2.1.

Nanoparticle Synthesis and Characterization

2.2.

Phantom Study

Gel phantoms (G9382, Sigma) were composed of 50% gelatin and deionized (DI) water. Nonaggregated nanoparticles were dispersed in DI water with gelatin at a concentration $1.2\times {10}^{11}$ nanoparticles per ml. Aggregated nanoparticles in phantoms were prepared from pellets of gold nanoparticles after overnight incubation with highly saturated NaCl solutions. Gold nanoparticles were mixed with 50% gelatin in a hot-water bath (60°C) to prepare final concentrations of $1.2\times {10}^{11}$ nanoparticles (for both aggregated and nonaggregated), and 25 μL of gel with nanoparticles were cover slipped on a glass microscopic slide, air-dried overnight, and imaged the following day.

2.3.

Peritoneal Macrophage Cell Culture Study

Nanoparticles were evaluated in primary peritoneal macrophage cultures harvested from C57/blk6 mice. Isolation of macrophages was performed as described.^24,25 After isolation macrophages were grown in Dulbecco’s modified Eagle’s medium, DMEM (Sigma, MO) with 10% FBS (Hyclone, Logan, UT), 2 mM L-glutamine, penicillin ($50\mathrm{U}/\mathrm{ml}$), and streptomycin ($100\mathrm{\mu g}/\mathrm{ml}$).Macrophages were grown on dual chamber slide for two to three days (Lab Tek, Rochester, NY) at 37°C and 5% ${\mathrm{CO}}_2$ before the addition of nanoparticles. Nanoparticles were incubated at $3\times {10}^{-9}$ nanoparticles per well for 24 h at 37°C. For time-course studies, macrophages were incubated with nanoparticles for 1, 4, and 24 h followed by washing with PBS $5\times$, fixation with 4% formaldehyde, and sealing of the coverslip with Fluoromount (Sigma-Aldrich, Saint Luis, MO) before imaging.

For TEM analysis peritoneal macrophages were isolated from the mice as previously described, placed in Teflon-coated Petri dishes (Welch Fluorocarbon, NH) with DMEM (Sigma, MO) with 10% FCS (Hyclone, Logan, UT), 2 mM L-glutamine, penicillin ($50\mathrm{U}/\mathrm{ml}$), and streptomycin ($100\mathrm{\mu g}/\mathrm{ml}$).Macrophages were grown on Teflon dishes for two to three days (Lab Tek, Rochester, NY) at 37°C and 5% ${\mathrm{CO}}_2$ before addition of nanoparticles. Nanoparticles were added in a concentration of $3\times {10}^{-9}$ particles per plate for 24 h. Before fixation cells were placed in 4°C for 1 h and after macrophages detached they were fixed with a mixture of 2.5% glutaraldehyde, 2% (para)formaldehyde in 100 mM cacodylate buffer (pH 7.0) with 2 mM ${\mathrm{CaCl}}_2$ by mixing equal volumes of fixative and cell suspension for two days at 4°C. Cells were postfixed with 1% ${\mathrm{OsO}}_4$ in 0.1 M PB for 1 h, washed several times with PBS, and dehydrated and embedded in epoxi-resin. Macrophage imaging was performed with transmission electron microscopy (TEM) using a Philips 208 $S$ system with digital imaging system. Determination of the number of nanoparticles per macrophage is described in the Supplementary section.

2.4.

Optical and TEM Imaging

Luminescence was detected with an Olympus IX FV1000 confocal microscope with a spectral scanning system, employing a 2 mW diode laser with excitation wavelength of 635 nm. Laser light was focused and collected with a UPLSAPO objective, $60\times \text{oil}$, and numerical aperture (NA) 1.46. Light was delivered to the sample with an $80/20$ beam splitter. Spectral luminescence profiles were collected between 600 and 800 nm, with 2 nm resolution. For visualization of luminescence from different types of nanoparticles, 0.4 mW laser power was used due to the high signal produced by the nanoshells. For visualization of different types of nanorose, 2 mW laser power was used. Finally, for differential interference contrast (DIC), images were collected in transmittance mode, while luminescence images were recorded in reflectance mode.

2.5.

Determination of the Number of Nanoparticles per Macrophage

A slightly modified formula²⁶ was applied to transform the two-dimensional (2-D) TEM determination of the number of nanoparticles per macrophage, to a three-dimensional (3-D) volume. Given a 2-D TEM image, the diameter of the section ($d$) will be much more than the average distance between nanoparticles ($L$). We can calculate the average distance between nanoparticles as:

If we employ a spherical configuration,$S=\frac{\pi }{4}{d}^2$, then Eq. (1) can be rewritten:

The number of nanoparticles per macrophage were determined by averaging results from $n=20$ macrophages per each type of nanoparticle.

2.6.

Statistical Analysis

ANOVA and paired $t$-tests were performed with the SPSS software package, version 13.0. Differences were considered as statistically significant when the $p$-value was $<0.05$.

3.1.

Phantom Studies of Nanoparticles

To determine the impact of nanoparticle aggregation on gold-induced luminescence, a phantom study using artificially aggregated and nonaggregated nanoroses in gelatin was performed. Laser-induced luminescence of nanoroses was measured at 635 nm with confocal microscopy and emission spectra between 600 and 800 nm wavelengths were collected for analysis. The extinction spectra for all nanoparticles are shown in Fig. 1, with typical phantom results shown in Fig. 2.

Fig. 1

Comparative extinction spectra of colloid nanoparticles.

Fig. 2

Phantom studies of nanoparticles. Two peaks of luminescence with confocal of nonaggregated and aggregated gold nanoparticles are shown.

No emission peaks were observed when the nanoroses were dispersed in the gelatin in a nonaggregated state ($n=5$). However, two emission peaks were observed at 650 to 670 nm and 745 to 760 nm ($n=5$) when the nanoroses were aggregated prior to embedding into the gelatin. The first emission peak detected at 650 to 670 nm was stronger than the second peak at 745 to 760 nm.

Next, we compared the luminescent properties of nanoroses with other commercially available gold nanoparticles of varying coating thickness and composition, including nanoshells, and nanorods with either a maximal extinction at 600 nm (nanorods/600) orat 750 nm (nanorods/750). The extinction spectra of these nanoparticles are shown in Fig. 1. Nanorods/600 and nanoshells produced the same two peaks of luminescence at the same wavelengths as nanoroses ($n=5$, Fig. 2). Further, nanoshells have the same two fluorescent peaks without aggregation ($n=5$). Nanorods/750 with an extinction maximum away from the excitation frequency of 635 nm did not produce luminescence when analyzed in a colloid state, but were weakly luminescent when aggregated.

3.2.

Studies of Nanoparticles Engulfed by Macrophages

To examine the impact of gold aggregation in a biological system, nanoparticles were examined after uptake by macrophages. Typical results are shown in Fig. 3(a) and quantitative results are shown in Fig. 3(b). All gold nanoparticles that aggregated within macrophages demonstrated the same two luminescent emission peaks as in the gelatin phantom studies. In Fig. 3(a), nanoshells produced a stronger luminescence compared to original nanoroses. The luminescence intensity per macrophage (2000 pixels analyzed per macrophage) was calculated and the mean luminescence from 20 macrophages was plotted. Figure 3(b) shows that nanoshells produced approximately four times stronger signals than original nanoroses. At an emission wavelength of 650 to 670 nm Cy5 conjugated iron oxide nanoparticles produced approximately 1.5-fold the signal intensity of original nanoroses, but did not have the second luminescent peak at 745 to 760 nm we observed for other gold nanoparticles. Nanorods/600 and nanorods/750 both produced two luminescent peaks, but the intensity of both peaks were weak and half that measured for original nanoroses. Despite being tuned to the excitation frequency of 635 nm, nanorods/600 were not superior to nanorods/750, because nanorods/600 do not have a surface coating that is as ideal for cellular uptake. In contrast, Nanorods/750 are coated with polyethylene glycol (PEG), which does not prevent uptake by macrophages in culture. Due to the weakness of the luminescence of both nanorods, they were not included in the quantitative comparison of nanoparticles presented in the next section.

Fig. 3

(a). Confocal images of various near-infrared nanoparticles in macrophages cell culture. Gray upper panels are differential interference contrast microscopy images (DIC) of macrophages, and two red lower panels represent luminescent images at two wavelengths at 650 to 670 and 745 to 760 nm. Second emission at 745 to 760 nm was not observed from Cy5 iron oxide nanoparticles. (b). The calculated luminescence intensity profile from $n=20$ macrophages/each nanoparticles, shows that nanoshells produced a signal approximately four times stronger than original nanoroses.

3.3.

Luminescent Intensity of Nanoparticles

To determine the impact of differing morphologies of surface gold on luminescence, the relative luminescent intensities of the two brightest gold nanoparticles (nanoshells and nanoroses) were compared in a more quantitative fashion to Cy5 conjugated nanoparticles (Table 3).The average number of nanoparticles per macrophage was determined by counting individual nanoparticles identified by transmission electron microscopy (TEM) with Image J. Twenty macrophages were analyzed per type of nanoparticles, and the mean results are presented. Because Cy5 conjugated nanoparticles and Cy5 molecules are below the resolution of TEM, the number of these nanoparticles and molecules per macrophage was taken from the literature.²⁷

Table 3

Luminescent intensity of nanoshells, original nanoroses, and Cy5 iron oxide nanoparticles and molecules.

Figure 4 shows the typical appearance of nanoparticles’ accumulation and distribution inside macrophages. Due to their larger size, nanoshells localized in the cytoplasm close to the cell plasma membrane, while the smaller original nanoroses and Cy5 conjugated iron oxide nanoparticles were located within deeper cellular structures. For original nanoroses, $7549\pm 236$ particles/macrophage was determined, whereas nanoshells were less concentrated in macrophages ($1025\pm 128$ particles/macrophage). For Cy5 conjugated nanoparticles and Cy5 molecules, published values of 96,000 and 288,000, respectively, were used per macrophage.²⁷

Fig. 4

Typical TEM images of different nanoparticles accumulated in a representative macrophage.

The luminescence intensity per macrophage at 650 to 670 nm emission was greatest for nanoshells, followed by Cy5 conjugated nanoparticles and Cy5 molecules, followed by original nanoroses (Table 3, $p<0.05$). Further, the brightness per nanoparticle or molecule was greatest for nanoshells, followed by original nanorose, and was less for Cy5 conjugated nanoparticles, and least for Cy5 molecules (Table 3). These results are consistent with the interpretation that luminescence increases when gold nanoparticles cluster because the overall surface of gold per nanoparticle increases (weight of gold per nanoparticle was greater for nanoshells compared to original nanoroses). However, when the brightness per gram of material was compared, the fluorescent dyes were more intense than nanoshells and original nanoroses, the intensity for the latter two being similar (Table 3).

3.4.

Time-Dependent Uptake of Nanoshells and Original Nanoroses by Macrophages

To further verify that gold nanoparticles gain luminescence after clustering, confocal imaging was performed to follow the accumulation of nanoshells and original nanoroses inside macrophages over time. Macrophages were incubated with nanoparticles and examined after 1, 4, and 24 h (Fig. 5). After the first hour of incubation, DIC imaging revealed no evidence of intracellular nanoparticles. Consistent with the phantom studies, individual nanoshells demonstrated luminescence without clustering, while the original nanorose did not.

Fig. 5

Time-dependent accumulation of original nanoroses and nanoshells in macrophages.

After 4 h, DIC imaging suggested that both nanoparticles had been internalized by macrophages, and there was a marked increase in the corresponding luminescence brightness. Similar results were obtained after 24 h, with greater visible luminescence intensity per macrophage for nanoshells compared to original nanoroses, as quantified in Table 3.

3.5.

Luminescence of Different Nanoroses with Varying Gold and Ironoxide Composition

To determine if the luminescence of the original nanorose could be enhanced by altering the consistency of the gold coating and its thickness, two sister nanoparticles were synthesized and examined. Representative drawings of the three different nanoroses are shown in Fig. 6, and their characterization and synthesis is compared in Tables 4 and 5. Descriptive names for these nanoparticles include thin knobby for the original nanorose, thin round for the more consistent thin gold coating, and thick knobby for the increased gold mass. Specifically, thin knobby (original nanorose) consists of more densely packed 5-nm iron oxide clusters with a variable gold coating. Thin round has a less densely packed iron oxide core and surface deposition of a more consistent thin layer of gold, coated with 20 kDa PEG. Thick knobby contains the greatest mass of gold per nanoparticle of the nanoroses, has a similar density of 5-nm iron oxide clusters to the thin round, and with a diameter of $179\pm 16\mathrm{nm}$, is the largest nanorose. Thick knobby is also coated with 20 kDa PEG polymer in contrast to the original nanorose, which is coated with dextran. The NIR shift and single photon luminescent signal is due to the gold coating, and not the iron oxide core or its density, and not due to PVA, dextran, citric acid, and/or PEG. The more gold on the surface of the nanoroses, the greater the NIR shift.

Fig. 6

The upper panel are TEM images of typical original nanoroses (thin knobby, left); thin round (middle) and thick knobby (right). The lower panel is a schematic drawing of composition and relative size of the nanoroses.

Table 4

Differences in synthesis of thin knobby (original nanoroses), thin round and thick knobby.

Table 5

Characterization of different iron oxide-gold nanoclusters (nanoroses).

All three nanorose extinction spectra tune to the NIR, with maximal extinction for thin round at 600 nm, thin knobby (original nanorose) at 700 nm, and thick knobby at 850 nm. As shown in Fig. 7 and Table 6, thin round and the original nanoroses (thin knobby) were taken up similarly by macrophages ($6032\pm 719$ versus $7549\pm 236$), but thick knobby to a lesser extent, $2643\pm 337$ ($p<0.05$, ANOVA). All three nanoroses demonstrated similar peaks of luminescent emissions at 650 to 670 and 745 to 760 nm but different intensity (Fig. 7). Figures 7(b) and 7(d) compare the relative luminescence of the three nanoroses, and demonstrates that the thinner consistent gold shell of the thin round generated greater luminescence intensity than the thick knobby with the greatest mass of gold per nanorose nanoparticle, or the thin knobby (original nanorose) with variation in the thin gold coating ($p<0.05$, ANOVA). Thick knobby produces less luminescence because 1. there are fewer per macrophage ($2643\pm 337$ versus $7549\pm 236$—thin knobby—and $6032\pm 719$—thin round), and 2. their extinction maximum was farthest from the excitation wavelength of 635 nm. In contrast, the extinction maximum of thin round has been tuned to 600 to 650 nm, which coincides with laser excitation wavelength.

Fig. 7

Comparison of the three varieties of nanoroses. (a) DIC and confocal images of the thick knobby and thin round in macrophage culture, with original nanorose (thin knobby) shown in Figs. 3 and 5. (b) Quantitative comparison of luminescence intensity of the three nanoroses, average spectrum from Fig. 7(d). (c) Extinctionspectrum of thin round, thick knobby and thin knobby (original nanoroses). (d) Maximum luminescence from individual experiments in A. M1 firstluminescence peak at 650 to 670 nm, M2 is the second luminescence peak at 745 to 760 nm. $*p<0.05$.

Table 6

Luminescent intensity of different types of nanoroses.