL of 1 M HCl and 100μL of the 15-nm NSps solution were added to 10 mL of 0.25 mM HAuCl4 water solution at room temperature under vigorous stirring. Subsequently, 100μL of 2 mM AgNO3 and 50μL of 0.1 M AA were added. The solution was stirred for 30 s while its color turned from light red to dark gray. Immediately afterward, 250μL of 0.36 mM PVP was added under gentle stirring. The solution was stirred for 15 min and centrifuged at a 7500 relative centrifugal force (RCF) in terms of acceleration (g) for 10 min twice. The solution was redispersed in 0.5 mL of water.

2.3.

Silica-Coating of Nanoparticles

A method from elsewhere43 was used to form a silica shell on PVP-covered nanostars and nanospheres. Under gentle stirring, 0.5 mL of the nanostars was added to 2.25 mL of IPA. Ninety microliters of a 30% aqueous ammonia solution and various amounts of TEOS (from 6.25 to 50μL) were added to the solution under continuous stirring to synthesize silica-coated nanostars or nanospheres with different thicknesses of silica shells. The reaction was allowed to proceed for 30 min at room temperature. The resulting solution was washed three times by centrifugation at 6000 RCF and the colloids were finally redispersed in 2 mL of water.

2.4.

Characterization of Nanostructures

The average size of the particles was obtained by analyzing transmission electron microscopy (TEM) images (LEO 912 OMEGA, energy-filtered transmission electron microscope operating at 120 kV). To prepare samples for TEM, one drop of 10-μL nanostructure water suspension was deposited onto a carbon-coated copper grid. Statistical calculations were performed using ImageJ free software. Extinction and absorption coefficients of suspensions of bare and silica-coated nanostars were estimated using a spectrophotometer system (Optronic Laboratories) with integrating spheres working in diffuse reflectance/transmittance and collimated transmittance regimes. The scattering and absorption coefficients were retrieved from the measurements by applying Beer’s law.

2.5.

Optical Coherence Tomography Experiments

A hyperion spectral-domain optical coherence tomograph (Thorlabs) operating at a 930-nm central wavelength with an axial (along the Z-axis) resolution of 5.8μm and a lateral resolution of 8μm (in air) was used for imaging of bare and silica-coated nanostars in a capillary. Aqueous colloidal suspensions of gold nanostars and composites with a concentration of 1mg/mL were added into 200-μm-thick plane glass capillaries. Two-dimensional images of the capillaries with the suspensions were recorded with the OCT system and later processed with MATLAB software.

2.6.

Cell Viability Test

Cell viability was examined using two modalities: a fluorescent cell viability assay with propidium iodide (PI) and an MTT assay. HeLa cells (Biocenter Oulu, Finland) were maintained at 37°C and 5% CO2 in a complete DMEM medium supplemented with 10% bovine serum, 1% penicillin–streptomycin.

For the fluorescent assay, cells at a concentration of 106mL1 were maintained in 48-well culture plates in 400 mL of medium and incubated overnight with NPs at final concentrations of 12.5, 25, 50, 100, 200, and 400μg/mL. After incubation, PI water solution was added to the cells to form the final concentration of 3μg/mL. The cells were stained for 20 min in the dark at 37°C. The fluorescence of cells with compromised membranes (and thus considered dead) was registered by an Olympus IX81 epifluorescent inverted microscope equipped with a TRITC 41002c filter cube (Chroma). The ratio of fluorescing cells to the total number of cells in the field of view was calculated. The number of live cells in the sample of pure cells was set at 100%. Each experiment was triplicated. Standard deviation was used as error estimation.

To determine the respiratory activity of cells after incubation with the NPs, an MTT test, as described by Nicks and Otto44 with minor modifications, was performed. The cells were maintained in 96-well culture plates in 200μL of medium and incubated overnight with nanostars and NCs at concentrations of 12.5, 25, 50, 100, 200, and 400μg/mL (6.25×104, 1.25×105, 2.5×105, 5×105, 106, 2×106NPs/cell, correspondingly). A 100-μL MTT solution (5mg/mL) was added to each well of the plate with cell suspensions and incubated for 1 h in the dark at 37°C in a humidified atmosphere with 5% CO2. After incubation, the supernatant was removed and 100μL of DMSO was added to each well. The precipitate was resuspended and incubated for 15 min in the dark at 37°C. The solution from each well was centrifuged at 12,000 RCF for 3 min. The resulting supernatant was placed in wells of a 96-well flat-bottomed plate; absorbance readings were obtained with a Power Wave microplate spectrophotometer (Bio-Tek Instruments) at a wavelength of 490 nm. The respiratory activity of the nonirradiated sample was set at 100%. Each experiment was repeated three times.

2.7.

Confocal Microscopy Experiments

Backscattering of the nanostructures was investigated by conventional laser confocal microscopy for both agarose and living cells. The samples were examined with a Zeiss LSM 780 laser scanning microscope with an EC Plan-Neofluar 10×/0.3 objective in a light scattering mode with an irradiating wavelength of 633 nm and a detection bandwidth of about 5 nm. To avoid saturation of the signal, the gain level was tuned to the same minimum value for all the samples. All the samples were scanned along the Z-direction in increments of 0.22μm. Sixty-seven optical slices were analyzed from each sample. The thickness of the slices was the same (0.22μm) for all the samples. The intensities of the signals were obtained for medial projections.

For agarose imaging, 20μL of an aqueous colloidal solution of the NPs (stock concentration of the particles 1mg/mL) was added to 100μL of hot 1% aqueous agarose solution, stirred rapidly, loaded onto a precleaned glass, and covered with a cover glass, achieving the same layer thickness for each sample.

For in vitro imaging, the HeLa cells were maintained at 37°C and 5% CO2 in a complete DMEM medium on a sterile microscopic cover glass and incubated overnight with gold nanostars and silica-coated gold nanostars at a gold concentration of 200μg/mL. After incubation, the cover glasses with attached cells were put on microscopy glasses, sealed to avoid airing, and analyzed immediately.

3.

Results and Discussion

3.1.

Synthesis and Characterization of Nanostructures

Synthesis of gold nanostars is based on the reduction of chloroauric acid with AA and silver nitrate at room temperature on presynthesized gold nanospheres (diameter 15 nm) in aqueous media. Such nanostars, first developed by Vo-Dinh’s research group,41 are supposed to be beneficial for biological applications and optical imaging due to their biocompatible surfactant-free synthesis and because of the presence of thin sharp tips that can intensively interact with NIR excitation. The synthesis is simple; however, according to our experience, the nanostars are not stable enough. Even after functionalization by m-PEG-SH, as described in Fales’ work,9 irreversible aggregation was observed. To avoid significant aggregation, the nanostars were functionalized with PVP instead of PEG. A preliminary study of stabilization and the optical properties of nanostars are described by the authors elsewhere.39 The PVP functionalization took 15 min, and the product proved to be stable for several months and allowed multiple centrifugation and redispersion cycles in different media.

The average diameter and shell thickness were obtained by analysis of TEM images of dried NP suspensions. To perform statistical analyses and calculations, we created a simple computation routine for image analysis using ImageJ software and estimated the dimensions of 500 particles on each given TEM image. Figure 1 shows representative TEM images of (a) NSts, (b) NCs, and (c and d) their corresponding size distribution diagrams.

Fig. 1

Representative transmission electron microscopy images of (a) nanostars (NSts), (b) nanocomposites (NCs), and their corresponding size distribution diagrams (c and d).

JBO_20_7_076017_f001.png

The multibranched morphology with thin sharp tips is typical for seed-mediated grown nanostars. The size and number of branches of nanostars may be adjusted by changing the concentration of silver ions41 and the ratio of auric chloride concentration to seed amount.4547 In a recent report by Khlebtsov et al., the size, morphology, and plasmon-resonant wavelength tunability of NSts were achieved by varying the initial size and concentration of seeds.48 Here, NSts with an average tip-to-tip size of 55±10nm [Fig. 1(a)] were synthesized, similar to those reported by Khlebtsov et al.48 and Yuan et al.41 for NSts grown on seeds 15 nm in diameter. The silica shells of the nanostars were synthesized following the method depicted by Khlebtsov et al.43 Shell thickness can simply be varied by changing the TEOS concentration and reaction time. Here, the amount of TEOS was 10μL. The final silica-coated nanostars have an average size of 105±16nm with a shell thickness of about 25±6nm [Fig. 1(b)].

The absorption and scattering behaviors of PRNP suspensions define their applicability for biomedical optics; thus, both properties were measured for different types of particles with integrating spheres49 and by OCT.50 He et al.51 used a monochromatic laser beam passing through the sample medium and then measured the absolute scattered energy within a small solid angle, and the total absolute scattering losses were finally obtained by integration over a 4π solid angle. Bogatyrev et al.52 used a collimated light beam passing through the sample and measured the scattering spectrum along the 90 deg direction. Here, we apply a spectrophotometer with integrating spheres in a collimated transmittance and diffuse reflectance/transmittance configuration to estimate the scattering and absorption coefficients of our nanostructure suspensions (Fig. 2).

Fig. 2

Schemes for measuring (a) collimated transmission and (b) absorption.

JBO_20_7_076017_f002.png

Under usual conditions, a spectrophotometer can be conveniently used to measure the collimated transmission of a suspension as a function of wavelength and to finally obtain the extinction spectrum of the sample. In this case, the extinction coefficient μt can be calculated as

Eq. (1)

μt=ln(Tc)l,
where Tc is the measured collimated transmittance and l is the cuvette’s internal thickness (path length). The measured extinction coefficient is the sum of the absorption and scattering coefficients, which cannot be retrieved without further analysis.

To measure TR, which is the sum of total transmittance T and reflectance R, we used an integrating sphere equipped with Spectralon™ (Labsphere, United States). Spectralon™ plays the role of a diffuse mirror due to its high reflectance, which is generally more than 95% for a wavelength range from 400 to 2500 nm.53 Since the light passes the double optical path within a sample, the absorption coefficient μa is

Eq. (2)

μa=ln(TR)2l.

Based on the above-mentioned equations, we can calculate the scattering coefficient as μs=μtμa. Figure 3 shows the retrieved extinction, absorption, and scattering coefficients of suspensions of bare and silica-coated nanostars [NSts, Fig. 3(a) and NCs, Fig. 3(b)], with initial and silica-coated 50-nm nanospheres [NSps, Fig. 3(c) and NCSps, Fig. 3(d)] given for comparison. For NCs and NCSps, the silica shell thicknesses were 25±6nm and 31±7nm, respectively.

Fig. 3

Retrieved normalized spectra of extinction μext, absorption μabs, and scattering μsct coefficients of (a) NSts, (b) NCs, (c) bare nanosphere suspensions, and (d) silica-coated (NCSps) suspensions.

JBO_20_7_076017_f003.png

According to the measured spectral data, the contribution of the scattering component to extinction is rather small, in line with data reported for nanocages and nanorods of comparable size.17 Since the scattering (Rayleigh) and absorption coefficients scale with particle size as54 μsr6 and μar3, larger nanostars scatter light stronger and are better suited for optical applications where the scattering intensity is critical. The stronger scattering ability of silica-coated nanostars can be theoretically explained by the use of nanospheres as a model, since similar scattering is an indicator of both NP types. Let us consider a simple two-layer model for gold nanospheres covered with a silica shell. Mie calculations (Fig. 4) for 30-, 40-, 50-, and 60-nm gold NPs show that silica coating in 30- and 60-nm layers results in a significant increase in differential scattering cross section in both forward and backward directions, whereas the scattering indicatrix retain their dipolar (Rayleigh) character. Physically, this enhanced scattering is explained by larger particle volume and, as a result, by large polarizability of composite particles. To the first approximation, the scattering intensity is proportional to the particle volume. As an example, after coating 60-nm Au particles with a 30-nm silica shell, the particle volume and scattering intensity roughly increased eight times.

Fig. 4

Mie calculations of differential scattering cross sections (Cdsca) of bare 30-, 40-, 50-, and 60-nm gold NPs and silica-coated NPs with a 30-nm or 60-nm silica shell. The calculations were done for both monodisperse (solid lines) and polydisperse (dashed lines) models. Standard Gaussian distribution with the normalized standard deviation = 0.3 was used for polydisperse model.

JBO_20_7_076017_f004.png

Figure 5 depicts separate normalized spectra of the extinction, absorption, and scattering coefficients of NP suspensions for a convenient comparison of their absorption and scattering contributions to the total extinction of bare and coated NPs. The average diameters of NSts and NSps were 55±10 and 50±7nm, respectively; the silica shell thicknesses of coated NSts were 25 nm (NCs-25) and 60 nm (NCs-60); and the silica shell thicknesses of coated nanospheres were 31 nm (NCSps-31) and 58 nm (NCSps-58).

Fig. 5

Normalized spectra of extinction coefficients for suspensions of nanostars and their derivatives; (b) nanospheres and their derivatives; (c) spectra of absorption coefficients of nanostars and their derivatives; (d) nanospheres and their derivatives; (e) spectra of scattering coefficients of nanostars and their derivatives; and (f) nanospheres and their derivatives.

JBO_20_7_076017_f005.png

The red shift of the spectral bands and the increase in the extinction value after silica-coating are well-known effects, associated with the change in the effective refractive index of the medium around the gold cores, demonstrated by experiments9,55,56 and also explained by theoretical models.57,58 The dependence of the plasmon-resonant peak on the dielectric environment for different types of PRNPs was investigated theoretically and experimentally. Our results, in good agreement with previous reports, show the expected red shift for the coated particles.

3.2.

Optical Coherence Tomography Experiments

Evaluation of the scattering properties of nanostars and their core–shell structures from OCT experiments was the next stage of our study. Bare (NSts) and silica-coated nanostars with shell thicknesses of 7 nm (NCs-7) and 52 nm (NCs-52) were synthesized, resulting in particles with an average diameter of 51±7nm for NSts; 64±10nm for NCs-7; and 155±20nm for NCS-52. Figure 6 depicts the normalized spectra of the obtained suspensions.

In addition, the results provide experimental evidence of the dependence of the shift on coating thickness (20 nm for NCs-7 and 30 nm for NCs-52 related to the uncoated nanostars).

Fig. 6

Spectra of extinction coefficients of nanostars and nanocomposites with different silica shell thicknesses. Adapted from Ref. 40.

JBO_20_7_076017_f006.png

A preliminary study on using nanostars for enhancement of the OCT image contrast of water flow in glass capillaries was demonstrated by the authors earlier.40 The plasmon resonance maximum of the nanostructures (840 to 870 nm) is close to the emission spectrum of our superluminescent diode light source with the central wavelength of 930 nm and spectral bandwidth of 100 nm employed in the OCT system. Briefly, we concentrated the suspensions of NSts, NCs-7, and NCs-52 40 times in comparison with as-prepared solutions. Figure 7 shows the OCT images of capillary cross section with NP suspensions of NSts (a), NCs-7 (b), and NCs-52 (c). The scattering medium in the middle of the image is the NP suspension, and the brighter blue color corresponds to the stronger OCT (backward-reflected) signal.

Fig. 7

Optical coherence tomography (OCT) images of capillary cross section with nanoparticle (NP) suspensions of (a) NSts, (b) NCs-7, and (c) NCs-52. Brighter color indicates higher scattering at λ=930nm. Inner capillary thickness is 200μm. Adapted from Ref. 40.

JBO_20_7_076017_f007.png

In-depth distribution of the intensity registered from NP suspensions was retrieved from the measured data (Fig. 8). The slope of the in-depth OCT signals correlates with the scattering coefficient of the medium.19 In the simplest case of single scattering, the scattering coefficient (or scattering cross section) could be directly calculated from the slope of the OCT signal using Beer’s law. However, the multiple scattering significantly complicates this relation.19,59

Fig. 8

In-depth distribution of the OCT signal over the capillary depth for the used NP suspensions. Adapted from Ref. 40.

JBO_20_7_076017_f008.png

In our case, the most intensive signal was registered from the silica-coated nanostars with the thickest silica shell. The spike on the left of all the curves is due to the glass–liquid interface. The backward scattering of the silica-coated nanostars, especially those with the thickest shell, is higher than the intensity of the signal from bare nanostars, which is in good agreement with previous reports26 and measured spectral data (see Fig. 4) showing signal enhancement of branched gold NPs in OCT imaging in water and agarose gel. It should be noted that the red shift of NCs plasmon resonance maximum closer to the OCT instrumental wavelength increases the intensity of the OCT signal, which makes the NCs promising contrast agents for OCT. In addition, the use of larger nanostars for better correspondence of the plasmon-resonant wavelength with the emission band of OCT will be the subject of further studies.

3.3.

Confocal Microscopy Experiments

For real-time visualization of the prepared NPs (NSts and NCs-52), the backscattered signal obtained with a conventional laser confocal microscope was analyzed in both agarose and cultured cells. To detect PRNPs’ backscattering by reflectance confocal microscopy, we superimposed the signal detection band with the excitation laser wavelength (633 nm) and reduced the laser power and photomultiplier voltage to minimize the parasitic backscattered signal (background) from agarose gel or cell organelles. The choice of an excitation laser wavelength of 633 nm is conditioned by instrument characteristics; therefore, the wide plasmon-resonant peak of nanostars allows one to still use it as a contrast label for confocal microscopy at this wavelength. For the bulk solution experiment, NSts and NCs-52 were mixed with high-density agarose gel to avoid Brownian motion of the nanostructures before the suspensions were applied onto microscopy slides. Scattering intensity was evaluated and statistical measurements (mean intensity and standard deviation) were calculated using a ZEN software (Carl Zeiss, Germany) application in a histogram view. First, images of the samples’ maximum intensity projection were obtained. Then the images were directly converted into binary images through a threshold. The binary image reveals whether a group of pixels in the image is recognized as a scattering spot. Finally, the intensity peak of each scattering spot was calculated and histograms of the intensity distribution were depicted. The whole process was realized in a ZEN black edition program. In Fig. 9, one can observe differences between the intensities of bare (a) and silica-coated nanostars (b) scattered signals. The silica-coated NPs have a higher scattering ability in comparison with bare nanostars, in agreement with the results obtained by OCT experiments. Relatively, large bright spots presented on the images may be caused by PRNPs’ polydispersity (the scattering intensity is proportional to the particle volume, as shown earlier).

Fig. 9

Images of maximal intensity projection of the (a) NSts and (b) NCs-52.

JBO_20_7_076017_f009.png

3.4.

Cell Studies

To demonstrate the applicability of bare and silica-coated nanostars for in vitro experiments, we examined the influence of nanostructures at various concentrations on a HeLa cell line using two modalities: an MTT test and a fluorescent cell viability assay with PI as a stain for dead cells. The toxicity of PRNPs was shown to be dependent on their size, morphology, concentration, and surface properties.60 Pernodet et al.61 investigated in vitro the influence of gold nanospheres on cell activity at concentrations of 200, 400, 600, and 800μg/mL. The high concentrations of NPs were toxic, since they penetrated through cell membranes and accumulated in vacuoles, affecting motility of the cells and their proliferative and adhesive abilities. An absence of toxicity has been shown by MTT assay for nanospheres up to a maximum particle concentration of 100μg/mL in several research papers.62,63 Here, we investigated the toxicity of NPs at concentrations of 12.5, 25, 50, 100, 200, and 400μg/mL or 6.25×104, 1.25×105, 2.5×105, 5×105, 106, 2×106NPs/cell, correspondingly. According to our results, all types of NPs have a significant impact on the metabolic activity of cells (about a 56% decrease in activity for NSts and about 44% for NCs) at a final concentration of gold equal to 400μg/mL [Fig. 10(a)]. Smaller concentrations of NPs are harmless to the investigated cells when the differences in the values are within the standard deviation. Staining experiments using PI confirm the nontoxicity of silica-coated NSts and NCs up to a concentration of 200μg/mL [Fig. 10(b)]. Similarly to the observations with the MTT assay, the amount of live cells decreased by 74% for NSts and by 50% for NCs in the case of a NP concentration of 400μg/mL. Thus, the safe concentration of NPs used for real applications, e.g., cell imaging, should not exceed 200μg/mL. It should be noted that due to the complexity and nonlinearity of biological systems such as living cells, the limited nontoxic concentration can vary up to five times, as was demonstrated in our previous study.39 In Ref. 40, we indicate concentrations of gold in stock solutions added to the cell medium.

Fig. 10

Cytotoxicity of silica-coated gold nanostars at different concentrations evaluated by (a) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and (b) fluorescent cell viability assay with propidium iodide.

JBO_20_7_076017_f010.png

Interestingly, the relatively spherical NCs showed a similar effect on cell viability as the nanostars with their sharp tips, which can be explained by the difference in the size and morphology of the nanostructures. It has recently been shown that NPs with sharp corners and edges can break endosomal membranes and reside in the cytoplasm for a long time, disrupting cellular organelles and leading to increased cytotoxicity. Spherical NPs, in contrast, remain in endosomes after endocytosis, evolve with endosome maturation, and can be easily excreted via exocytosis.64

For intracellular experiments with confocal microscopy, we selected NSts as scattering particles and HeLa cells as model objects. Figure 11 shows images of living cells incubated with nanostars in (a) scattering, (b) transmitted, and (c) combined regimes. The difficulty of localizing the scattering NPs is due to the strong scattering interference of cells and their organelles, resulting in parasitic background illumination from the sample. Therefore, it is preferable to simultaneously observe cells in scattering and transmitted illumination modes.

Fig. 11

(a) Medial slides of living cells incubated with NSts obtained in scattering mode, (b) transmission light mode and (c) combined regimes. The nanostructures are concentrated inside the cells (red arrows) and seen as bright/dark dots in the scattering/transmission light modes, respectively.

JBO_20_7_076017_f011.png

Real-time visualization of living cells incubated with gold nanostructures allows monitoring of PRNP uptake and localization and focuses on the influence of plasmonic particles and complexes on cells, necessary for biomedical applications.

4.

Conclusion

In summary, gold nanostars and silica-coated core–shell NPs with different coating thicknesses were synthesized and characterized to assess their plasmon resonance and scattering properties, particularly important for applications in theranostics. We pioneered optical characterization of the gold nanostructures by decomposition of the attenuation into scattering and absorption. Both scattering and absorption were evaluated by using collimated transmittance and diffuse reflectance/transmittance measurements. The higher scattering properties of the silica-shell-coated NPs were exploited in OCT imaging of capillaries and also in reflectance confocal microscopy experiments in living cells. Nontoxicity of the investigated nanostructures, at least up to 200μg/mL, is suggested by colorimetric and fluorescent cell viability assays.

To the best of our knowledge, this is the first time conventional laser confocal microscopy was applied in combined scattering and transmitted light modes to detect the backscattered signal of gold nanostars, which is useful for direct real-time observation of NP uptake and localization by living cells. The absence of potentially toxic fluorescent dyes in the samples enables focusing on the influence of plasmonic nanostructures exclusively, particularly important for biomedical and theranostics applications.

Acknowledgments

This research was supported by Infotech Oulu Graduate School and the Academy of Finland project 277697. We also acknowledge the support from BM1205 COST Action, the Government of the Russian Federation (Grant No. 14.Z50.31.0004 to support scientific research projects implemented under the supervision of leading scientists), and the Russian Presidential Grant NSh-703.2014.2. The work by NGK and VAB was supported by the Russian Scientific Foundation (Project No. 14-13-01167). We thank I. Skovorodkin and V.-P. Ronkainen for the collaborative work described in this paper.

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Biographies for the authors are not available.

© 2015 Society of Photo-Optical Instrumentation Engineers (SPIE) 1083-3668/2015/$25.00 © 2015 SPIE
Olga Bibikova, Alexey P. Popov, Alexander V. Bykov, Artur Prilepskii, Matti T. Kinnunen, Krisztián Kordás, Vladimir Bogatyrev, Nikolai Khlebtsov, Seppo Vainio, and Valery V. Tuchin "Optical properties of plasmon-resonant bare and silica-coated nanostars used for cell imaging," Journal of Biomedical Optics 20(7), 076017 (31 July 2015). https://doi.org/10.1117/1.JBO.20.7.076017
Published: 31 July 2015
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