2.1.

Nanoparticles

The Ag NPs were synthetized by the group of Matthias Epple, as described by Mahl et al.³² The particles were coated with PVP (Ag NPs size: $70\pm 20\mathrm{nm}$ silver core in diameter) and soaked in the liquid suspension (ultrapure water). They had a zeta potential of $-25\mathrm{mV}$.³³ The Ag NPs’ concentration in suspension was $1.2\mathrm{mg}/\mathrm{mL}$.

2.2.

Skin Sample Preparation

Fresh porcine ears were obtained on the day of slaughter from a local butcher. Three porcine ears were selected, and three samples of $2\times 2\mathrm{cm}$ were excised from each of them. Before the measurement started, the ears were cleaned with cold flowing water and dried with paper towels. The bristles were carefully removed so as not to affect the integrity of the SC.

After the Ag NPs–containing solution was applied on the surface of porcine ears ($40\mu \mathrm{L}$ for $4{\mathrm{cm}}^2$ area), the skin samples were stored in a wet chamber in an incubator for 24 h at a temperature of 37°C, 5% ${\mathrm{CO}}_2$, and 100% humidity for passive penetration.

2.3.

Two-Photon Tomography with Fluorescence Lifetime Imaging Technique

The two-photon tomograph (DermaInspect, Jenlab GmbH, Jena, Germany) equipped with a tunable femtosecond titanium sapphire laser (Mai Tai XF, Spectra Physics, USA, 710 to 920 nm) was used for ex vivo measurements of porcine ear skin.^34,35 The laser was operated at 760 nm and generated 100-fs pulses at a repetition rate of 80 MHz. Employing the time-correlated single photon counting technology, one channel FLIM module with a temporal resolution of 250 ps was attached to the DermaInspect tomograph. The femtosecond laser is focused on the sample within a femtoliter volume by a high-NA oil-immersed objective lens (magnification $40\times$; NA: 1.3). In the DermaInspect, the optics of the signal-receiving channel consists of one each short bandpass filter 409 nm and long bandpass filter 680 nm (HC 680/SP). The maximal scanning range is $350\times 350\mu \mathrm{m}$ in the $X-Y$ direction and $200\mu \mathrm{m}$ in the $Z$ direction. The lateral and axial resolutions of the DermaInspect system in the skin are 0.4 to $0.6\mu \mathrm{m}$ and 1.2 to $2.0\mu \mathrm{m}$, respectively.³⁶ The FLIM and AF intensity signals can be registered through the same objective lens by different photomultiplier tubes. For intensity images, the resolution is typically $512\times 512\text{pixels}$, and for FLIM images, the typical resolution is $128\times 128\text{pixels}$. The immersion oil, the measurement window, and the skin all have the same refractive index of around 1.45; therefore, all depths measured using this method are real geometrical depths.

Fluorescence lifetime analysis was performed with software (SPCImage 4.2) incorporated into the DermaInspect system. The fluorescence lifetime is the average time from absorbance to emission of a photon. Since the number of photons is linearly proportional to the fluorescence intensity, the fluorescence lifetime can be determined from the fluorescence intensity decay as the $1/e$ of the maximum value. The fluorescence decay is often multiexponential. A biexponential fit function was utilized as shown in Eq. (1)

After pulsed excitation, the lifetime of AF will decay exponentially as described in Eq. (1). The width of the instrumental response function has to be taken into account, and in our case, it is small compared to the width of the time channel ($250\mathrm{ps}/\text{channel}$) in the fluorescence lifetime histogram. The measured fluorescence intensity, $I(t)$, follows the description presented in Eq. (3)

Skin samples were fixed onto the cover glass. The excitation wavelength was set at 760 nm. To image the Ag NPs–treated skin with SC or even stratum spinosum (SS), the FLIM measured depth reached $18\mu \mathrm{m}$, based on the Ag NPs’ signal detection. The acquisition time was equal to 9.6 s for one image. The depth increments were set at $2\mu \mathrm{m}$ for the FLIM stack measurements. For each of the three ear samples, three different positions were measured.

2.4.

Confocal Raman Microscopy

CRM measurements were performed using a model 3510 Skin Composition Analyzer (River Diagnostics, Rotterdam, The Netherlands), which is commonly implemented for in vivo/ex vivo skin investigations. A near-infrared laser (785 nm, 26 mW on the skin surface) was used to analyze the skin samples in the fingerprint region (400 to $2000{\mathrm{cm}}^{-1}$). The 785 nm excitation wavelength has been widely used for Raman measurements in the field of dermatology due to the reduced absorption and scattering by the skin and, as a result, the high penetration ability.^37,38 The Raman fingerprint spectra were recorded from the skin surface down to a depth of $40\mu \mathrm{m}$ at increments of $2\mu \mathrm{m}$. The immersion oil, the measurement window, and the skin all have the same refractive index around 1.45; therefore, all depths measured using CRM are real geometrical depths. The acquisition time for one Raman spectrum was 5 s. In this case, the detailed Raman profiles were acquired within the epidermis. According to the CRM spectrum of the Ag NPs–treated skin, the penetration profile can be determined by the nonrestricted multiple least square fit (NMF) method using Skin Tools 2.0 software provided by River Diagnostics.^39,40 For each porcine ear skin sample, 10 different points were measured in order to obtain the penetration profile. In total, three different porcine skin ear samples were measured. The utilized Raman microscope was described in detail elsewhere.^37,41–43

2.5.

Surface-Enhanced Raman Scattering Microscopy

As Ag NPs are able to generate an SERS signal under the excitation of 785 nm,²² the CRM described above was used for analyzing the Ag NPs’ penetration into the skin by SERS microscopy. The same measurement procedure was applied, i.e., a measurement depth of $40\mu \mathrm{m}$ at increments of $2\mu \mathrm{m}$, 5 s acquisition time for one spectrum, and excitation wavelength of 785 nm at 26 mW on the skin surface. Again, 10 different points were measured on each porcine skin ear sample. In total, three different porcine skin ear samples were measured. The penetration profile could be easily calculated as the shape of the SERS spectrum differs strongly from that of the intact skin Raman spectrum, and its intensity is much higher.

3.1.

TPT-FLIM Experiment

To understand the spectroscopic properties of the PVP-coated Ag NPs, the AF intensity was investigated, depending on the excitation wavelength in the range between 710 and 920 nm. The maximal fluorescence efficiency of Ag NPs was found in the range between 730 and 780 nm. Thus, the wavelength of 760 nm was chosen as the excitation wavelength of the TPT-FLIM system in our experiments. Separately, the average lifetime (${\tau }_m$) distributions of PVP-coated Ag NPs, SC, and SS of porcine skin were acquired by the TPT-FLIM system. They are shown in Fig. 1(a). From Fig. 1(a), one can see that the peak value of the Ag NPs’ fluorescence lifetime distribution is at $\sim 75\mathrm{ps}$, and SC’s and SS’s mean lifetimes based on the maximum values of the lifetime distribution lie in the range of 1300 and 1700 ps, respectively. Therefore, the Ag NPs and skin layers can be clearly distinguished according to their own lifetime distributions. In this case, we continued our experiments on the Ag NPs–treated porcine skin after 24 h. Starting from the SC, two prominent peaks are visible in the lifetime distribution curve, which are shown in Fig. 1(b). One peak is at $\sim 150\mathrm{ps}$ and the other at $\sim 1275\mathrm{ps}$. The first peak is caused by Ag NPs, but its lifetime value is higher than that of independent Ag NPs’ AF, which may have been induced by the interaction between the NPs and skin components. Thus, we can track the peaks of Ag NPs’ FLIM on the skin layers to determine the NPs’ penetration depth. The lifetime peak of the initial testing position (at the depth of $0\mu \mathrm{m}$) appears at ${\tau }_m=159\mathrm{ps}$. In this way, the penetration depth can be confirmed in the position where the Ag NPs’ FLIM signal disappears with respect to the ${\tau }_m=159\mathrm{ps}$ peak position. According to our experimental analysis, the Ag NPs’ signal at ${\tau }_m=159\mathrm{ps}$ disappears at a depth of 12 to $14\mu \mathrm{m}$ on average. The NP penetration depth could, therefore, be determined to be 12 to $14\mu \mathrm{m}$, thus not exceeding the SC thickness. Figure 2 represents the Ag NPs’ penetration profile inside the porcine skin. The Ag NPs’ signal intensity decreases strongly from 0 to $4\mu \mathrm{m}$, and below $4\mu \mathrm{m}$, it continues to decline, having completely disappeared at 12 to $14\mu \mathrm{m}$ in depth. Below $14\mu \mathrm{m}$ in depth, the Ag NPs’ signal approaches zero, showing that no Ag NPs exist in these depths.

Fig. 1

The average lifetime (${\tau }_m$) distributions of silver nanoparticles (Ag NPs), stratum corneum (SC), and stratum spinosum (SS) (a) and Ag NPs–treated skin at a depth of 0 to $2\mu \mathrm{m}$ (b).

Fig. 2

The penetration profile of Ag NPs into porcine skin obtained using fluorescence lifetime imaging microscopy technique.

Furthermore, according to the literature,^13,44 normal skin has the amplitude coefficient, $a_1$, values of AF between 50 and 90%, whereas values of $a_1>90\%$ denote the NP signals. Accordingly, the $a_1$ distribution curves could be obtained for Ag NPs, SC, and SS, which are shown in Fig. 3. The Ag NPs’ $a_1$ values are distributed in the range between 85 and 100%. Both SC’s and SS’s $a_1$ values vary between 40 and 85%, which is basically identical to the values in the reference literature.

Fig. 3

The $a_1$ distribution maps of Ag NPs, SC, and SS.

Using SPCImage 4.2 software, $a_1$ values were coded by a discrete color scale ranging from blue ($\le 40\%$), green ($>40\%$, $\le 85\%$) to red ($>85\%$), which are shown in Fig. 4. Figure 4(a) is the amplitude $a_1$ distribution map on the skin surface, i.e., at the depth of $0\mu \mathrm{m}$. The green areas represent the SC FLIM information and the red is the Ag NPs. In Fig. 4(a), the Ag NPs are densely distributed on the surface of the skin. Figure 4(b) shows the amplitude $a_1$ distribution maps at a depth of $18\mu \mathrm{m}$. It can be seen that the Ag NPs’ signal has completely disappeared.

Fig. 4

$a_1$ value distributions coded by a discrete color scale ranging from blue ($\le 40\%$), green ($>40\%$, $\le 85\%$) to red ($>85\%$) on the skin surface measured at a depth of $0\mu \mathrm{m}$ (a) and at the depth of $18\mu \mathrm{m}$ (b).

3.2.

CRM Experiment

Figure 5 shows the Raman spectra of the PVP-coated Ag NPs and the PVP coating material. As can be seen, the PVP is a Raman active substance that does not contain any fluorescence signal. The PVP-coated Ag NPs, characterized by the strong fluorescence background, originated from the Ag NPs itself and not from the PVP coating. Prominent Raman peaks at 956, 1400, and $1638{\mathrm{cm}}^{-1}$ are visible on the fluorescence background, which are shifted in comparison to the PVP’s Raman spectrum. The Raman peak at $956{\mathrm{cm}}^{-1}$ is the shifted peak of the PVP’s breathing ring mode at $935{\mathrm{cm}}^{-1}$.⁴⁵ The Raman peak at $1400{\mathrm{cm}}^{-1}$ can be interpreted as the vibration of the carbonate,^46,47 which may have been formed by the chemical interaction of Ag NPs’ and PVP’s $\mathrm{C}═\mathrm{O}$ atomic group. The peak at $1638{\mathrm{cm}}^{-1}$ is the Raman signal that comes from PVP’s carbonyl bond stretching mode.^45,48 Normally, this peak is observed at $1666{\mathrm{cm}}^{-1}$ for PVP’s solid state, but its position is shifted in the solution, which can be explained by the partial donating of lone electrons of PVP to the vacant orbitals of the Ag atom.^49,50

Fig. 5

Raman spectra of the poly-N-vinylpyrrolidone (PVP)-coated Ag NPs (solid line) and PVP coating material (dotted line).

Figure 6 shows the Raman spectra of intact and PVP-coated Ag NPs’ pretreated porcine ear skin samples. The significant difference between these spectra is observed in the range of $950\pm 40{\mathrm{cm}}^{-1}$, which corresponds to the Raman peak at $956{\mathrm{cm}}^{-1}$ of the PVP-coated Ag NPs. Other peaks at 1400 and $1638{\mathrm{cm}}^{-1}$ could not be evaluated in the skin due to their complete superposition with the skin Raman peaks.

Fig. 6

Raman spectra of the PVP-coated Ag NPs–treated skin and intact skin at a depth of $4\mu \mathrm{m}$ and the difference between them.

The NMF method is applied for analyzing the penetration depth of PVP-coated Ag NPs into the skin. The NMF method includes the fitting of the skin Raman spectra by the known model spectra of SC’s compounds, such as cholesterol, ceramide, keratin, urea, water, etc. Thus, the depth-dependent coefficients of each individual compound are determined, minimizing the residual fitting error. For the skin pretreated with PVP-coated Ag NPs, the same skin coefficients and an additional coefficient of PVP-coated Ag NPs could be determined. This depth-dependent coefficient serves as the PVP-coated Ag NPs’ relative concentration in the skin. The maximum of the PVP-coated Ag NPs’ concentration is observed near the skin surface. Then their concentration is exponentially decreased, disappearing completely at a depth of $11.1\pm 2.1\mu \mathrm{m}$. The penetration profile of PVP-coated Ag NPs is shown in Fig. 7. Thus, the results obtained using CRM show that the majority of PVP-coated Ag NPs remains on the skin surface and in the superficial layers, and does not penetrate into the viable layers of the epidermis.

Fig. 7

The penetration profile of Ag NPs into porcine skin obtained using confocal Raman microscopy.

3.3.

SERS Experiment

Figure 8 shows the exemplary SERS spectra of a PVP-coated Ag NPs’ pretreated porcine ear skin sample measured at the three different depths. The SERS spectra look completely different compared to the intact skin Raman spectrum, showing an almost five times higher intensity and another spectrum shape. As can be seen from Fig. 8, some small SERS peaks exist and disappear depending on the measured depth, but for all the skin depths, the three strong SERS peaks are always observed, e.g., $650{\mathrm{cm}}^{-1}$, which corresponds to C-S stretching mode of the cystine component of keratin,^25,51,52 $1003{\mathrm{cm}}^{-1}$, which corresponds to the stretching mode of the C-C backbone of the phenylalanine and urea,^53,54 and $1600{\mathrm{cm}}^{-1}$, which corresponds to the $\mathrm{C}═\mathrm{O}$ stretching mode of proteins.⁵⁵ As SERS peaks at 1003 and $1600{\mathrm{cm}}^{-1}$ are superimposed on the intact skin Raman peaks, the peak position at $650{\mathrm{cm}}^{-1}$ was used for determining the penetration profiles of Ag NPs into the skin. By detecting the SERS signal intensity at $650{\mathrm{cm}}^{-1}$, the penetration depth of Ag NPs is determined to be $15.6\pm 8.3\mu \mathrm{m}$. The high standard deviation is explained by the fluctuation of SERS intensities and by the lack of SERS signal reproducibility. The generation of reproducible SERS spectra from the Ag NPs–treated skin will be a promising task for the future. The existence of a SERS signal in the skin shows that PVP coating material was dissolved in the SC and that Ag NPs were released. The optimal release conditions were not investigated in the present study and serve as a topic for future work.

Fig. 8

Surface-enhanced Raman scattering spectra of the PVP-coated Ag NPs–treated skin at different depths and Raman spectrum of intact skin at a depth of $4\mu \mathrm{m}$.

The intensity of SERS spectra at different depths could change significantly, showing a blinking effect that was mentioned in the literature.²² In fact, the SERS generation can be affected by many factors, such as NPs release efficiency, the aggregation of NPs, the shape, size, and periphery of NPs,^29,56,57 and the coating influence. The depth-dependent intensity fluctuation could be caused by the discrete aggregation of Ag NPs in the skin tissue, by the differences in combining organic molecules to Ag NPs, and by the non-optimal release of Ag NPs from the PVP coating. Therefore, the penetration profile based on SERS analyses is not presented in this paper, but the maximal penetration depth was successfully determined. The appearance frequency of the SERS signal in the skin was determined at $\sim 14\%$ of the total measurement points. The obtained SERS spectra are similar to the SERS spectra of human tissue presented in the literature.^22,25