Distribution of label free cationic polymer-coated gold nanorods in live macrophage cells reveals formation of groups of intracellular SERS signals of probe nanoparticles

Abstract

Gold nanorods coated with poly(diallydimethylammonium chloride) (PDAC-GNRs) were used to observe the distribution of surface-enhanced Raman signals in live cells and generate distinct groups of surface-enhanced Raman scattering (SERS) spectra in different regions of the cells. Spectra with unique features were clustered into sets of molecules in live murine macrophage cells (Raw 264.7). The distribution of biological substances detected by SERS signals of PDAC-GNRs is also discussed.

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The use of gold nanorods (GNRs) in various biomedical applications both in vitro and in vivo has recently expanded because of their unique optical properties.¹ It is well known that GNRs display both transverse and longitudinal surface plasmon resonance bands. The transverse plasmon resonance band is similar to that of spherical-shaped gold nanoparticles, where light absorption and scattering are due to electronic oscillations across the short axis of the GNR. For the longitudinal band, light absorption and scattering are due to oscillation of electrons along the long axis, resulting in a high absorption intensity.^2,3 Owing to such strong resonance effects, GNRs are highly suitable for enhancement of light absorption and scattering when compared with other shapes of gold nanoparticles.^4,5 Furthermore, the surface of GNRs is easy to functionalize with various chemical and biological molecules. All these properties make GNRs a suitable material for biomedical applications.⁶

Recently, the combination of Raman spectroscopy and enhancement by gold nanoparticles to produce surface enhanced Raman scattering (SERS) has shown a high potential to detect low concentrations of biological molecules and analyse compositions in live cells.^7–11 When compared with spherical gold nanoparticles, GNRs provide a higher SERS signal than gold nanospheres owing to the properties of GNRs mentioned above.^12,13 Thus, GNRs are of interest as a Raman signal enhancer. It has been reported that GNRs were used as a SERS substrate for various types of biological detection.^14,15 However, to our knowledge, studies on molecular distributions in cells probed by SERS from label-free GNRs are rare. Investigation of SERS probing in live macrophages is a crucial step towards the wide application of GNRs enhancement for intracellular SERS detection of cellular components, which normally display weak Raman scattering signals. The study is also highly relevant to intracellular sensing applications. Thus, we are interested in investigating the generation of SERS signals in live cells by label-free GNRs, the types of SERS signals produced, and the occurrence pattern of the signals (random or the result of a myriad of potential intracellular targets).

The biocompatibility of GNRs is important for this investigation. GNRs are commonly prepared in the presence of cationic surfactant cetyltrimethylammonium chloride (CTAB) that is toxic to cells. To this effect, various methods have been used to modify the surface of GNRs to be suitable for biological and therapeutic applications.^16–20 We recently reported the effect of cationic polymer (poly(diallydimethylammonium chloride); PDAC)-coated GNRs on macrophage cells where we found that at a PDAC-GNR concentration of 5 μg mL⁻¹ had no effect on cell viability. Besides toxicity, it is important to determine other effects on the cells such as the development of an inflammatory response. Likewise, PDAC-GNRs at a concentration of 5 μg mL⁻¹ do not cause any significant effect on the immune response. Furthermore, the rate of uptake of PDAC-GNRs in the cells is higher than that of other types of surface-modified GNRs reported in our previous work.²¹ Thus, in the present study, PDAC-GNRs at a concentration of 5 μg mL⁻¹ were investigated for evaluating the intracellular distribution of SERS signals in macrophage cells (Raw 264.7). To our knowledge, this is the first report to demonstrate the distribution of label-free PDAC-GNRs in the vesicles (i.e., endosomes, vacuoles, lysosomes) of live macrophage cells and formation of clusters of intracellular SERS signals interaction of the PDAC-GNRs with the cellular components in those vesicles. Furthermore, we demonstrate the distribution of distinct or identical groups of SERS spectra from different areas of the cells using an automated spectral detection and classification method, as reported in.²²

GNRs (width: ∼28.8 ± 1.4 nm and length: ∼60.8 ± 2.6 nm) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and coated with PDAC using the same approach as reported in.²¹ The PDAC-coated GNRs (PDAC-GNRs) particles are shown in Fig. 1. The Raw 264.7 cells (1 × 10⁵ cells) were cultured in a glass Petri dish at 37 °C in a 5% CO₂-humidified incubator for 24 h. PDAC-GNRs were then added to the Raw 264.7 cells, and incubated under the same conditions for 24 h. Following incubation, the cells were washed with phosphate-buffered saline solution (PBS) four times before the SERS measurements.

Fig. 1 Transmission electron microscopy image of PDAC-GNRs.

The internalization of PDAC-GNRs in Raw 264.7 cells was observed by transmission electron microscopy (TEM), as reported previously.²¹ The Raw 264.7 cells were first cultured in a well containing polystyrene coverslips (1 × 10⁵ cells per well). Thereafter, the cells were incubated with 5 μg mL⁻¹ PDAC-GNRs for 24 h. Following incubation, the cells were first washed and fixed with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Next, cells were washed again with 0.1 M cacodylate buffer solution containing 7% sucrose, and post-fixed with 0.1 M cacodylate buffer solution containing 1% osmium tetroxide and 0.5% potassium ferrocyanide. Following post fixation, the cells on the coverslip were dehydrated, embedded in Epon812 (TAAB), sectioned into slices with a thickness of 80 nm, and stained. All sections were imaged by a JEM-1011 transmission electron microscope (Fig. 2). TEM images of the Raw 264.7 cells incubated with PDAC-GNRs show that most of the particles were in the vesicles located in the cytoplasm (Fig. 2a). Some particles were observed during the endocytosis (Fig. 2b) and exocytosis (Fig. 2c) processes. Aggregation of the PDAC-GNRs in the cells was also observed in various areas (Fig. 2d–f). The intracellular environment contains various salts that may influence aggregation. Additionally, during endocytosis, particles are wrapped by endosomal membrane and trapped in endosomal vesicles, which may result in the aggregation of particles. Also, aggregation of the PDAC-GNRs could possibly be induced by the fusion of endosomes with acidic cellular organelles (lysosomes) (Fig. 2d). Aggregation of PDAC-GNRs can significantly enhance and improve detection of Raman signals generated from molecular components in the cells.²³ The PDAC-GNR particle distribution, as observed in the TEM images, also provides indications of the development of the expected SERS signals. For instance, because the particles were commonly observed in the endosomes (as indicated by spots 1 and 2 in Fig. 2e), other intracellular vesicles such as lysosomes²⁴ (Fig. 2f), and vacuoles (Fig. 2e, spot 3), we could expect the detected SERS signals to comprise spectral signatures from the molecules in these vesicles. The detection of the intracellular SERS signals was performed by Raman micro-spectroscopy (Nanophoton). A 785 nm diode laser was used as the excitation source with a power of ∼2.0 mW μM⁻². A near infrared (NIR) microscope objective lens (Nikon Apo 60×/1.0 W water immersion, 1.0 NA, 2.8 mm working distance) was used to focus on the sample and collect the back-scattered Raman spectra generated from the cells.

Fig. 2 TEM images of Raw 264.7 cells incubated with PDAC-GNRs for 24 h. (a) Aggregation of PDAC-GNRs in intracellular vesicles located in cytoplasm of Raw 264.7 cell. (b) Endocytosis and (c) exocytosis of PDAC-GNRs by Raw 264.7 cells. Distribution and aggregation of PDAC-GNRs (d) by fusion of endosome with lysosome, (e) in endosomes (spots 1 and 2) and vacuoles (spot 3), and (f) lysosomes.

The integration/collection time for each spectrum was 2 s per line for all scans. Spectral data were processed and extracted from the measurements by an automated spectral detection and classification method by employing a peak count criterion, as described in.²² A baseline correction was performed on selected spectra and then spectra were grouped in clusters according to their similarity in spectral properties using Pearson correlation–correlation coefficients above 0.7. Further information is provided in the electronic supporting information (ESI).†

The most important issues to address are the occurrence of the detected signals (single or multiple) and their spatial distribution in the cells. The SERS signals generated from different Raw 264.7 cells displayed one of several spectral groups following incubation with PDAC-GNRs for 24 h. Some of the peaks in these spectral groups (Fig. 3 and 4) can be assigned to the prominent bands observed in PDAC-GNRs spectrum (i.e., 756 cm⁻¹, 996 cm⁻¹, 1073 cm⁻¹, 1442 cm⁻¹, and 1605 cm⁻¹, as noted in black in Fig. 3 and 4). Detailed peak positions of PDAC-GNRs are shown in ESI.† Other peaks that corresponded to weak bands originally observed in the PDAC-GNRs spectrum (i.e., 496 cm⁻¹, 530 cm⁻¹, 890 cm⁻¹, 1130 cm⁻¹, 1295 cm⁻¹, and 1369 cm⁻¹, as noted in blue in Fig. 3 and 4) became more prominent when the nanoparticles were in the cells. New peaks also appeared after incubation of the Raw 264.7 cells with PDAC-GNRs. These new peaks are assigned accordingly, as shown in Table 1, and discussed further later. The observation of signals corresponding to the coated GNRs and new signals following incubation of the cells with the coated GNRs has been reported.^25,26 Based on the TEM images, most PDAC-GNRs were located in the intracellular vesicles, especially endosomes and lysosomes, as discussed previously. Therefore, differences in the intracellular SERS signals could be due to the inhomogeneity of molecules in the vesicles of the Raw 264.7 cells. It is worth noting that PDAC-GNRs were not observed in the nucleus.

Fig. 3 Multiple occurrence spectra and spatial distribution of average SERS spectra occurring multiple times in Raw 264.7 cells incubated with PDAC-GNRs for 24 h. Visible images show the region (yellow dots) in the cells from which the SERS spectra were obtained. The same spectral profiles were detected in each image, as denoted by the red circles. The corresponding spectral profiles are shown to the right of each visible image. The green and the red lines show that the peaks at 996, 1130, 1345, 1590, 1745 cm⁻¹ were detected in all spectra (a–f). The number of collected spectra and the frequencies of occurrences are provided in ESI.†

Fig. 4 Single occurrence spectra, spatial distribution and average SERS spectra for single occurrence spectra detected inside Raw 264.7 cells incubated with PDAC-GNRs for 24 h. Visible images show the region (yellow dots enclosed in red circles) in the cells from which the SERS spectra were obtained. The corresponding spectral profiles of each image are shown on the right. The red and brown lines show that the peaks at 1130 and 1190 cm⁻¹ were detected in all spectra (a–d). The number of collected spectra and the frequencies of occurrences are provided in ESI.†

Table 1 Assignment of SERS features for multiple and single occurrence spectra in intracellular PDAC-GNRs distributions in Raw 264.7 cells

Assignment for Raman features	Multiple occurrence SERS spectra (cm^−1)						Single occurrence SERS spectra (cm^−1)
	a	b	c	d	e	f	a	b	c	d
Cystine: S–S stretching vibration^26,35	511				511		511
Phospholipids: P–O–C bending vibration^32				551	553		540
Possibly C–S stretching vibration^26				651				652	659
Cysteine: C–S stretching vibration^26	681	691	684				667		683	678
Unassigned									702
Lipid, adenine: C–N stretching vibration^30,31						733	728		726
Tryptophan: indole symmetric breathing vibration^28	773		766	774		776		782	771	782
Lipids: O–P–O anti symmetric stretching vibration^31,36	811	800		821	805	821	816		814	803
Tryptophan^37			874			874			874
Amino acids, proteins: C–C stretching vibration^28	909	916				908
Proteins: backbone vibration^38			933				937			932
Proteins: C–C skeletal stretching vibration^37,39	949			950	959			957
Phenylalanine: C–H in plane bending vibration^30	1022		1023				1030			1034
Carbohydrates: C–C, C–O stretching vibration^33	1063		1070	1078		1055				1062
Phosphate: O–P–O; carbohydrates C–C, C–O–C vibration^33									1095
PSS–PDAC complex	1135	1126	1122	1126	1127	1124	1137	1139	1129	1137
Threonine: CH3 rocking vibration^28,40						1193	1188	1190	1192	1198
Proteins: amide III^30,32,37	1213	1220		1208	1232			1225	1233
Proteins: amide III^30,32,37	1270	1269	1261	1264				1257		1249
Proteins: CH2 wagging vibration^28,33			1310					1304
Proteins: CH3 deformation, C–H deformation vibration^31,33,41	1338	1342	1350	1342	1351	1346		1354	1333	1334
Proteins: COO^− symmetric stretching vibration^28,33	1418		1410					1415	1423	1420
Lipids/proteins: CH2, CH3 gamma vibration^33,41		1468							1479
Lipid stretches^30			1529	1553				1529		1524
Proteins/lipids: amide II^23,28,32–34						1550	1556
Phenylalanine/tyrosine: C=C vibration^28,30,31,33,41	1582	1590	1591	1591	1591	1598		1577	1578	1596
Proteins/lipids: amide I, C=C stretching vibration^41	1684					1691		1692
Lipids: C=O stretch from ester region vibration^31	1753	1745	1744	1740	1749	1740		1750	1745	1752

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Based on the above results, the SERS signals were categorised in two main groups according to the distribution of spectra in the macrophages using the automated clustering method described in ESI.† Spectral clusters observed at more than one occurrence were classified as ‘multiple occurrence’ spectra (Fig. 3) and spectral clusters observed at a single occurrence and lacking a distribution of SERS signals in the studied areas of cells were denoted as ‘single occurrence’ spectra (Fig. 4). Most importantly, the group of multiple occurrence spectra shows that the SERS spectra of individual areas of the same cell and those in different cells were similar. This implied that formation of aggregated PDAC-GNRs occurred in the same type of vesicle, as indicated by the generation of the same SERS spectral profiles that could occur from similar molecular components in the vesicles. As noted earlier, most PDAC-GNRs aggregated in the vesicles, especially lysosomes and endosomes, after incubation for 24 h (Fig. 2). This also confirmed that PDAC-GNRs were detecting the same type of molecule, possibly because of their presence in the same type of vesicle. Previous work by Kneipp et al.²⁷ also reported that gold nanoparticles were present in lysosomes rather than other structures following overnight incubation with cells. Also, the observed similarity in the spectral profiles could indicate the presence of similarly formed PDAC-GNRs aggregates in the cells.

It is interesting to note that in the cluster of multiple occurrence spectra, some peaks were detected in all spectra i.e., ∼996 cm⁻¹ (assigned to the PDAC-GNRs), ∼1345 cm⁻¹ (CH₃ deformation or C–H deformation of proteins),²⁸−1590 cm⁻¹ (phenylalanine, tyrosine),^27,28 and ∼1745 cm⁻¹ (lipids: ν(C=O) ester region),²⁹ as shown in Fig. 3a–f (these peaks are denoted by the green lines). Together with the TEM analysis, this finding may evaluate the endocytotic entrance and endo-lysosomal pathway of the particles. Moreover, the results obtained from this technique allow observation of the distribution of the intracellular components that relates to the distribution of the PDAC-GNRs probe. In contrast, for the cluster of single occurrence spectra, these peaks were not detected in all spectra, as shown in Fig. 4. This may be due to the generation of single occurrence SERS signals from particles in different locations or those interacting with some fundamentally different intracellular molecules.

Fig. 3a and b show two SERS spectra obtained from the same area (area 1) after clustering. The spectra were collected from the clusters as identified by the yellow dots in the red circles. The important finding is that the same spectra were obtained in different areas of the same cell and different cells. Approximately half of the peaks detected in the spectrum panel of Fig. 3a were observed in the spectrum panel of Fig. 3b. The same findings were observed for area 2 (Fig. 3c and d). In contrast, only one type of spectral profile was obtained by the cells shown in Fig. 3e and f following clustering. This implies that the distribution and/or the degree of aggregation of PDAC-GNRs in these two studied areas (areas 3 & 4) were more homogeneous than the studied areas 1 & 2 (Fig. 3a–d). Different Raw 264.7 cells apparently show differences in the homogeneity of the detected SERS signals, thereby indicating that different Raw 264.7 cells react to the particles differently. Also, the uptake ability and cellular activities might be different in each Raw 264.7 cell, which could affect the distribution of the PDAC-GNRs, subsequently leading to different distributions of the intracellular SERS signals. Physically, the homogeneous or inhomogeneous SERS signal distributions and multiple or single occurrence spectra could result from differences in the location of the PDAC-GNRs in the cells and degree of aggregation of the PDAC-GNRs in the cell vesicles. As seen in Fig. 2f, only two PDAC-GNRs particles were located in the first lysosome, whereas numerous PDAC-GNRs clumped together in the other lysosome. This finding confirms the different degrees of distribution and aggregation of particles inside intracellular vesicles.

Around 90% of the peaks detected in the spectra of Fig. 3e and f were also observed in the spectra of Fig. 3a–d. For the spectra in Fig. 3f, additional peaks at 733 cm⁻¹ (C–N stretch in lipids/amino acid)^28,30,31 and 1550 cm⁻¹ (lipid stretches/amide II)^28,32–34 that correspond to lipids and proteins were observed (these bands are denoted in purple). Overall, the main peaks detected in the spectra in Fig. 3a–f can be assigned to proteins, lipids, and carbohydrates. The results are summarized in Table 1; the peaks are assigned according to the literature references given earlier.

In addition to evaluating the formation of clusters of spectra in cells and their spatial distribution by nanoparticles-based SERS, one benefit of nanoparticles-based SERS is that it has the ability to detect molecules that appear rarely. Even a single occurrence of a molecular signature may be biologically relevant, and our spectral detection algorithm discriminates only on the spectral quality rather than the number of occurrences. A number of single occurrence spectra were detected in the intracellular SERS signals (Fig. 4a–d) in the different studied areas. This implies that PDAC-GNRs aggregate in different types of vesicles and organelles. The peak assignments of the spectra after clustering of all studied areas in Fig. 4 are shown in Table 1. Most peaks detected in the single occurrence spectra (Fig. 4) were also observed in the multiple occurrence spectra in Fig. 3. The peaks that were detected in Fig. 4 but were absent in Fig. 3 were 702 cm⁻¹ (unassigned) and 1095 cm⁻¹ (phosphates/carbohydrates)³³ (Fig. 4c, bands denoted in purple). Interestingly, the peak at 1190 cm⁻¹ (CH₃ rocking vibration from threonine, this peak is denoted by the brown line)^28,40 is seen in all areas of single occurrence SERS spectra, but only in one region of the multiple occurrence SERS spectra (Fig. 3f). Additionally, peaks corresponding to C–S stretching vibrations (652–691 cm⁻¹)^35,42 were detected in all single occurrence spectra but likewise, not in all the multiple occurrence SERS spectra (Fig. 3, Table 1). Therefore, our findings show that the multiple and single occurrence SERS spectra have unique features in terms of peaks and do not randomly vary across the cells. Additionally, the results show that the intensities of SERS signals are different. This implies that the number of molecules interacting with the particles and the degree of particle aggregation influence the strength of the SERS signal.

Conclusions

Herein, we demonstrate that PDAC-GNRs can enhance intracellular Raman signals and provide spectral characteristics related to the PDAC-GNRs distribution inside live macrophage cells. TEM was used to observe the location of the PDAC-GNRs in the macrophage cells, and to evaluate the uptake of the particles by cells and form of the particles following entrapment in the cells. The SERS spectra were generated according to the interaction between the aggregated PDAC-GNRs and molecular components inside the vesicles (endosomes, lysosomes, and vacuoles) of live macrophage cells. The detected spectra were categorised into two groups based on the distribution of particles. Multiple occurrence spectra were observed from different cells and different regions of aggregated nanoparticles. These multiply occurring spectra featured certain spectral properties that differ from those of the singly occurring spectra, suggesting a difference in intracellular location and/or aggregation of the particles. These results show how the distribution of the signals in the cell and their similarities. However, they cannot be used to identify specific molecules inside the cells because of the difficulty in precisely identifying the biological molecular components and the diverse range and complexity of molecular components in the cell. Nevertheless, our approach provides an understanding of the types of spectra that can be detected in macrophage cells. It also shows that the use of automated clustering, as reported,²² can reveal correlations between (i) the distributions of PDAC-GNRs particles and (ii) the distribution and number of occurrences of the SERS signals and the SERS spectral fingerprints from the molecules in the cells. These distributions are also related to the aggregation of PDAC-GNRs in the cells. Although there are limitations in controlling the aggregation and distribution of particles inside cells, our results show that the SERS signals of PDAC-GNRs inside macrophage cells produce distributions whereby the spectra generated from different cells and different areas of the same cell are essentially the same (Fig. 3a–f) with some unique peaks appearing in specific spectra. It is also worth noting that the peak at∼1130 cm⁻¹ from PDAC-GNRs (possibly C–H in plane bending vibration) is observed in all studied areas of the cells in both the single and multiple occurrence spectra (Fig. 3 and 4 denoted as a red line). This peak can be assigned to the complex formation of PSS–PDCA.⁴⁰ Because of this peak occurrence in all areas, it could be used as a reference for detecting changes in the environment of the cells or distribution of intracellular molecules of interest using PDAC-GNRs. Overall, this technique could be used as a biosensor tool to evaluate differences in the cell condition or the environment inside cells.

Acknowledgements

We acknowledge funding from the Japan Society for the Promotion of Science (JSPS), a Grant-in-aid for Young Scientist B (no. 24700481), the JSPS World Premier International Research Center Initiative Funding Program, and the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra45556d

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