Analysis of the average spectra of live cells and the isolated cytoplasm

The average SERS spectra of the endolysosomal compartments (live cell SERS mapping) and of the isolated cytoplasm are presented in Figure 8.1. The average spectra of the endolysosomal compartments and of the isolated cytoplasm were calculated from 883 and 613 SERS spectra, respectively. The endolysosomal spectra originated from five different J774 cells with identical incubation conditions to compensate for cell-to-cell variation.

The two average spectra exhibit different traits. The main differences appear in the 1580-1440 cm^-1, 1320-1220 cm^-1, 1180-1120 cm^-1, and the 680-460 cm^-1 regions. The average SERS spectrum of the extracted cytoplasm shows a higher intensity in the 1580-1440 cm^-1 and the 1320-1220 cm^-1 regions, which include many vibrations assigned to the amide II and amide III bands [253], as shown in Table 8.1. Some spectral bands corresponding to vibrations of tryptophan at 1352 cm^-1, C-C and C-N stretching at 1170 cm^-1, 1130 cm^-1, and 1085 cm^-1, vibrations of tyrosine at 838 cm^-1, and C-S and S-S stretching vibrations at 670-466 cm^-1 are more intense in the average of the endolysosomal spectra (compare black spectrum in Figure 8.1 and tentative assignments

97 in Table 8.1). The prominent presence of the vibrational bands of tryptophan and tyrosine and those of C-C stretching allow for the conclusion that the nonpolar residues of proteins can be exposed in the endolysosomal compartments. An increased exposure of the nonpolar side chains to the aqueous environment can result from the denaturation and fragmentation of some protein molecules, as proteins tend to fold into structures with hydrophobic cores in polar solvents [47]. The increased contribution of nonpolar residues to the SERS spectra can further indicate the interaction of the gold nanoparticles with the membrane of the compartments they reside in, which has been discussed previously [39].

Since the laser intensity was lower in the SERS data collection of live cells than in solution, it can be excluded that the denaturation of those proteins results from photothermal damage.

Figure 8.1. The average of 613 SERS spectra from the isolated cytoplasm (red trace) and of 883 SERS spectra from five live J774 cells (black trace). 24 h incubation, excitation wavelength: 785 nm, acquisition time: 1 s, excitation intensity: 2.3×10⁵ W/cm² and 5.7×10⁵ W/cm² in the endolysosomal compartments and the isolated cytoplasm, respectively.

The presence of S-S stretching vibrations in the 500-600 cm^-1 region in the average spectra of both the endolysosomal compartments and the isolated cytoplasm indicates that

98

despite the suggested denaturation and fragmentation of some protein molecules, other protein chains preserve their secondary and tertiary structures at least partially (Figure 8.1 and Table 8.1). The higher intensity of the C-S and S-S stretching vibrations between 466 cm^-1 and 675 cm^-1 in the endolysosomal spectra (Figure 8.1 black spectrum and Table 8.1) can come from the revealed disulfide bridges during protein digestion or from proteins with a high number of sulfur bridges [254, 255]. Based on the results of Chapter 7 (see Table S7.2), the latter can be explained by the high amount of histones in the protein corona, as nucleosomes have been shown to stabilize with disulfide bonds [256] between the N-terminal cysteine of histone H4 and a cysteine in the α-helix 2 of histone H2A [257, 258].

Table 8.1. Raman shifts and their tentative assignments in Figure 8.1. ν stretching, δ deformation, symm symmetric, wag wagging, rock rocking, br breathing, R benzene ring, r pyrrole ring. Band assignments were based on refs. [40, 104, 171, 172, 215, 219, 253].

Raman shift

(cm^-1) Tentative assignment Raman shift

(cm^-1) Tentative assignment

1744 ν(C=O) 1021 ν(C-O) of ribose

99 8.1.2. Principal component analysis of live cell and isolated cytoplasm spectra Principal component analysis (PCA) was performed to gain information about the differences that are characteristic of the two samples. The scores plot and the loadings of the first and second principal components (PC1 and PC2, respectively) are shown in Figure 8.2.

Figure 8.2. Scores plot (top) and the loadings of the first (middle) and second principal components (bottom) as results of the PCA of the SERS data of five live J774 cells (883

100

spectra) and the isolated J774 cytoplasm (613 spectra), incubated with gold nanoparticles for 1.5 h.

The incubation of living J774 cells was continuous during the 1.5 h incubation time, which could result in gold nanoparticles residing in intracellular compartments that are in different states of the endolysosomal pathway: some particles had just been internalized, while others had been processed for the whole incubation time. This intracellular heterogeneity is well reflected in the scores plot (Figure 8.2 top). While the SERS spectra collected from the homogeneous solution of isolated cytoplasm show low variance along PC1 and PC2, the endolysosomal spectra are spread along both principal components. As the PC1 loading shows (Figure 8.2 middle), the PCA separation of the data was based on the variances at the wavenumbers 1310 cm^-1 assigned to vibrations of guanine, amide III, and C-H deformation; 1280 cm^-1 assigned to vibrations of amide III, DNA, RNA, and CH2 wagging; 1240 cm^-1 assigned to vibrations of amide III, adenine, RNA, and PO2

asymmetric stretching; 1123 cm^-1 assigned to C-C and C-N stretching of proteins and C-O stretching of carbohydrates; 834 cm^-1 assigned to vibrations of tyrosine, proline, and O-P-O asymmetric stretching; 653 cm^-1 assigned to C-S stretching and C-C twisting vibrations of tyrosine and phenylalanine; and 500 cm^-1 assigned to S-S stretching vibrations (for tentative assignments, see Table 8.1). The highest variances were observed in the bands at 1123 cm^-1 and 500 cm^-1. It can be seen by comparing the PC1 loadings with the average spectra (Figures 8.2 middle and 8.1) that all the bands with high variance are more characteristic to the spectra collected from the endolysosomal compartments, except for the 1240 cm^-1 band, which is more prominent in the spectra of the isolated cytoplasm. These bands were assigned to vibrations of nonpolar protein residues, which suggests a more hydrophobic environment of the gold nanoparticles. This could partially be due to their interaction with the membrane of the intracellular compartment [39], since the band at 1123 cm^-1 can be further assigned to (phospho)lipid vibrations [144, 253], and also to the denaturation of the probed proteins in the close proximity of the gold nanoparticles. Moreover, most of the bands can be assigned to different vibrations of nucleic acids as well, which suggests their higher abundance in the close proximity of the gold nanoparticles inside the cells than in the solution of isolated cytoplasm. The loadings of the second principal component (Figure 8.2 bottom) reflect that the band at 785 cm^-1 is more prominent in the dataset of living cells, which can be attributed to nucleic acids

101 (for tentative assignments, see Table 8.1). Due to the general association of nucleic acids with histones, these findings are in agreement with the mass spectrometry data of the hard protein corona presented in Chapter 7 and Tables S7.1 and S7.2, and further support that the interaction of nanoparticles with the histone molecules in the hard protein corona contributes to the high abundance of the C-S and S-S stretching vibrations in the endolysosomal spectra.

8.1.3. Band occurrences in the spectra of live cells and the isolated cytoplasm To understand the variances in the datasets, the band occurrences were analyzed in every spectrum as described in Section 4.11.3. The results of this analysis are independent of the relative band intensities and inform of whether an interaction takes place between the nanoparticle and the given functional group or not.

In Figure 8.3, the band occurrences are shown with respect to whole datasets comprising all the spectra of all five cells and of the extracted cytoplasm, respectively. The analysis of band occurrences was described in details in Section 4.11.3. The relative band occurrence graphs are the summaries of these analyses in the whole datasets, where the peak at a chosen Raman shift refers to the number of spectra in which the band at the chosen wavenumber appeared. The band occurrences of datasets belonging together were presented in the same figure so that the occurrence of individual bands in the whole datasets could be related. Hence, the figures are graphs of relative band occurrences, where comparing the intensity of a chosen band in relevant datasets sheds light on the likeliness of the corresponding nanoparticle-biomolecule interaction in the given datasets.

In the relative band occurrence curve interpreting the SERS data of the isolated cytoplasm (Figure 8.3 red curve), so-far undetected vibrational bands appear with low relative abundance at 1340 cm^-1 assigned to vibrations of guanine, amide III, and C-H deformation; 815 cm^-1 assigned to vibrations of proline, tyrosine, and C-C stretching; and at 785 cm^-1 assigned to various vibrations of nucleic acids, such as O-P-O stretching and the C6-ring breathing vibrations of cytosine, uracil, and thymine (Table 8.1). In the average spectra (Figure 8.1), these bands only appear as weak shoulders due to their low abundance in the whole dataset.

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Figure 8.3. The relative band occurrences in the dataset of 613 SERS spectra from the isolated cytoplasm (red trace) and in the dataset of 883 SERS spectra from five live J774 cells (black trace).

The abundance of several bands attributed to components of the amide II vibration decreases in the endolysosomal spectra compared to the spectra of the isolated cytoplasm, e.g., at 1560 cm^-1, 1520 cm^-1, 1492 cm^-1, and amide III bands, e.g., at 1239 cm^-1 (Figure 8.3). At the same time, bands of nonpolar group vibrations are more abundant in the endolysosomal spectra than in the spectra of the isolated cytoplasm, such as at 1352 cm^-1 attributed to vibrations of tryptophan, 1153 cm^-1, 1130 cm^-1 attributed to C-C and C-N stretching, and 830 cm^-1 attributed to vibrations of tyrosine (Table 8.1 and Figure 8.3). Similar to the average spectra and the PCA results (Figures 8.1 and 8.2), the C-S stretching vibration at 650 cm^-1 and the S-S stretching vibration at 502 cm^-1 are more common in the endolysosomal spectra (Figure 8.3 black curve). The amide bands derive

103 from vibrations of the polypeptide backbone [105], and their loss potentially indicates the decreased number of peptide bonds, denoting the cleavage of the protein chains that interact with the gold nanoparticle. Based on these results, it can be deduced that the spectral differences were the results of protein denaturation rather than the nanoparticle-membrane interactions.

The data presented above suggest the interactions of the gold nanoparticles with protein fragments resulting from enzymatic cleavage. To support the presence of these interactions, the enzymatic digestion of bovine serum albumin (BSA) by bovine trypsin (which process is further referred to as trypsinization) was studied to model the arising differences between the interaction of gold nanoparticles with intact BSA molecules and with their digested fragments, respectively.

8.2. Comparison of the SERS datasets of BSA, trypsin, and trypsinized BSA solutions

The SERS spectra of the pure proteins were collected from gold nanoparticle agglomerates aggregated by BSA or trypsin, respectively (see Sections 4.5.1 and 4.8 for experimental details). After the BSA spectra were collected, 5 µL of the pure trypsin solution was added to the sample carefully, while observing the steadiness of the agglomerate used for the spectrum acquisition of BSA molecules previously. Acquiring SERS spectra from the same sample position before and after the addition of trypsin allowed for the collection of highly comparable data, which represented the changes that occur in the agglomerate upon digestion.

8.2.1. Average SERS spectra of BSA, trypsin, and trypsinized BSA

The average of the SERS spectra of BSA, bovine trypsin, and the trypsinized BSA are shown in Figure 8.4. The three average spectra, each calculated from ~100 representative SERS spectra, showed significant differences. It is important to see that the strongest band in the average spectrum of trypsin at 1145 cm^-1, corresponding to C-N and C-C stretching vibrations, only has a minor contribution to the average spectrum of trypsinized BSA (see Table 8.2 for the tentative band assignments). The strongest bands in the pure BSA average spectrum, e.g., at 681 cm^-1, corresponding to the imidazole ring deformation in

104

histidine [219], have a much lower relative intensity in the average spectrum of trypsinized BSA. Several bands appear in the SERS data of trypsinized BSA that are not present in the average spectra of BSA and trypsin.

Figure 8.4. The averages of ~100 respective SERS spectra collected from BSA, trypsin, and from trypsinized BSA solutions in the presence of gold nanoparticles. The spectra are stacked for clarity. Excitation wavelength: 785 nm, acquisition time: 1 s, excitation intensity: 5.7×10⁵ W/cm².

105 Table 8.2. Raman shifts and their tentative assignments in Figure 8.4. ν stretching, δ deformation, symm symmetric, rock rocking, wag wagging, tor torsion, br breathing, bend bending, R benzene ring, r pyrrole ring. Band assignments were based on refs. [40, 104, 171, 172, 215, 219, 253].

Raman shift

(cm^-1) Tentative assignment Raman shift

(cm^-1) Tentative assignment

1735 δ(COOH) of Asp, Glu 1060 ν(C-C), Pro 490 cm^-1, mostly corresponding to nonpolar group vibrations and C-S and S-S stretching vibrations (see Table 8.2 for tentative assignments) are more intense in the average spectrum of the trypsinized BSA than in the average spectra of the pure proteins. These

106

results allow for the conclusion that the BSA molecules adsorbed on the surface of gold nanoparticles did not exchange to trypsin molecules during the time of the SERS experiments (~5 min). The data show that due to the addition of trypsin, major changes have occurred in the interaction of gold nanoparticles with the BSA molecules, which can be attributed to the enzymatic digestion of the BSA molecules. This was supported by the observed changes on the micrometer-scale: after ~5 min, much smaller aggregates appeared on the surface of the CaF2 slide, which can be interpreted as the successful digestion of BSA resulting in the agglomerates falling apart.

8.2.2. PCA analysis of the SERS spectra of BSA, trypsin, and trypsinized BSA To further understand the differences in the three datasets, PCA analysis was performed.

The scores plot and the loadings of PC1 and PC2 are shown in Figure 8.5. As the scores plot shows in Figure 8.5, the trypsin dataset is the most homogeneous, which can derive from the fact that over half of the trypsin molecule is natively folded into random coils and turns [179, 259], which has previously been shown to facilitate specific residue-nanoparticle interactions rather than non-specific adsorption [172]. The most heterogeneous dataset was the one collected from the sample with trypsinized BSA, which is possibly the result of the many possible interactions the protein fragments can have with the gold nanoparticles. It is important to observe that the intact BSA and trypsin data partially overlap, while the SERS data of the trypsinized BSA strongly separate along PC1 and PC2, the two principal components explaining the highest variance. Since the mixture originally contained the same proteins as the pure solutions of BSA and trypsin, the separation of the trypsinized BSA data from the other two datasets must be based on the new interactions resulting from the enzymatic cleavage of BSA.

The loadings of PC1 (Figure 8.5 middle) reveal that the highest variance in the three datasets can be found in the bands at 1376 cm^-1 assigned to vibrations of tryptophan and CH3 deformation, 1021 cm^-1 assigned to vibrations of phenylalanine and C-C stretching, 976 cm^-1 assigned to C-C stretching, 643 cm^-1 assigned to C-S stretching and the C-C twisting of tyrosine and phenylalanine, 596 cm^-1 assigned to benzene ring deformation, and 552 cm^-1 and 490 cm^-1 assigned to S-S stretching vibrations (Table 8.2). These bands are more abundant in the data collected from the solution of trypsinized BSA. As

107 discussed before, the exposure of nonpolar residues and the disulfide bridges to a polar environment suggests protein denaturation [47, 260].

Figure 8.5. Scores plot (top) and the loadings of PC1 (middle) and PC2 (bottom) of the PCA analysis of the SERS datasets collected from BSA, trypsin, and trypsinized BSA solutions in the presence of gold nanoparticles. Each sample was represented by ~100 SERS spectra.

The loadings of PC2 (Figure 8.5 bottom) reveal further basis of the separation of the trypsinized BSA SERS data from the SERS data of pure protein solutions. The separation of the datasets along PC2 was based on the variances at the wavenumbers 1618 cm^-1

108

assigned to an amide I vibration and to the C=C stretching in tyrosine, tryptophan, and phenylalanine; 1467 cm^-1 and 1389 cm^-1 respectively assigned to CH2 and CH3

deformations; 1064 cm^-1 assigned to vibrations of proline and C-C stretching; 987 cm^-1 assigned to C-C stretching; 694 cm^-1 assigned to C-S stretching; 660 cm^-1 assigned to a C-S stretching vibration and to the C-C twisting of tyrosine and phenylalanine; 601 cm^-1 assigned to COO^– and benzene ring deformation; and 487 cm^-1 assigned to vibrations of S-S stretching, benzene ring torsion and bending (Table 8.2). These bands all appear more prominently in the SERS data of trypsinized BSA than in those of the pure protein solutions.

The bands at 1536 cm^-1 assigned to an amide II vibration, 1486 cm^-1 assigned to vibrations of NH3+ and amide II, and 1244 cm^-1 assigned to an amide III vibration and CH2 wagging were more characteristic to the datasets of the pure BSA and trypsin solutions (Table 8.2). It can be observed that the separation of SERS spectra collected in the trypsinized BSA solution was not only based on the higher abundance of nonpolar group vibrations and the C-S and S-S stretching vibrations, which suggest the denaturation of proteins in a polar environment, but also on the loss in amide II and amide III bands, similarly to the separation of the SERS spectra of the isolated cytoplasm and the endolysosomal compartments of live cells. Contrary to the biochemical environment in the endolysosomal compartments, no other processes occur in the solution of trypsinized BSA that could cause these spectral signatures. Therefore, the loss in amide bands must be the result of the fragmentation of the polypeptide backbone of proteins in cells.

8.2.3. Band occurrences in the spectra of BSA, trypsin, and trypsinated BSA Some of the bands based on which the datasets separated do not appear in the average spectra, because even though the data were background corrected and vectornormalized, some very intense and frequently occurring bands can overpower other, less frequent or less intense bands in the average spectra. The analysis of the band occurrences can help to gain further understanding of the data.

The occurrence of spectral bands in the SERS data of BSA, trypsin, and trypsinized BSA, respectively, are shown in Figure 8.6. The 1170 cm^-1 and 1150 cm^-1 bands, the 955 cm^-1

109 and 920 cm^-1 bands, and the 865 cm^-1 and 840 cm^-1 bands in the relative band occurrence curve of the pure BSA spectra, which show low abundance in Figure 8.6, appear as single bands in the average SERS spectrum of pure BSA at 1161 cm^-1, 935 cm^-1, and 846 cm^-1, respectively (see tentative assignments in Table 8.2).

Figure 8.6. Relative band occurrence in the SERS data sets collected from the solutions of BSA, trypsin, and trypsinized BSA, respectively. Each analyzed dataset consisted of

~100 individual spectra.

The data suggest the absence of the significant exchange of BSA molecules on the surface of gold nanoparticles by trypsin, as observed in the average spectra of the three datasets

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as well. This is supported, e.g., by the highly abundant 1145 cm^-1 band in the trypsin spectra, which is nearly completely absent in the spectra collected from the trypsinized BSA solution.

Nonpolar group vibrations are more abundant in the spectra of trypsinized BSA compared to the spectra of pure BSA, as seen in the bands at 1618 cm-1 assigned to the C=C stretching vibrations of tyrosine, tryptophan, and phenylalanine, and an amide I vibration;

1560 cm^-1 assigned to vibrations of tryptophan, tyrosine, amide II, and COO^– deformation; 1380 cm^-1 assigned to CH3 bending; 1145 cm^-1 assigned to C-C and C-N stretching vibrations; 1004 cm^-1 assigned to a vibration of phenylalanine; 985 cm^-1 assigned to C-C stretching; and 600 cm^-1 assigned to COO^– and benzene ring deformations (tentative assignments were based on Table 8.2). This suggests the denaturation of BSA in the presence of trypsin [47].

This is further supported by the appearance of a new C-S stretching vibration at 655 cm^-1 and the higher abundance of the S-S stretching vibration at 490 cm^-1, given the often nonpolar environment of disulfide bridges inside the protein structures [260]. The abundance of contributions by components of the amide II, e.g., at 1535 cm^-1, and amide III bands, e.g., at 1320 cm^-1 and 1300 cm^-1, is reduced in the spectra of the trypsinized

This is further supported by the appearance of a new C-S stretching vibration at 655 cm^-1 and the higher abundance of the S-S stretching vibration at 490 cm^-1, given the often nonpolar environment of disulfide bridges inside the protein structures [260]. The abundance of contributions by components of the amide II, e.g., at 1535 cm^-1, and amide III bands, e.g., at 1320 cm^-1 and 1300 cm^-1, is reduced in the spectra of the trypsinized