In this paper, we present a very simple and rapid method for the creation of a new class of SERS substrates able to generate highly reproducible SERS spectra for molecules of biomedical interest, possessing a very low Raman cross section (e.g., pharmaceutical compounds, cell lysates). The here-proposed plasmonic substrates have the capacity to operate even in the case of using a NIR excitation laser, which represents a sine qua non condition for SERS measurements performed on bio analytes. The plasmonic substrates consist of silver nanostars, synthesized using an original method developed in MedFuture's Research Center laboratory. The only chemical precursors employed in this wet-chemical procedure are silver nitrate and trisodium citrate, with no addition of any other reagents. The microwave-assisted synthesis of the nanostars has been performed in a sealed bottom flask, which turned out to be a critical parameter in the synthesis process. The chemical reaction took place inside of a microwave oven capable of generating an almost uniform temperature in the whole volume of the liquid in a very short period. By the end of the synthesis process, the colloidal solution was immersed in a water bath at 35 ◦C. The SERS activity of the here-proposed AgNS has been evaluated on test molecules (Methylene Blue) as well as on pharmaceutical compounds with low Raman cross section (doxorubicin, atenolol & metoprolol), cell lysates and amino acids (cysteine & methionine), by pouring a very small volume of colloidal solution (~1.5 µL) onto a MgF2 glass plate heated at 50 ◦C, prior to the deposition of the analytes. The quality of the vibrational spectra collected using the here-proposed plasmonic substrates represents strong experimental evidence of their versatility, especially in the case of bioanalytes.

#### **2. Results and Discussion**

In this study we proposed an original procedure for the microwave-assisted synthesis of silver nanostars by involving trisodium citrate as a reducing and stabilizing agent of Ag+ ions. The role of microwave heating is to create a more homogenous thermal environment for the synthesis procedure and to speed up the process as shown in literature [9]. Once the synthesis procedure was completed, the nanostars' morphology was analyzed by microscopic complementary techniques (TEM/STEM, AFM). Their plasmonic properties have been tested on several biological analytes by means of SERS using a NIR excitation wavelength (785 nm). The SERS experiments were carried out on interconnected AgNS that can be regarded as solid plasmonic substrates, as well as on individual nanostars that can be easily localized on the MgF2 port-probe using an optical microscope.

#### *2.1. Characterization of AgNS Colloids by UV-Vis Spectroscopy*

UV-Vis absorption spectroscopy is the first experiment that can be employed for the characterization of colloidal solutions containing nanoparticles of different types and shapes. The UV-Vis absorption spectrum of the colloidal solution containing silver nano stars is presented in Figure 1 together with the optical images of the colloids, right after synthesis (inset a) and after purification (inset b) through centrifugation, respectively.

The spectrum is characterized by the occurrence of a strong absorption peak located around 420 nm, specific for silver nanoparticles. The broadness of this peak is quite high with respect to isotropic silver nanoparticles [10–12], and this is a direct consequence of the presence of anisotropic silver nanostars in the solution. The full-width half-maximum (FWHM) value is ~200 nm in this case, which is more than double as compared to silver spherical nanoparticles. However, this property allows the use of NIR excitation lasers

(785/830 nm) for SERS analysis of biological samples, strongly decreasing their intrinsic fluorescence and facilitating the recording of highly qualitative SERS spectra of pharmaceutical compounds, amino acids or even cell lysates. *Int. J. Mol. Sci.* **2022**, *23*, 8830 3 of 15

*Int. J. Mol. Sci.* **2022**, *23*, 8830 3 of 15

**Figure 1.** UV-Vis absorption spectra of the colloidal solution containing AgNS. The inset shows an optical image of the colloidal solutions before (**a**) and after purification through centrifugation (**b**) respectively. **Figure 1.** UV-Vis absorption spectra of the colloidal solution containing AgNS. The inset shows an optical image of the colloidal solutions before (**a**) and after purification through centrifugation (**b**) respectively. ceutical compounds, amino acids or even cell lysates. *2.2. Transmission Electron Microscopy (TEM) and Scanning Transmission Electron Microscopy*

#### The spectrum is characterized by the occurrence of a strong absorption peak located *2.2. Transmission Electron Microscopy (TEM) and Scanning Transmission Electron Microscopy (STEM) Characterization (STEM) Characterization*  TEM together with STEM has been employed for a proper characterization of AgNS.

around 420 nm, specific for silver nanoparticles. The broadness of this peak is quite high with respect to isotropic silver nanoparticles [10–12], and this is a direct consequence of TEM together with STEM has been employed for a proper characterization of AgNS. A typical TEM image of an individual nanostar is presented in Figure 2a, together with a zoom image of its central part (Figure 2b). A typical TEM image of an individual nanostar is presented in Figure 2a, together with a zoom image of its central part (Figure 2b).

**Figure 2.** TEM image of a typical individual silver nanostar (**a**). TEM image of the central zone of the nanostar (**b**). **Figure 2.** TEM image of a typical individual silver nanostar (**a**). TEM image of the central zone of the nanostar (**b**).

One can clearly distinguish that our AgNS consist of a central nanoparticle interconnected with several highly one-dimensional individual arms. The Energy Dispersive One can clearly distinguish that our AgNS consist of a central nanoparticle interconnected with several highly one-dimensional individual arms. The Energy Dispersive Analysis (EDS) confirmed that AgNS are composed of silver atoms (Figure S1). The STEM images of similar AgNS are presented in Figure 3.

**Figure 2.** TEM image of a typical individual silver nanostar (**a**). TEM image of the central zone of

One can clearly distinguish that our AgNS consist of a central nanoparticle interconnected with several highly one-dimensional individual arms. The Energy Dispersive

the nanostar (**b**).

**Figure 3.** STEM image of an individual silver nanostar (**a**). STEM image of the central zone of the nanostar (**b**). **Figure 3.** STEM image of an individual silver nanostar (**a**). STEM image of the central zone of thenanostar (**b**). **Figure 3.** STEM image of an individual silver nanostar (**a**). STEM image of the central zone of the nanostar (**b**).

Analysis (EDS) confirmed that AgNS are composed of silver atoms (Figure S1). The STEM

Analysis (EDS) confirmed that AgNS are composed of silver atoms (Figure S1). The STEM

The STEM images revealed the same structure as the one obtained from TEM images. The AgNS appear as branched 3D structures with high electron-scattering capacities. The structure points towards a {1 1 1} oriented crystalline structure [13] with a single center seed particle being the origin of the branching arms. Differences in the contrast of the outer section of the nanostars (Figure 3b) are also indicative of a {1 0 0} preferential growth of the crystals as presented in [14] and, thus, could be interpreted as having a 2D structure. These results are complementary to the TEM morphological analysis of the nanostars presented in Figure 2. The STEM images revealed the same structure as the one obtained from TEM images. The AgNS appear as branched 3D structures with high electron-scattering capacities. The structure points towards a {1 1 1} oriented crystalline structure [13] with a single center seed particle being the origin of the branching arms. Differences in the contrast of the outer section of the nanostars (Figure 3b) are also indicative of a {1 0 0} preferential growth of the crystals as presented in [14] and, thus, could be interpreted as having a 2D structure. These results are complementary to the TEM morphological analysis of the nanostars presented in Figure 2. The STEM images revealed the same structure as the one obtained from TEM images. The AgNS appear as branched 3D structures with high electron-scattering capacities. The structure points towards a {1 1 1} oriented crystalline structure [13] with a single center seed particle being the origin of the branching arms. Differences in the contrast of the outer section of the nanostars (Figure 3b) are also indicative of a {1 0 0} preferential growth of the crystals as presented in [14] and, thus, could be interpreted as having a 2D structure. These results are complementary to the TEM morphological analysis of the nanostars presented in Figure 2.

#### *2.3. Atomic Force Microscopy (AFM) Characterization 2.3. Atomic Force Microscopy (AFM) Characterization 2.3. Atomic Force Microscopy (AFM) Characterization*

images of similar AgNS are presented in Figure 3.

images of similar AgNS are presented in Figure 3.

*Int. J. Mol. Sci.* **2022**, *23*, 8830 4 of 15

The evolution of the AgNS deposited onto the heated MgFଶ glass slides was evaluated by means of AFM measurements (Figure 4). All the measurements were performed on semi-contact operating mode. The 2D and 3D topographic images (height images) were recorded together with phase contrast ones (data not shown). The phase contrast images gave us a clear indication regarding the substrate's composition which is a very homogenous one (very small phase variations have been detected on the samples). On the other hand, the topographic images confirmed the presence on the substrate of interconnected AgNS that coexist with individual ones (Figure 4a). Their sizes vary between a few hundred nanometers and a few microns. The cross-sectional analysis of the individual nanoflowers revealed they have a maximum height of 600 nm, which is much smaller (~one order of magnitude) than their lateral dimensions. The evolution of the AgNS deposited onto the heated MgF2 glass slides was evaluated by means of AFM measurements (Figure 4). All the measurements were performed on semicontact operating mode. The 2D and 3D topographic images (height images) were recorded together with phase contrast ones (data not shown). The phase contrast images gave us a clear indication regarding the substrate's composition which is a very homogenous one (very small phase variations have been detected on the samples). On the other hand, the topographic images confirmed the presence on the substrate of interconnected AgNS that coexist with individual ones (Figure 4a). Their sizes vary between a few hundred nanometers and a few microns. The cross-sectional analysis of the individual nanoflowers revealed they have a maximum height of 600 nm, which is much smaller (~one order of magnitude) than their lateral dimensions. The evolution of the AgNS deposited onto the heated MgFଶ glass slides was evaluated by means of AFM measurements (Figure 4). All the measurements were performed on semi-contact operating mode. The 2D and 3D topographic images (height images) were recorded together with phase contrast ones (data not shown). The phase contrast images gave us a clear indication regarding the substrate's composition which is a very homogenous one (very small phase variations have been detected on the samples). On the other hand, the topographic images confirmed the presence on the substrate of interconnected AgNS that coexist with individual ones (Figure 4a). Their sizes vary between a few hundred nanometers and a few microns. The cross-sectional analysis of the individual nanoflowers revealed they have a maximum height of 600 nm, which is much smaller (~one order of magnitude) than their lateral dimensions.

**Figure 4.** AFM topological image of several interconnected nanostars self-organized on the MgF2 surface (**a**). Corresponding cross-sectional analysis of AgNS (**b**). The scale bar represents 2 µm. **Figure 4.** AFM topological image of several interconnected nanostars self-organized on the MgF2 surface (**a**). Corresponding cross-sectional analysis of AgNS (**b**). The scale bar represents 2 µm. **Figure 4.** AFM topological image of several interconnected nanostars self-organized on the MgF2 surface (**a**). Corresponding cross-sectional analysis of AgNS (**b**). The scale bar represents 2 µm.

As a result of the slow heating process, the space between the individual one-directional arms (identified in the TEM images) has been completely filled, as can be noticed in the high-resolution topographic image presented in Figure 5. The formation of the 2D network has been also observed.

network has been also observed.

network has been also observed.

**Figure 5.** High resolution AFM topographical image of silver nanostars. The scale bar represents 2 µm. **Figure 5.** High resolution AFM topographical image of silver nanostars. The scale bar represents 2 µm. All SERS measurements included in this study were performed on plasmonic substrates obtained by pouring colloidal solutions containing silver nanostars on Raman

As a result of the slow heating process, the space between the individual one-directional arms (identified in the TEM images) has been completely filled, as can be noticed in the high-resolution topographic image presented in Figure 5. The formation of the 2D

As a result of the slow heating process, the space between the individual one-directional arms (identified in the TEM images) has been completely filled, as can be noticed in the high-resolution topographic image presented in Figure 5. The formation of the 2D

*Int. J. Mol. Sci.* **2022**, *23*, 8830 5 of 15

#### *2.4. SERS and Raman Analysis*  All SERS measurements included in this study were performed on plasmonic sub-*2.4. SERS and Raman Analysis*

strates obtained by pouring colloidal solutions containing silver nanostars on Raman transparent MgFଶ glass slides heated at 50 °C. The as-obtained plasmonic substrates allowed the recording of very reproducible SERS spectra on different regions of the substrate for all the bioanalytes here presented. All Raman measurements were performed on MgFଶ transparent glass using a 785 nm All SERS measurements included in this study were performed on plasmonic substrates obtained by pouring colloidal solutions containing silver nanostars on Raman transparent MgF2 glass slides heated at 50 ◦C. The as-obtained plasmonic substrates allowed the recording of very reproducible SERS spectra on different regions of the substrate for all the bioanalytes here presented. transparent MgFଶ glass slides heated at 50 °C. The as-obtained plasmonic substrates allowed the recording of very reproducible SERS spectra on different regions of the substrate for all the bioanalytes here presented. All Raman measurements were performed on MgFଶ transparent glass using a 785 nm laser excitation.

laser excitation. The SERS spectrum of the pristine silver substrate were recorded under the same All Raman measurements were performed on MgF2 transparent glass using a 785 nm laser excitation. The SERS spectrum of the pristine silver substrate were recorded under the same

experimental conditions and can be observed in Figure 6. The intensity of the vibrational bands is very low as can be seen in the figure, emphasizing, the capacity of the here-proposed plasmonic substrates to be employed for SERS analysis of various bioanalytes. The SERS spectrum of the pristine silver substrate were recorded under the same experimental conditions and can be observed in Figure 6. The intensity of the vibrational bands is very low as can be seen in the figure, emphasizing, the capacity of the hereproposed plasmonic substrates to be employed for SERS analysis of various bioanalytes. experimental conditions and can be observed in Figure 6. The intensity of the vibrational bands is very low as can be seen in the figure, emphasizing, the capacity of the here-proposed plasmonic substrates to be employed for SERS analysis of various bioanalytes.

**Figure 6.** Raman spectra of AgNS recorded using a 785 nm laser excitation.

The enhancement capabilities of the plasmonic substrate were first tested on rhodamine 6G (R6G). This analyte was used in a first instance for testing the reproducibility of the recorded spectra. Finally, the Raman and SERS spectra of R6G were employed for the calculation of the substrate's enhancement factor (EF). In the case of SERS, 1 µL solution of 1 mM R6G was deposited on the plasmonic substrate and left to dry. In the case of Raman, 1 µL solution of 1 mM R6G was poured on a MgF2 glass slide and left to dry. The SERS spectrum of R6G is presented in Figure S2b. A value of ~8 × 103 was calculated for the substrate's enhancement factor (EF). This value was obtained by comparing Raman (Figure S2a) and SER spectra of R6G (Figure S2b). the recorded spectra. Finally, the Raman and SERS spectra of R6G were employed for the calculation of the substrate's enhancement factor (EF). In the case of SERS, 1µL solution of 1 mM R6G was deposited on the plasmonic substrate and left to dry. In the case of Raman, 1µL solution of 1 mM R6G was poured on a MgFଶ glass slide and left to dry. The SERS spectrum of R6G is presented in Figure S2b. A value of ~8 × 103 was calculated for the substrate's enhancement factor (EF). This value was obtained by comparing Raman (Figure S2a) and SER spectra of R6G (Figure S2b). For a proper assessment of the substrates' plasmonic properties, we performed SERS measurements using as analytes a standard test molecule (methylene blue-MB) and three

The enhancement capabilities of the plasmonic substrate were first tested on rhodamine 6G (R6G). This analyte was used in a first instance for testing the reproducibility of

For a proper assessment of the substrates' plasmonic properties, we performed SERS measurements using as analytes a standard test molecule (methylene blue-MB) and three pharmaceutical compounds having different Raman cross sections: doxorubicin (DOX), atenolol (ATE), and metoprolol (MET). The capacity of our AgNS to perform as very efficient plasmonic substrates was further tested on cell lysates (DLD1 cells) and two amino acids (methionine and cysteine) The spectra were recorded using NIR excitation laser (785 nm). pharmaceutical compounds having different Raman cross sections: doxorubicin (DOX), atenolol (ATE), and metoprolol (MET). The capacity of our AgNS to perform as very efficient plasmonic substrates was further tested on cell lysates (DLD1 cells) and two amino acids (methionine and cysteine) The spectra were recorded using NIR excitation laser (785 nm). In the case of MB and DOX, previous studies reported comprehensive analysis of their SERS spectra collected on different plasmonic substrates together with a complete

In the case of MB and DOX, previous studies reported comprehensive analysis of their SERS spectra collected on different plasmonic substrates together with a complete vibrational band assignment. Given their great Raman cross section, they can be considered "standard" analytes for the evaluation of the plasmonic properties for new substrates [10,12,15]. The MB Raman and SERS spectra, collected on our substrates, are presented in Figure 7. vibrational band assignment. Given their great Raman cross section, they can be considered "standard" analytes for the evaluation of the plasmonic properties for new substrates [10,12,15]. The MB Raman and SERS spectra, collected on our substrates, are presented in Figure 7.

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**Figure 6.** Raman spectra of AgNS recorded using a 785 nm laser excitation.

**Figure 7.** SERS spectrum of dried MB solution (1 µM) using a 785 nm excitation laser (**a**). Raman spectrum of dried MB solution (1 µM) using a 785 nm excitation laser (**b**). **Figure 7.** SERS spectrum of dried MB solution (1 µM) using a 785 nm excitation laser (**a**). Raman spectrum of dried MB solution (1 µM) using a 785 nm excitation laser (**b**).

The major vibrational peaks of MB, as described in the literature, are clearly visible even for an analyte concentration of µM. The main vibrational bands of MB are located at 451, 504, 677, 773, 1178 and 1395 cm−1. The most visible ones can be assigned to C-N-C skeletal deformations (447 and 500 cm−1) and asymmetric C-N stretching (1395 cm−1). The assignment of the most important bands is presented in Table S1. The spectrum is similar with the one recorded using other types of plasmonic substrates, suggesting a similar geometry of interaction between the substrate and the analyte. The major vibrational peaks of MB, as described in the literature, are clearly visible even for an analyte concentration of µM. The main vibrational bands of MB are located at 451, 504, 677, 773, 1178 and 1395 cm−1 . The most visible ones can be assigned to C-N-C skeletal deformations (447 and 500 cm−1 ) and asymmetric C-N stretching (1395 cm−1 ). The assignment of the most important bands is presented in Table S1. The spectrum is similar with the one recorded using other types of plasmonic substrates, suggesting a similar geometry of interaction between the substrate and the analyte.

The first pharmaceutical compound that was employed for testing the plasmonic substrate was doxorubicin. The typical vibrational frequencies of DOX are presented in Figure 8. The SERS spectrum of DOX included in this figure were collected using the The first pharmaceutical compound that was employed for testing the plasmonic substrate was doxorubicin. The typical vibrational frequencies of DOX are presented in Figure 8. The SERS spectrum of DOX included in this figure were collected using the individual nanostar highlighted in the inset of Figure 8. This spectrum is very similar to those reported in the scientific literature [16] recorded using other plasmonic substrates. The majority of DOX vibrational bands originate from the conjugated aromatic chromophore of

*Int. J. Mol. Sci.* **2022**, *23*, 8830 7 of 15

marized in Table S1.

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the drug molecule. A tentative assignment of the most important bands is summarized in Table S1. phore of the drug molecule. A tentative assignment of the most important bands is summarized in Table S1.

individual nanostar highlighted in the inset of Figure 8. This spectrum is very similar to those reported in the scientific literature [16] recorded using other plasmonic substrates. The majority of DOX vibrational bands originate from the conjugated aromatic chromo-

individual nanostar highlighted in the inset of Figure 8. This spectrum is very similar to those reported in the scientific literature [16] recorded using other plasmonic substrates. The majority of DOX vibrational bands originate from the conjugated aromatic chromophore of the drug molecule. A tentative assignment of the most important bands is sum-

**Figure 8.** SERS spectrum of dried DOX solution (1 mM) collected on an isolated nanostar using a 785 nm excitation laser (**a**). The inset shows an optical image of the individual nanostar that has been used as plasmonic substrate. Raman spectrum of dried DOX solution (1 mM) collected using a 785 nm excitation laser (**b**). **Figure 8.** SERS spectrum of dried DOX solution (1 mM) collected on an isolated nanostar using a 785 nm excitation laser (**a**). The inset shows an optical image of the individual nanostar that has been used as plasmonic substrate. Raman spectrum of dried DOX solution (1 mM) collected using a 785 nm excitation laser (**b**). been used as plasmonic substrate. Raman spectrum of dried DOX solution (1 mM) collected using a 785 nm excitation laser (**b**). In order to prove the capacity of the here-reported AgNS to be used as plasmonic

substrates for biomolecules with low Raman cross section, we have performed SERS

In order to prove the capacity of the here-reported AgNS to be used as plasmonic substrates for biomolecules with low Raman cross section, we have performed SERS measurements on Metoprolol (Figure 9) and Atenolol (Figure 10). The spectra were recorded on an individual AgNS (MET) and on a solid substrate containing interconnected AgNS (ATE), respectively. The Raman spectra of MET and ATE are also presented (Fig-In order to prove the capacity of the here-reported AgNS to be used as plasmonic substrates for biomolecules with low Raman cross section, we have performed SERS measurements on Metoprolol (Figure 9) and Atenolol (Figure 10). The spectra were recorded on an individual AgNS (MET) and on a solid substrate containing interconnected AgNS (ATE), respectively. The Raman spectra of MET and ATE are also presented (Figures 9b and 10b, respectively). measurements on Metoprolol (Figure 9) and Atenolol (Figure 10). The spectra were recorded on an individual AgNS (MET) and on a solid substrate containing interconnected AgNS (ATE), respectively. The Raman spectra of MET and ATE are also presented (Figures 9b and 10b, respectively).

**Figure 9.** SERS spectrum of MET 1mM (**a**) collected on individual AgNS using a 785 nm laser. The **Figure 9.** SERS spectrum of MET 1mM (**a**) collected on individual AgNS using a 785 nm laser. The inset in figure (**a**) shows an optical image of the individual nanostar that has been used for recording the spectra. Raman spectrum of MET 1 mM (**b**) collected using a 785 nm laser. **Figure 9.** SERS spectrum of MET 1mM (**a**) collected on individual AgNS using a 785 nm laser. The inset in figure (**a**) shows an optical image of the individual nanostar that has been used for recording the spectra. Raman spectrum of MET 1 mM (**b**) collected using a 785 nm laser.

inset in figure (**a**) shows an optical image of the individual nanostar that has been used for record-

ing the spectra. Raman spectrum of MET 1 mM (**b**) collected using a 785 nm laser.

**Figure 10.** SERS spectrum of ATE 1 mM (**a**) collected on individual AgNS using a 785 nm laser. Raman spectrum of ATE 1 mM (**b**) collected using a 785 nm laser. **Figure 10.** SERS spectrum of ATE 1 mM (**a**) collected on individual AgNS using a 785 nm laser. Raman spectrum of ATE 1 mM (**b**) collected using a 785 nm laser.

In the scientific literature, there are very few studies reporting a SERS analysis of these two pharmaceutical compounds belonging to the class of beta-blockers [11]. These two drug molecules have a very similar chemical structure. The central part of both molecules consists of a phenyl ring linked to an amine alkanol side chain containing the asym-In the scientific literature, there are very few studies reporting a SERS analysis of these two pharmaceutical compounds belonging to the class of beta-blockers [11]. These two drug molecules have a very similar chemical structure. The central part of both molecules consists of a phenyl ring linked to an amine alkanol side chain containing the asymmetric carbon atom.

metric carbon atom. As expected, both spectra included in Figures 9 and 10 are very similar, being dominated by the bands assigned to the vibrational breathing mode of the central phenyl ring located at 852 cm−1 (MET) and 859 cm−1 (ATE), respectively. Other significant vibrational bands with their corresponding attributions are listed in Table S1. According to Moskovits' surface selection rules, this is a direct proof of a similar geometry of interaction between these two molecules and the plasmonic substrates [17]. On the other hand, the quality, reproducibility, and ease of recording of the SERS spectra can be attributed to the unique As expected, both spectra included in Figures 9 and 10 are very similar, being dominated by the bands assigned to the vibrational breathing mode of the central phenyl ring located at 852 cm−1 (MET) and 859 cm−1 (ATE), respectively. Other significant vibrational bands with their corresponding attributions are listed in Table S1. According to Moskovits' surface selection rules, this is a direct proof of a similar geometry of interaction between these two molecules and the plasmonic substrates [17]. On the other hand, the quality, reproducibility, and ease of recording of the SERS spectra can be attributed to the unique plasmonic properties of AgNS.

plasmonic properties of AgNS. Very recently it has been shown that Raman/SERS spectroscopy has the potential to provide very useful information related to the nanoscale molecular interactions between Very recently it has been shown that Raman/SERS spectroscopy has the potential to provide very useful information related to the nanoscale molecular interactions between analytes and plasmonic substrate [5,18].

analytes and plasmonic substrate [5,18]. As such, cell lysates were used for testing the plasmonic properties of our AgNS. A typical SERS spectrum of DLD1 cell lysate is presented in Figure 11a, while the Raman As such, cell lysates were used for testing the plasmonic properties of our AgNS. A typical SERS spectrum of DLD1 cell lysate is presented in Figure 11a, while the Raman spectrum is presented in Figure 11b.

spectrum is presented in Figure 11b. The spectrum from Figure 11 is dominated by a very intense vibrational peak at 725 cm−1 . This peak can be assigned to the presence of adenine and adenosine as it has been recently shown by Genova et al. [19]. The presence of adenosine triphosphate in relatively high concentration could be an explanation for the strong intensity of this vibrational band. In the spectral fingerprint region (600–900 cm−1 ) one can detect the presence of another two distinct peaks at 627 cm−1 and 656 cm−1 . The latest peak can be assigned to guanine ring breathing vibrational mode. This behavior represents a strong proof of the fact that, in the case of untreated cells, one can detect the presence of the same biomolecular species responsible for the occurrence of specific vibrational bands. According to Barhoumi et al. [20], the rest of the prominent vibrational bands observed in the lysate spectrum can be assigned to vibration modes of PO2 (1094 cm−1 ) and adenine (1328 & 1586 cm−1 ), respectively.

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**Figure 11.** SERS spectra of DLD1 cell lysate obtained using a 785 nm excitation laser (**a**). Raman spectra of DLD1 cell lysate obtained using a 785 nm excitation laser (**b**). **Figure 11.** SERS spectra of DLD1 cell lysate obtained using a 785 nm excitation laser (**a**). Raman spectra of DLD1 cell lysate obtained using a 785 nm excitation laser (**b**). In the spectral fingerprint region (600–900 cm−1) one can detect the presence of another two distinct peaks at 627 cm−1 and 656 cm−1. The latest peak can be assigned to guanine ring breathing vibrational mode. This behavior represents a strong proof of the fact that,

The spectrum from Figure 11 is dominated by a very intense vibrational peak at 725 cm−1. This peak can be assigned to the presence of adenine and adenosine as it has been recently shown by Genova et al. [19]. The presence of adenosine triphosphate in relatively high concentration could be an explanation for the strong intensity of this vibrational band. The last class of bio analytes that were used for testing the Raman signal enhancing capacities of our plasmonic AgNS were the amino acids. We have chosen Cysteine (Cys) and Methionine (Met) because their sidechain contains one sulfur atom that has the capacity to interact directly with the silver surface [21]. SERS and Raman spectra of Cys and Met are presented in Figures 12 and 13, respectively. in the case of untreated cells, one can detect the presence of the same biomolecular species responsible for the occurrence of specific vibrational bands. According to Barhoumi et al. [20], the rest of the prominent vibrational bands observed in the lysate spectrum can be assigned to vibration modes of PO2 (1094 cm−1) and adenine (1328 & 1586 cm−1), respectively.

In the spectral fingerprint region (600–900 cm−1) one can detect the presence of another two distinct peaks at 627 cm−1 and 656 cm−1. The latest peak can be assigned to guanine ring breathing vibrational mode. This behavior represents a strong proof of the fact that, in the case of untreated cells, one can detect the presence of the same biomolecular species responsible for the occurrence of specific vibrational bands. According to Barhoumi et al. [20], the rest of the prominent vibrational bands observed in the lysate spectrum can be The strong affinity of Sulphur atoms for silver surface is confirmed by the presence of two very intense vibrational bands (665 cm−1 for Cys & 682 cm−1 for Met) that have been assigned to C-S stretching vibrations of the two amino acid, in SERS spectra of Cys (Figure 12a) and Met (Figure 13a). Nevertheless, the presence of a strong vibrational peak around 1050 cm−1 (1054 cm−1 for Cys/1049 cm−1 in the case of Met) indicates a possible interaction of amino acids with the silver substrate through the amino group. The last class of bio analytes that were used for testing the Raman signal enhancing capacities of our plasmonic AgNS were the amino acids. We have chosen Cysteine (Cys) and Methionine (Met) because their sidechain contains one sulfur atom that has the capacity to interact directly with the silver surface [21]. SERS and Raman spectra of Cys and Met are presented in Figures 12 and 13, respectively.

**Figure 12.** SERS (**a**) and Raman (**b**) spectra of Cysteine (1 mM) recorded using a 785 nm excitation laser.

**Figure 13.** SERS (**a**) and Raman (**b**) spectra of Methionine (1 mM) (**a**) recorded using a 785 nm excitation laser. **Figure 13.** SERS (**a**) and Raman (**b**) spectra of Methionine (1 mM) (**a**) recorded using a 785 nm excitation laser.

**Figure 12.** SERS (**a**) and Raman (**b**) spectra of Cysteine (1 mM) recorded using a 785 nm excitation

The strong affinity of Sulphur atoms for silver surface is confirmed by the presence of two very intense vibrational bands (665 cm−1 for Cys & 682 cm−1 for Met) that have been Table S1 represents a tentative assignment of the main vibrational bands for all the samples that were analyzed in this study.

#### assigned to C-S stretching vibrations of the two amino acid, in SERS spectra of Cys (Figure 12a) and Met (Figure 13a). Nevertheless, the presence of a strong vibrational peak around **3. Materials and Methods**

#### 1050 cm−1 (1054 cm−1 for Cys/1049 cm−1 in the case of Met) indicates a possible interaction *3.1. Materials*

laser.

of amino acids with the silver substrate through the amino group. Table S1 represents a tentative assignment of the main vibrational bands for all the samples that were analyzed in this study. **3. Materials and Methods**  *3.1. Materials*  All chemical compounds employed in this paper were of analytical grade. Silver nitrate (AgNOଷ) and trisodium citrate were purchased from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. The aqueous solutions were prepared in Milli-Q water (Milli-Q® Direct Water Purification System, Darmstadt, Germany) . MgFଶ polished glasses (Crystran Ltd., Poole, UK), having a diameter of 20 mm, were used All chemical compounds employed in this paper were of analytical grade. Silver nitrate (AgNO3 ) and trisodium citrate were purchased from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. The aqueous solutions were prepared in Milli-Q water (Milli-Q®Direct Water Purification System, Darmstadt, Germany). MgF2 polished glasses (Crystran Ltd., Poole, UK), having a diameter of 20 mm, were used as port probes for the creation of the solid SERS substrates. The analytes and the pharmaceutical compounds used for SERS measurements (methylene blue MB, doxorubicin DOX, atenolol ATE, metoprolol MET, cysteine Cys and methionine Met) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture products: Roswell Park Memorial Institute (RPMI 1640) cell culture medium, Fetal Bovine Serum (FBS), Glutamine, Penicillin/Streptomycin and Phosphate Buffered Saline solution (PBS 1X) were purchased from Gibco (Grand Island, NY, USA).

#### as port probes for the creation of the solid SERS substrates. The analytes and the pharma-*3.2. Cell Culture and Lysates*

ceutical compounds used for SERS measurements (methylene blue MB, doxorubicin DOX, atenolol ATE, metoprolol MET, cysteine Cys and methionine Met) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell culture products: Roswell Park Memorial Institute (RPMI 1640) cell culture medium, Fetal Bovine Serum (FBS), Glutamine, Penicillin/Streptomycin and Phosphate Buffered Saline solution (PBS 1X) were purchased from DLD1 (ATCC® CCL-221™) colorectal carcinoma cells were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in RPMI 1640 cell culture medium, supplemented with 10% FBS, 1% Glutamine and 1% Penicillin/Streptomycin. During the experiment, the cells were stored in a humidified incubator at 37 ◦C and 5% CO2.

Gibco (Grand Island, NY, USA). *3.2. Cell Culture and Lysates*  DLD1 (ATCC® CCL-221™) colorectal carcinoma cells were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in RPMI 1640 cell culture medium, supplemented with 10% FBS, 1% Glutamine and 1% Penicillin/Strep-For obtaining the cell lysates, the cells were detached from the culture flasks and washed three times with fresh complete medium, followed by three washing steps with PBS 1X. Then the cell pellet was washed three times with UltraPure DNAse/RNAse free water. The washing steps were performed by centrifugation of the cells at 600× *g*, for 5 min at room temperature. After the washing procedures, the cell pellet was resuspended in 333 µL of UltraPure DNAse/RNAse free water and stored at −80 ◦C until further use.

tomycin. During the experiment, the cells were stored in a humidified incubator at 37 °C and 5% CO2. The lysate processing involved mechanical lysis steps. The samples were removed from −80 ◦C and placed in the heating block at 37 ◦C for 15 min. The samples were sonicated with the following settings for the EpiShear 5/640 (2 mm) Probe Sonicator (Active Motif, Carlsbad, CA, USA): 18 s with pulse of 3 s and 80% amplitude (from a maximum intensity level of 200 µm). After the sonication process, the samples were stored at −80 ◦C for 15 min, and the samples were sonicated and frozen two more times.

The cell lysate, processed as mentioned above, was stored at −80 ◦C and thawed only when the sample was analyzed.

#### *3.3. Fabrication of Silver NanoStars (AgNS)*

Colloidal suspensions of silver nanostars (AgNS) were prepared using an original method developed in MedFuture's laboratories. The chemical reduction of Ag+ ions by trisodium citrate molecules were performed in a sealed-bottom flask introduced in the center of a microwave oven having a nominal power of 2100 W, operating at 2450 MHz capable of inducing water boiling (100 ◦C) after 30 s of irradiation. After 60 s of heating, the vials were removed from the oven, immersed in a water bath at 35 ◦C and let to accommodate at this temperature for 5 min, with no stirring. In a typical synthesis procedure, 10 mg of AgNO3 were dissolved in 50 mL of Milli-Q water and introduced in an empty 100 mL bottom flask. Trisodium citrate solution (1%, 1 mL) was added to the flask. The two solutions were gently stirred for 20 s, and then the bottom flask was sealed and introduced in the microwave oven. No sign of chemical reaction between the silver ions and the citrate molecules was observed at this stage. Afterwards, the solution was exposed to microwaves for 1 min. It was noted that, after 30 s of microwave exposure, the aqueous solution started to boil in the whole volume. After 15 more seconds, the solution suddenly changed its color from colorless to milky grey, indicating the successful formation of silver nanostars. By the end of the process, the colloidal solution was cooled down at room temperature using a two-step process. First the vials were immersed in a water bath at 35 ◦C for 5 min, and then they were brought to room temperature and left for another 5 min in air. The whole synthesis procedure took no more than 15 min.

#### *3.4. Preparation of SERS Substrates*

The SERS substrates were prepared by pouring 1.5 µL of as-synthesized AgNS solution onto a MgF2 glass slide heated at 50 ◦C. After 2 min from the deposition, the glass slides were removed from the heated plate and the substrates were let to accommodate at room temperature for 15 min. Once this final procedure was completed, the SERS substrates containing self-aggregated as well as individual nanostars were ready for use.

#### *3.5. Preparation of Samples for SERS and Raman Investigation*

Stock aqueous solutions of standard Raman-test molecules (methylene blue MB) as well as of therapeutic agents (doxorubicin DOX, atenolol ATE, metoprolol MET) and amino acids (cysteine Cys and methionine Met) were prepared. The concentration of all stock solution was 1 mM. Further dilutions were prepared using Milli-Q water. Once the drying process of the solid SERS substrates was completed, a very small volume of analytes aqueous solution (1 µL) was poured in the center of the solid substrate. The solution was left to dry for 30 min at room temperature. By the end of this process the samples were ready for SERS measurements, using a NIR excitation laser (785 nm).

The Raman spectra were recorded on dry solutions poured over Raman transparent MgF2 glass, using the same 785 nm laser excitation.

#### *3.6. Characterization of Silver Nanostars and Solid Plasmonic Substrates*

The colloidal solutions containing silver nanostars and the solid plasmonic sub-strates they formed were characterized by UV-VIS, STEM, TEM, AFM and SERS.

UV-Vis absorption measurements of the silver colloid were performed using a T92+ Spectrophotometer (PG Instruments, Lutterworth, UK) in the 190–900 nm wavelength range with a 2 nm resolution.

Electron microscopy measurements were performed on a Hitachi HT7700 Transmission Electron Microscope (Hitachi, Tokyo, Japan) operating at 120 kV, in the highresolution mode.

The topography of the substrates was investigated by an Atomic Force Microscope (NT-MDT NTEGRA, NT-MDT Spectrum Instruments, Zelenograd, Russia) coupled with a micro-Raman system. The topographic images presented in this paper were acquired in the semi-contact operating mode.

The SERS spectra were recorded using a multilaser confocal Renishaw InVia Reflex Raman spectrometer. The wavelength calibration was performed on an internal reference sample (silicon). The 785 and 830 nm laser lines were employed as the excitation sources, but preliminary tests have been performed also on 532 and 633 nm laser lines. The emit-ting laser power was measured for each laser and for each objective on top of the sample. The SERS spectra presented in this paper were recorded using a 50× objective and different acquisition times ranging from few seconds to 50 s. The spectral resolution of the spectrometer was 0.5 cm−1 . Baseline correction was applied to all SERS spectra, in order to eliminate the background. Each spectrum represents the average of a minimum of 30 spectral acquisitions. The baseline correction was performed by using the Wire 4.2 software provided by Renishaw (Gloucestershire, UK), with the inVia spectrometer. For all the spectra included in this study, the intensity of the vibrational bands is represented in kconts/(mW × s) units.

The calculations of EF have been performed according to the procedure developed by Gupta and Weimar [22], using the following equation:

$$\text{EF} = \frac{\mathbf{M}\_{\text{Raman}} \times \mathbf{S}\_{\text{Surf}} \times \mathbf{I}\_{\text{Surf}}}{\mathbf{M}\_{\text{Surf}} \times \mathbf{S}\_{\text{Raman}} \times \mathbf{I}\_{\text{Raman}}}$$

where MSurf and MRaman are the numbers of molecules, SSurf and SRaman are the geometrical areas of the molecular films and ISurf and IRaman are the SERS/Raman intensities of the most intense vibrational band that was used for the calculation of EF (1510 cm−1 ). Both measurements were performed using a 50× objective and an excitation laser of 785 nm. In the case of Raman measurements, a 100% laser power was used, the acquisition time was 10 s and the number of spectral acquisitions was 4. In the case of SERS measurement, the only modification that was made was the laser power which was set to 0.1%. The laser intensity, measured on sample surface, was 113 mW (100 % laser power) and 0.22 mW (0.1% laser power,), respectively. The intensities of all vibrational bands included in this study were plotted in kcounts/(mW × s) units. In the case of Raman/SERS measurements employed for EF calculation, 1 mM aqueous solutions of R6G were used. The diameter of the circular spot was ~2 mm in both cases. The Raman/SERS values of 1510 cm−1 band intensities were 43.3 and 0.0054 kcounts/(mW × s), respectively.

#### **4. Conclusions**

In this research, we report a very simple, rapid and reproducible microwave-aassisted method for the fabrication of anisotropic silver nanostars (AgNS) that can be successfully used as highly efficient SERS substrates for a broad range of bioanalytes: pharmaceutical compounds, cell lysates and/or amino acids. The AgNS consist of a central nanoparticle interconnected with several highly one-dimensional individual arms having a star shape. The microwave-assisted synthesis of the nanostars was performed in a sealed- bottom flask in a very short time interval (less than 3 min). The only chemical precursors employed in this wet-chemical procedure were silver nitrate and trisodium citrate, with no addition of any other reagents. Based on their unique properties, our plasmonic substrates have the capacity to generate very intense SERS spectra even in the case of using a NIR excitation laser, which represents a sine qua non condition for SERS measurements on biological samples.

The high quality and reproducibility of the vibrational spectra collected using the hereproposed plasmonic substrates represents strong experimental evidence of their versatility and application potential, especially in the case of bioanalytes.

**Supplementary Materials:** The supporting information can be downloaded at: https://www.mdpi. com/article/10.3390/ijms23158830/s1. References [11,16,22–44] are cited in the supplementary materials.

**Author Contributions:** Conceptualization, R.N.R., R.I.S, . and G.F.S, .; methodology, V.T., A.B.T, ., A.M., E.F.-F. and R.I.S, .; software, V.T., E.F.-F. and R.T.; validation, E.B., G.F.S, . and R.I.S, .; formal analysis, V.T.; investigation, A.B.T, . and E.F.-F.; resources, R.I.S, .; data curation, V.T., G.F.S, . and A.O.; writing original draft preparation, G.F.S, ., A.B.T, ., A.M., A.O., V.T. and R.I.S, .; writing—review and editing, G.F.S, . and R.I.S, .; visualization, A.M. and V.T.; supervision, R.N.R., E.B., R.T. and R.I.S, .; project administration, R.N.R.; funding acquisition, R.I.S, . All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was part of grant no. PN-III-P4-ID-PCCF-2016-0112 funded by Executive Agency for Higher Education, Research, Development and Innovation Funding (UEFISCDI), Romanian Ministry of Education and Research.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was granted by project PDI-PFE-CDI 2021, entitled Increasing the Performance of Scientific Research, Supporting Excellence in Medical Research and Innovation, PROGRES, no. 40PFE/30.12.2021.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

## *Review* **Reporter Genes for Brain Imaging Using MRI, SPECT and PET**

**Tianxin Gao 1 , Pei Wang 1 , Teng Gong 2 , Ying Zhou 1 , Ancong Wang 1 , Xiaoying Tang 1,3, Xiaolei Song 2,\* and Yingwei Fan 3,\***

**Abstract:** The use of molecular imaging technologies for brain imaging can not only play an important supporting role in disease diagnosis and treatment but can also be used to deeply study brain functions. Recently, with the support of reporter gene technology, optical imaging has achieved a breakthrough in brain function studies at the molecular level. Reporter gene technology based on traditional clinical imaging modalities is also expanding. By benefiting from the deeper imaging depths and wider imaging ranges now possible, these methods have led to breakthroughs in preclinical and clinical research. This article focuses on the applications of magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) reporter gene technologies for use in brain imaging. The tracking of cell therapies and gene therapies is the most successful and widely used application of these techniques. Meanwhile, breakthroughs have been achieved in the research and development of reporter genes and their imaging probe pairs with respect to brain function research. This paper introduces the imaging principles and classifications of the reporter gene technologies of these imaging modalities, lists the relevant brain imaging applications, reviews their characteristics, and discusses the opportunities and challenges faced by clinical imaging modalities based on reporter gene technology. The conclusion is provided in the last section.

**Keywords:** reporter gene; MRI; radionuclide imaging; brain imaging

### **1. Introduction**

Molecular imaging is an imaging technique that visualizes, characterizes, and measures biological processes in vivo at the molecular and cellular levels [1]. Reporter gene imaging is a critical technical route for molecular imaging, which introduces or expresses imaging agents into cells through so-called reporter genes. Reporter genes are those genes that, when introduced into target cells (e.g., brain tissues, cancer, and circulating white cells), produce a protein receptor or enzyme that binds, transports, or traps a subsequently injected imaging probe, which becomes the contrast agent for reporter gene imaging [2]. Reporter gene imaging is developing very rapidly for monitoring cell therapy and gene therapy by providing critical information on the biodistributions, magnitudes, and durations of viral gene expressions. Imaging the brains of large animals or humans on a large scale has become the next challenge of reporter gene imaging.

Among multiple imaging modalities, fluorescence reporter genes have drawn great attention; however, penetration depths limit their in vivo application [3]. Recently, other imaging modalities, including magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), and ultrasound (US) imaging, have been explored in the field of reporter genes [2]. The advantages of genetically

**Citation:** Gao, T.; Wang, P.; Gong, T.; Zhou, Y.; Wang, A.; Tang, X.; Song, X.; Fan, Y. Reporter Genes for Brain Imaging Using MRI, SPECT and PET. *Int. J. Mol. Sci.* **2022**, *23*, 8443. https://doi.org/10.3390/ ijms23158443

Academic Editor: Raghvendra Singh Yadav

Received: 30 June 2022 Accepted: 25 July 2022 Published: 30 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

encoded fluorescent imaging are high spatiotemporal resolution, high sensitivity, and high molecular specificity, while some conventional imaging modalities that use modern reporter gene indicators are effective in clearly examining the brains of larger subjects in deep organs and on large scales. Compared with optical reporter gene imaging, other types of reporter gene imaging have a variety of properties, as shown in Table 1. For the imaging depths and scales, clinical imaging techniques provide better performance, while fluorescence reporters are excellent in terms of their temporal–spatial resolution, noninvasiveness, molecular and cell specificity, and sensitivity.

**Table 1.** Pros and cons of reporter gene brain imaging techniques.

Brain imaging includes imaging of diseases, such as neurodegenerative disease and glioma, and foundational brain research, which can be divided into two categories: macroscopic, noninvasive human cognitive neuroscience and invasive reductionist neurobiology [4]. Reporter gene base brain imaging includes both of these categories. Nonoptical imaging modalities are commonly used in clinics, which means that such studies involve direct human applications.

Observing brain activity is a dream of researchers. Gene-encoded fluorescent indicators that are used to dynamically monitor neurotransmitters and neuromodulators with fluorescence imaging have recently achieved great breakthroughs and have been reviewed in excellent studies [3]. We note that, for clinical imaging modalities, such as MRI, radionuclide imaging, and US, some breakthroughs have been achieved in reporter gene-based brain studies. A number of positive reviews have summarized reporter gene materials [5], the history of MRI in brain activity detection [4], transgene-based strategies in nuclear-based imaging [6], nanoparticle-mediated brain imaging [7], and reporter gene imaging in oncolytic virotherapy and gene therapy [2]. Here, we focus on the recently developed nonoptical techniques of reporter gene imaging used in brain imaging.

#### **2. Principle of Reporter Gene Imaging**

The two basic elements of reporter gene imaging consist of reporter genes and their respective imaging probes (which are also the referred to as imaging agents, substrates, or imaging reporters in other references). The accumulation of imaging probes directly depends on the protein products of reporter gene expression, thereby imaging the reporter

gene. Because it only monitors living cells, it can accurately provide important information such as that of survival, proliferation, migration, differentiation, and functional integration of transplanted cells in vivo [1,8,9].

#### *2.1. Reporter Gene Imaging with MRI*

MRI employs the resonance properties of atomic nuclei subjected to strong magnetic fields and radiofrequency pulses to generate signals and reconstruct images. MRI reporter genes can directly or indirectly produce magnetic resonance contrast signals that are based on the expressions of coding enzymes, receptors, metalloproteins, etc., which can specifically combine with MRI contrast agent [10–13]. MRI reporter genes can be used to longitudinally monitor the cell migration process and gene expressions by using noninvasive imaging [14]. The main types of existing MRI reporter genes include reporter genes that encode enzymes (e.g., tyrosinase), reporter genes that encode receptors on cells (e.g., transferrin receptor (TfR)), endogenous reporter genes (e.g., ferritin and aquaporin 1 (AQP1)), and reporter genes that express CEST-detectable proteins (e.g., lysine rich-protein (LRP)) [15].

As shown in Figure 1A,B, the most classic contrast agent for MRI reporter genes is iron. As a ubiquitous protein in the cells of various organisms, ferritin is assembled by two subunits, a heavy chain and a light chain. The proportions of light and heavy chains vary in different tissues. The ferritin heavy chain (FTH1) contains ferrous oxidase and can combine with iron oxide to transform unstable Fe2+ into stable, insoluble, and nontoxic Fe3+ forms. The light chain mainly increases the activity of FTH1 and stabilizes ferritin. Ferritin can specifically bind to iron, which results in accumulations of intracellular iron particles and decreases in T2 signals [16], and it is one of the most commonly used reporter genes in MRI (Figure 1A). The transferrin receptor (TfR) is another commonly used MRI reporter gene. TfR can bind to transferrin and transfer iron into cells via endocytosis, thus reducing the T2 relaxation time (Figure 1B) [14,17]. Lysine-rich protein (LRP) is an artificially designed gene. Due to the uniquely high chemical exchange rate of poly-Llysine, LRP can be used as a reporter gene in chemical exchange saturation transfer (CEST) MRI (Figure 1C) [18,19]. The CEST mechanism occurs because exchangeable protons have chemical shifts that are different from water. These protons are selectively saturated and exchanged with water molecules, thus reducing the water signal. Magnetic resonance diffusion-weighted imaging (DWI) is an imaging method that uses MRI to observe the microdiffusion movements of water molecules in living tissues. It can noninvasively image the structures and physiological functions of living brain tissues. The apparent diffusion coefficient (ADC) is used to describe the diffusion rate of water molecules in DWI. A positive correlation exists between them. When water molecules with freer diffusion are dephasing, the level of signal loss is greater, the signal is weaker, and it appears darker in DWI, and vice versa [20,21]. Aquaporins mediate the selective exchange of water-conducting molecules across plasma membranes in many cell types, and their expressions are related to water diffusivity and DWI signals in several disease states [22,23]. Previous studies have shown that overexpression of aquaporin can increase tissue water diffusivity without affecting viability, and contrasts are observed in diffusion-weighted MRI (Figure 1D) [24].

#### *2.2. Reporter Gene Imaging with Radionuclides*

Radionuclide imaging refers to SPECT and PET. SPECT detects gamma rays that are produced by the decay of the radioactive isotopes used in imaging, and it has been developed to elucidate the basic molecular neurodegeneration mechanism in PD, AD, and drug addiction, as well as to improve therapeutic strategies with minimum adverse effects. PET uses the annihilation of positrons (emitted by decaying radioisotopes in the imaging agent) and electrons to generate 510 keV collinear photons, which are detected simultaneously to generate a three-dimensional map of radioactivity distributions in the body [6]. Radionuclide imaging has very high sensitivity and good penetration ability in

tissues, and it can be used in clinical practice. It has been widely used for noninvasive tracing and monitoring of living cells.

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 4 of 20

**Figure 1.** The principle of MRI reporter genes commonly used in brain imaging. (**A**) The expression of ferritin will cause intracellular iron particles to aggregate, resulting in a decrease in the T2 signal in MRI images. (**B**) The expression of TfR increases the iron uptake of cells and shows low signal **Figure 1.** The principle of MRI reporter genes commonly used in brain imaging. (**A**) The expression of ferritin will cause intracellular iron particles to aggregate, resulting in a decrease in the T2 signal in MRI images. (**B**) The expression of TfR increases the iron uptake of cells and shows low signal intensity on T2 images of MRI. (**C**) LRP has a high chemical exchange rate and can be used in CEST MRI. (**D**) Aquaporin can increase water diffusivity and produce contrast in diffusion-weighted MRI.

intensity on T2 images of MRI. (**C**) LRP has a high chemical exchange rate and can be used in CEST

MRI. (**D**) Aquaporin can increase water diffusivity and produce contrast in diffusion-weighted MRI. *2.2. Reporter Gene Imaging with Radionuclides*  Radionuclide imaging refers to SPECT and PET. SPECT detects gamma rays that are produced by the decay of the radioactive isotopes used in imaging, and it has been developed to elucidate the basic molecular neurodegeneration mechanism in PD, AD, and drug addiction, as well as to improve therapeutic strategies with minimum adverse effects. PET PET imaging of reporter gene expression utilizes reporter gene imaging agents that are labeled by positron radionuclides. Currently, there are three types of commonly used radionuclide reporter gene imaging systems based on enzymes, receptors, and transporters, as shown in Figure 2 [6,25]. The most common reporter genes of enzymes are herpes simplex virus 1 thymine kinase (HSV1-tk) [9] and human ∆-mitochondrial thymine kinase type 2 (h∆tk2) [26]. The most common reporter genes are human somatostatin receptor type 2 (hSSTR2) [27] and dopamine D2 receptor (D2R) [28]. The most common reporter genes of transporters are human sodium–iodide symporter (hNIS) [29] and human norepinephrine transporter (hNET) [2,30].

uses the annihilation of positrons (emitted by decaying radioisotopes in the imaging agent) and electrons to generate 510 keV collinear photons, which are detected simultaneously to generate a three-dimensional map of radioactivity distributions in the body [6]. Radionuclide imaging has very high sensitivity and good penetration ability in tissues, One of the first and hence most intensively studied reporter genes, HSV1-tk, is also a suicide gene that adds an extra layer of control to ensure safety. NIS imaging is the most mature reporter gene imaging method used in human clinical trials and is more sensitive and longer lasting than HSV1-tk.

and it can be used in clinical practice. It has been widely used for noninvasive tracing and monitoring of living cells. PET imaging of reporter gene expression utilizes reporter gene imaging agents that are labeled by positron radionuclides. Currently, there are three types of commonly used radionuclide reporter gene imaging systems based on enzymes, receptors, and transporters, as shown in Figure 2 [6,25]. The most common reporter genes of enzymes are herpes The SPECT imaging system was developed to elucidate the basic molecular neurodegeneration mechanism in PD, AD, and drug addiction, as well as to improve therapeutic strategies with minimum amounts of adverse effects. PET imaging can provide diagnosis and treatment guidance for tumors and cardiovascular and brain diseases. PET imaging of reporter gene expressions is capable of monitoring gene and cell therapy [6].  The most common reporter genes are human somatostatin receptor

genes of transporters are human sodium–iodide symporter (hNIS) [29] and human nore-

One of the first and hence most intensively studied reporter genes, HSV1-tk, is also a suicide gene that adds an extra layer of control to ensure safety. NIS imaging is the most mature reporter gene imaging method used in human clinical trials and is more sensitive

pinephrine transporter (hNET) [2,30].

and longer lasting than HSV1-tk.

Endogenous reporter genes

Ferritin T2WI

**Figure 2.** The major types of reporter gene expression in radionuclide imaging, based on (**A**) en-**Figure 2.** The major types of reporter gene expression in radionuclide imaging, based on (**A**) enzymes, (**B**) receptors, and (**C**) transporters.

#### zymes, (**B**) receptors, and (**C**) transporters. **3. Reporter Gene Imaging in Brain Studies**

*3.1. Brain Imaging of Reporter Genes with MRI*

The SPECT imaging system was developed to elucidate the basic molecular neurodegeneration mechanism in PD, AD, and drug addiction, as well as to improve therapeutic strategies with minimum amounts of adverse effects. PET imaging can provide diagnosis and treatment guidance for tumors and cardiovascular and brain diseases. PET imaging of reporter gene expressions is capable of monitoring gene and cell therapy [6]. In brain studies, brain cancer and neurodegenerative disorders are the major diseases diag-In view of the diversity, high resolution, and noninvasive nature of MRI, MRI imaging of the brain can be used in a variety of applications, such as observing the process of virus infection through in vivo imaging, longitudinally monitoring cell migration and proliferation during cell therapy, noninvasive detection of neural connections, and monitoring neurogenesis. The majority of studies of MRI reporter genes in brain imaging are listed in Table 2.

nosed and monitored by reporter gene expression PET imaging, as discussed thoroughly **Table 2.** Reporter genes in brain imaging of MRI.

Receptor TfR–FTH T2WI Shows increased contrast on T2-weighted brain images. [31]

Ferritin–EGFP T2WI, FI Synaptically connected neural network can be detected by ex vivo

FTH–EGFP T2WI, FI Tracks the tropism and fate of MSCs after systematic transplantation

FTH1 DWI, SWI, T2WI Allows in vivo detection of BMSCs transplanted due to cerebral

MRI and fluorescence imaging. [32]

into orthotopic gliomas [34]

ischemia/reperfusion injury and to treat intervention. [35]

[33]

Realizes the accumulation of iron ions, resulting in a change in MR signal in the infected regions.

detect the neural circuits of living animals.

**Table 2.** *Cont.*

Visualization of neural networks helps provide a better understanding of the mechanisms of some brain functions and brain diseases. In the study of Wang et al. [32], vesicular stomatitis virus (VSV), a neurovirus that can spread sequentially in synaptic networks, was used to carry chimeric genes that encode ferritin and enhanced green fluorescent protein (EGFP). After recombinant VSV (rVSV) was injected into the somatosensory cortex (SC) of mice, the structural nerve connections were detected by MRI and fluorescence imaging. However, due to the high toxicity of VSV, mice infected with rVSV cannot survive for long periods, and in vivo MRI research is not allowed. The MRI and fluorescence images obtained after the death of mice are shown in Figure 3. In another study conducted by the team [33], hypotoxicity virus adeno-associated virus (AAV) was used as a vector to integrate the ferritin coding gene to obtain a ferritin coding viral vector (e.g., rAAV2-retro– CAG–Ferritin), which was injected into the caudate putamen (CPu) of mice to achieve noninvasive detection of neural networks in vivo. The CPu connection area was displayed by MRI at different time points after rAAV2-retro–CAG–ferritin injection (Figure 4). The team then focused on describing the activity of astrocytes, which are a major component of the central nervous system. They used the EGFP–AQP1 fusion gene of EGFP and aquaporin 1 (AQP1) as the reporter gene, detected astrocytes by fluorescence imaging and diffusion-weighted MRI, and established a new technique for the noninvasive detection of astrocytes in vivo for the first time [39]. In the newly published work [40] of the team, a tool virus rAAV-retro–AQP1–EGFP expressing nonmetallic magnetic resonance reporter gene AQP1 was prepared and used for in vivo brain-wide neural network detection. Three weeks after microinjection of virus rAAV-retro–AQP1–EGFP into the CPU brain area of mice, the changes in magnetic resonance signals in multiple brain regions (CPU, Ctx, BLA, Ins, Tha, HIP, etc) were observed by diffusion-weighted MRI, and the rapid imaging of specific brain region-related brain networks was successfully realized (increase from 60 days [33] to 21 days). The project also combined with the Cre–loxP system to prepare a brain network expressing Cre-dependent AQP1-related tool virus rAAV-retro–DIO–AQP1–EGFP for in vivo detection of specific neuronal types in specific

brain regions. This strategy provides a solid foundation for the visualization of neural networks in rodents and nonhuman primates. *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 7 of 20

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 7 of 20

**Figure 3.** Brain MRI (lower in both groups) and fluorescence images (upper). Four days after rVSV-EGFP or rVSV–Ferritin–EGFP was injected into the SC (red box) of mice, the mice were killed, and the images were obtained. Reproduced with permission from Ref. [32], 2019, Elsevier. **Figure 3.** Brain MRI (lower in both groups) and fluorescence images (upper). Four days after rVSV-EGFP or rVSV–Ferritin–EGFP was injected into the SC (red box) of mice, the mice were killed, and the images were obtained. Reproduced with permission from Ref. [32], 2019, Elsevier. **Figure 3.** Brain MRI (lower in both groups) and fluorescence images (upper). Four days after rVSV-EGFP or rVSV–Ferritin–EGFP was injected into the SC (red box) of mice, the mice were killed, and the images were obtained. Reproduced with permission from Ref. [32], 2019, Elsevier.

**Figure 4.** After different times (10 days, 30 days, or 60 days) of the rAAV2-retro–CAG–Ferritin injection, the CPU connection area is displayed by MRI in vivo (expressed by the change in T2 relaxation times). A color change (red–yellow) is used to indicate a change in T2 relaxation time (4–12 **Figure 4.** After different times (10 days, 30 days, or 60 days) of the rAAV2-retro–CAG–Ferritin injection, the CPU connection area is displayed by MRI in vivo (expressed by the change in T2 relaxation times). A color change (red–yellow) is used to indicate a change in T2 relaxation time (4–12 ms). Reproduced with permission from Ref. [33], 2021, Wiley. **Figure 4.** After different times (10 days, 30 days, or 60 days) of the rAAV2-retro–CAG–Ferritin injection, the CPU connection area is displayed by MRI in vivo (expressed by the change in T2 relaxation times). A color change (red–yellow) is used to indicate a change in T2 relaxation time (4–12 ms). Reproduced with permission from Ref. [33], 2021, Wiley.

ms). Reproduced with permission from Ref. [33], 2021, Wiley.

Mesenchymal stem cells (MSCs) can cross the blood–brain barrier and tend to accumulate in tumors [43]; hence, they can be developed as cell carriers to treat gliomas [44–46]. Longitudinal in vivo monitoring of the migration and fate of MSCs is very important for the development of MSCs as cell carriers. Cao et al. [34] used a lentivirus as a vector to carry the ferritin heavy chain (FTH) and EGFP genes and transferred it to MSCs. MCSs expressing reporter genes were injected into a rat glioma model using different injection methods (e.g., arterial injection, intravenous injection, and stereotactic injection), and the homing and migration behaviors of MSCs were detected by MRI. The results showed that arterial injections of MSCs had a clear ability to treat glioma. MRI based on the ferritin reporter gene can be used to trace the tendency of MSCs to accumulate in glioma in vivo. Mao et al. [37] constructed MSCs with high expressions of interferon-β (IFNβ) and FTH in a similar manner. MRI was used to evaluate whether MSCs can be used as cell carriers to carry IFNβ to treat brain tumors, which provides a new option for treating brain tumors. Studies have shown that FTH-based MRI can monitor this treatment process. Mesenchymal stem cells (MSCs) can cross the blood–brain barrier and tend to accumulate in tumors [43]; hence, they can be developed as cell carriers to treat gliomas [44– 46]. Longitudinal in vivo monitoring of the migration and fate of MSCs is very important for the development of MSCs as cell carriers. Cao et al. [34] used a lentivirus as a vector to carry the ferritin heavy chain (FTH) and EGFP genes and transferred it to MSCs. MCSs expressing reporter genes were injected into a rat glioma model using different injection methods (e.g., arterial injection, intravenous injection, and stereotactic injection), and the homing and migration behaviors of MSCs were detected by MRI. The results showed that arterial injections of MSCs had a clear ability to treat glioma. MRI based on the ferritin reporter gene can be used to trace the tendency of MSCs to accumulate in glioma in vivo. Mao et al.[37] constructed MSCs with high expressions of interferon-β (IFNβ) and FTH in a similar manner. MRI was used to evaluate whether MSCs can be used as cell carriers to carry IFNβ to treat brain tumors, which provides a new option for treating brain tumors. Studies have shown that FTH-based MRI can monitor this treatment process.

 Oncolytic viruses can be used to treat malignant tumors, such as glioblastoma [47,48]. The infection process is expected to be observed by MRI. In clinical trials, Christian et al. [18] integrated the lysine-rich protein (LRP) gene into a herpes simplex virus-derived oncolytic virus G47∆ virus. CEST MRI was used to detect gliomas in rats before and 8 h after injection of G47∆-LRP or a control G47∆-empty virus. The contrast increased in tumors of CEST images after infection with G47∆-LRP virus. This study shows that LRP can be used as a reporter gene for real-time monitoring of virus transmission, but the highly repeated gene sequence of LRP may lead to DNA recombination events and expression of a series of truncated LRP protein fragments, which limits the sensitivity of CEST imaging. To address this problem, Perlman et al. [41] redesigned an LRP reporter (rd LRP) without DNA repeat sequences and improved its CEST MRI contrast. The in vivo CEST MRI of brain tumors in mice is shown in Figure 5.  Oncolytic viruses can be used to treat malignant tumors, such as glioblastoma [47,48]. The infection process is expected to be observed by MRI. In clinical trials, Christian et al. [18] integrated the lysine-rich protein (LRP) gene into a herpes simplex virus-derived oncolytic virus G47∆ virus. CEST MRI was used to detect gliomas in rats before and 8 h after injection of G47∆-LRP or a control G47∆-empty virus. The contrast increased in tumors of CEST images after infection with G47∆-LRP virus. This study shows that LRP can be used as a reporter gene for real-time monitoring of virus transmission, but the highly repeated gene sequence of LRP may lead to DNA recombination events and expression of a series of truncated LRP protein fragments, which limits the sensitivity of CEST imaging. To address this problem, Perlman et al. [41] redesigned an LRP reporter (rd LRP) without DNA repeat sequences and improved its CEST MRI contrast. The in vivo CEST MRI of brain tumors in mice is shown in Figure 5.

**Figure 5.** In vivo imaging of the rd LRP. (**a**,**b**) The amide proton CEST amplitudes of glioma cells that did not express rd LRP (**a**) and expressed rd LRP (**b**) in mouse brain. (**c**) Comparison of the T2 relaxation time of the two groups. The results showed no significant difference, and there was edema in the tumor area. (**d**,**e**) T2-weighted imaging corresponding to (**a**,**b**) respectively. (**f**,**g**) Overlay of the amide proton CEST contrast (obtained using a frequency selective saturation pulse (**a**,**b**)) on the (**d**,**e**) image. (**h**) Comparison of CEST amplitudes of amide proton. CEST signal increased significantly in rd LRP group. Reproduced with permission from Ref. [41], 2020. **Figure 5.** In vivo imaging of the rd LRP. (**a**,**b**) The amide proton CEST amplitudes of glioma cells that did not express rd LRP (**a**) and expressed rd LRP (**b**) in mouse brain. (**c**) Comparison of the T2 relaxation time of the two groups. The results showed no significant difference, and there was edema in the tumor area. (**d**,**e**) T2-weighted imaging corresponding to (**a**,**b**) respectively. (**f**,**g**) Overlay of the amide proton CEST contrast (obtained using a frequency selective saturation pulse (**a**,**b**)) on the (**d**,**e**) image. (**h**) Comparison of CEST amplitudes of amide proton. CEST signal increased significantly in rd LRP group. Reproduced with permission from Ref. [41], 2020.

Additional innovative studies have been reported. Hyla et al. [42] developed a system called GeneREFORM, calculated and designed a group of two-color reporter genes and probes, and established a two-color gene imaging system. With the aid of existing MRI technology, GeneREFORM can accurately locate and achieve noninvasive two-color imaging of multiple genes in the deep tissues of living animals. The GeneREFORM system is also applicable to nontumor models.

There are also potential MRI reporter genes that can be used for brain imaging, notably gas vesicles (GVs). GVs [49] are gas-filled protein nanostructures that are originally located in the cells of some bacteria and archaea that regulate cell buoyancy in aqueous environments [50,51]. GVs are gene-encoded nanoscale probes composed of the primary structural protein, GvpA, the optional external scaffolding protein, GvpC, for structure reinforcement, and several secondary proteins that function as essential minor constituents or chaperones [52]. GVs consists of external hydrophilic and internal hydrophobic protein structures, which cause their interiors to form gas cavities filled with gas that is separated from the surrounding medium and realizes the simultaneous free exchange of internal and external gas [53]. In the biological world, photosynthetic bacteria use the contents of gas contained in vesicles to regulate buoyancy and accomplish their own floating behavior. The magnetic susceptibility of GVs is quite different from that of water, which can produce large contrasts in magnetic resonance imaging, even at sub-nanomolar concentrations. The gas cavities of the vesicles can scatter sound waves and produce ultrasonic contrasts. When the pressure on the air wall is greater than the threshold, the vesicles collapse; thus, background-free imaging can be achieved by acoustically modulated magnetic resonance imaging. The mechanical and surface characteristics of GvpC can be genetically modified by replacing the natural external GvpC with its recombinant variant, thus changing its magnetic susceptibility and collapse pressure, with the potential to obtain multichannel imaging. George et al. [54] showed that background-free imaging can be achieved by acoustic modulation MRI after injection of GVs into the striatum of mice. When using the same method, MRI contrast could not be obtained after injections of phosphate buffer without GVs. These results indicate that GVs are expected to become an MRI reporter gene for brain imaging.

Vasoactive peptides are another potential MRI reporter gene for use in brain imaging. Their expression can cause vasodilation and facilitate hemodynamic imaging. Designed probes based on vasodilating peptides can image brain regions [55] and can be used to detect important molecules in the brain, such as neurochemicals [56]. Its use provides the potential to examine a wide variety of molecular phenomena in the brain and other organs.

#### *3.2. Brain Imaging of Reporter Genes with Radionuclide*

The majority of radionuclide imaging studies in the brain are related to cell/gene therapy monitoring, as shown in Table 3 [57–74]. They must be able to address challenges such as penetrating the blood–brain barrier (BBB), imaging in regions of high endogenous gene expressions in the central nervous system (CNS), low specificity, and endogenous expressions of reporter genes in microglia [73]. Shimojo [57] used bacterial dihydrofolate reductase (ecDHFR) as a reporter gene and [18F]FE-TMP as an imaging probe, which functioned as a dual probe in both fluorescence and PET imaging to image the CNS system. As a result, PET could analyze mammalian brain circuits at the molecular level.

**Table 3.** Reporter genes and corresponding radiotracer in radionuclide brain imaging.

**Table 3.** *Cont.*

ecDHFR, bacterial dihydrofolate reductase; D2R, the dopamine type 2 receptor; [11C]NMSP, [11C]Nmethylspiperone; D2R80A, a mutant of the dopamine type 2 receptor; hCB(2), human type 2 cannabinoid receptor; [11C]-GW405833, [11C]-labeled CB(2) ligand; DMT1, divalent metal transporter 1; FIAU, fluoro-5-iodo-1 beta-D-arabinofuranosyluracil; FEAU, fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil; FLT, fluoro-30deoxy-30 -Lfluorothymidine; [52Mn2+], Mn based PET contrast agents.

SPECT and PET are useful in neuroscience research, especially in studies of neurodegeneration and neuro-oncology [64,75]. Stem = cell therapy offers new strategies for treating neurological diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis caused by the loss of different types of neurons and glial cells in the brain. SPECT [65,72] and PET [68,69,73] can trace and evaluate the function of stem cells in the nervous system [76]. Multimodality imaging using several reporter genes used dual [77,78] or triple [79] fusion reporter vectors to enable high-sensitivity detection of cells in living animals. A dual-membrane protein positron and gamma-imaging reporter system using sodium iodide symporter and mutant dopamine D-2 receptor transgenes was developed for brain tumor detection.

HSV1-TK using the imaging probe, FHBG, has been used in glioma treatments to monitor chimeric antigen receptor (CAR) T-cell biodistributions and proliferation [58,59]. A study of the imaging probe, FIAU, in patients showed that FIAU cannot penetrate an intact BBB [63,64]. After gene therapy, substantial levels of FIAU may be detected within areas of BBB disruption; hence, clinically relevant levels of HSV-1-tk gene expression in brain tumors can be detected [63]. FBAU [66] is another promising imaging probe that has been studied in glioma imaging based on a mouse model.

In addition to glioma, bone marrow stem cells (BMSCs) used in experimental middle cerebral artery occlusion (MCAO) rat models have been imaged with a reporter gene–probe system [65], consisting of the HSV1-tk and [131I]FIAU pair. BMSCs were introduced into MCAO rat models via local injections into the brain or via injections into the lateral ventricle, carotid artery, or tail vein. The quantity of injected dose per gram in infarcted brain tissue in rats receiving injections into the brain was significantly higher than that in rats receiving injections elsewhere. No differences were seen in the other cell transplantation groups. SPECT imaging with [131I]FIAU 24 h after injection provides peak target-to-nontarget count ratios. Neural stem cells have also been traced by SPECT [72]. The human sodium iodide symporter (hNIS) has been used as a reporter gene to track neural stem cells after transplantation in the brains of rats by using SPECT/CT imaging with technetium-99m to indicate the effectiveness and lack of interference with neural stem cell functioning. Dopamine type 2 receptor (D2R) and its mutant (D2R80A) have been used for neural stem cell tracing in the central nervous system. [11C]*N*-methylspiperone microPET has been proven useful in imaging neural stem-cell-induced D2R expressions in a rat model of traumatic brain injury [68]. It has also been proven in athymic rats that D2R80A is an effective reporter gene for human mesenchymal stem-cell detection in vivo [69]. In another study with mice and cats, a separate adeno-associated virus type 1 vector with identical gene expression control elements was co-injected with the D2R80A vector. This dual-vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring [70].

The BBB penetration ability of imaging probes hampers the usage of reporter gene imaging. FHBT was studied to improve BBB permeability [61], but there were no significant improvements compared with the traditional probe, FHBG. It was demonstrated that the novel scaffold proposed in this study supports the development of a new imaging probe with better BBB permeability for HSV1-tk and its mutant in the future. This imaging probe combined with reporter genes other than HSV1-TK provides a better solution for crossing the intact BBB. The human-type 2 cannabinoid receptor (hCB(2)) related ligand, [ 11C]GW405833, for example, is readily distributed across the BBB. hCB(2)(D80N) was locoregionally overexpressed in the rat striatum by stereotactic injections of lentiviral and adeno-associated viral vectors. Kinetic PET revealed specific and reversible CB(2) binding of [11C]GW405833 in the transduced rat striatum. The hCB(2) expressions were followed for 9 months, which demonstrates the potential future clinical use of CB(2) as a PET reporter in the intact brain [71]. In another study [73], the PKM2 reporter gene was delivered to the brains of mice by adeno-associated virus (AAV9) via stereotactic injection. PET imaging at 8 weeks post AAV delivery showed that the AAV-injected mice had increases in [18F]DASA-23 brain uptake in the transduced sites. PKM2 can be used in the central nervous system to monitor gene and cell therapy without breaking the BBB.

Shimojo et al. [57] used bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to visualize the neuronal circuit activities elicited by chemogenetic manipulation in the mouse hippocampus. In addition to mice, a 3.4 year old common marmoset underwent PET scans 45 days after AAV injection, when ecDHFR-EGFP was expressed in the brain. The PET results and postmortem fluorescence images are shown in Figure 6.

Biological variables are the byproducts of reporter gene expression PET imaging. Xu et al. [80] described a method that minimizes both the design and variability of vector delivery vehicles for alternative PET reporter genes (PRG) and the biological variability of the in vivo target when comparing the efficacy, sensitivity, and specificity of alternative PET reporter gene/PET reporter probe (PRP) combinations for in vivo PRG imaging. The Xu group described the process of comparing the standard HSVsr39TK/[18F]FHBG PRG/PRP reporter system to four other PRGs, which were all coupled with the same PRP, 18F-L-FMAU.

**Figure 6.** PET imaging of ecDHFR/TMP reporters in primate brain. (**A**) Representative coronal PET images generated by averaging dynamic scan data at 60–180 min after i.v. injection of [18F]FE-TMP. Note that reporter molecules were densely distributed in thalamus and substantia nigra pars compacta, which are connected to the neocortex and putamen via direct neuronal tract, respectively. PET images are overlaid with an individual MRI. Scale bar represents the SUV. (**B**) Time–radioactivity curves for [18F]FE-TMP in the putamen and neocortex carrying ecDHFR-EGFP or control AAV, and in the hippocampus are displayed in the upper panel. Curves in the thalamus, substantia nigra, and cerebellum along with hippocampus data are also shown in the lower panel. (**C**) Postmortem analysis of ecDHFR-EGFP expression in brain slices of different part with high-magnification image frames in inserts. Reproduced with permission from the authors (2021) [57]. **Figure 6.** PET imaging of ecDHFR/TMP reporters in primate brain. (**A**) Representative coronal PET images generated by averaging dynamic scan data at 60–180 min after i.v. injection of [18F]FE-TMP. Note that reporter molecules were densely distributed in thalamus and substantia nigra pars compacta, which are connected to the neocortex and putamen via direct neuronal tract, respectively. PET images are overlaid with an individual MRI. Scale bar represents the SUV. (**B**) Time–radioactivity curves for [18F]FE-TMP in the putamen and neocortex carrying ecDHFR-EGFP or control AAV, and in the hippocampus are displayed in the upper panel. Curves in the thalamus, substantia nigra, and cerebellum along with hippocampus data are also shown in the lower panel. (**C**) Postmortem analysis of ecDHFR-EGFP expression in brain slices of different part with high-magnification image frames in inserts. Reproduced with permission from the authors (2021) [57].

#### Biological variables are the byproducts of reporter gene expression PET imaging. Xu **4. Opportunities and Challenges**

#### et al. [80] described a method that minimizes both the design and variability of vector *4.1. About the Modalities*

delivery vehicles for alternative PET reporter genes (PRG) and the biological variability of the in vivo target when comparing the efficacy, sensitivity, and specificity of alternative PET reporter gene/PET reporter probe (PRP) combinations for in vivo PRG imaging. The Xu group described the process of comparing the standard HSVsr39TK/[18F]FHBG PRG/PRP reporter system to four other PRGs, which were all coupled with the same PRP, 18F-L-FMAU. Four clinical imaging modalities have the potential for conducting reporter gene imaging. MRI is the most common modality in brain detection and study. The limitations of MRI are the sensitivity of available imaging agents, the cost, and the imaging time. PET and SPECT have been used in gene and cell therapy because of their high sensitivity. SPECT needs strict collimation to maintain resolution, which may cause an increase in radioisotope dose. PET is more sensitive and has higher resolution than SPECT. SPECT and PET are often combined with CT for co-registered anatomical and functional imaging. The reporter genes for ultrasound, namely, the acoustic reporter gene (ARG), are exclusively

GVs. Studies in prokaryotic cells and mammalian cells have been carried out. Traditionally, imaging with probes that directly interact with target cells or molecules is called direct imaging. Imaging with probes that react to surrogate components that are related to the target cells or molecules is called indirect imaging. Imaging based on reporter genes is indirect. In existing brain studies, GVs can be used in brain imaging as probes but are not produced by target cells through transgenic technology. Further studies are needed to indirectly correlate GVs with target cells or molecules. The future of GVs as reporter genes or imaging contrast or therapy methods is very promising.

#### *4.2. MRI*

The high spatial resolution of MRI leads to its wide use in brain imaging research. The use of targeted contrast agents [81] has further improved its signal-to-noise ratio, and reporter gene technology has further improved the sensitivity and specificity of imaging. Some MR reporter genes (e.g., β-galactosidase) need to be used together with contrast agents to be detected, while some MR reporter genes (e.g., ferritin) do not need to add exogenous contrast agents, which has unique advantages compared with targeted contrast agents. On the one hand, the use of only reporter genes avoids the biological barriers that need to be overcome by injecting contrast media and the clearance barriers that contrast agents retain in the blood and tissue. Moreover, these reporter genes are generally not diluted with cell division-like targeted contrast agents (e.g., SPIO [82,83]), which can achieve more long-term and effective MRI monitoring [84,85]. On the other hand, reporter genes are generally expressed only in living cells, and they generally do not report both living and dead cells like imaging probes (e.g., the perfluorocarbon tracer), resulting in false-positive results [86–88].

Reporter gene-based MRI has great potential for use in brain imaging applications, but there are still many limitations to be overcome. Low sensitivity is a major limitation of MRI, while reporter genes based on CEST need high expression levels to achieve observable contrasts, which greatly limits the sensitivity of CEST. Reporter genes based on metal proteins or metal ion transporters may be hindered by the bioavailability or toxicity of metal ions. Therefore, there is a great demand for new MRI reporter genes that do not require metals and can be detected at low expression levels. The emergence of the aquaporin reporter gene improves these problems to some extent, but a potential limitation of aquaporin is its negative contrast enhancement.

In recent years, with the rapid development of MRI reporter genes, many innovative studies have been conducted. An interesting research direction is to integrate several types of reporter genes and combine the advantages of multiple imaging methods to achieve multimodal imaging. The search for new MRI reporter molecules is also an important research direction. In recent years, some studies have suggested that additional new molecules can be used as MRI reporter genes. For example, gas vesicles may become unique and powerful reporter genes in background-free MRI [54]. The ability to map several transgenes expressed at the same time by MRI will further enrich the gene transform palette and add additional "colors", thus expanding the "multicolor" imaging toolbox to image previously inaccessible deep tissues [42]. However, most current research is currently in the animal experiment stage, and there is still a long way to go before they can be applied in clinics.

#### *4.3. Radionuclide Imaging*

Radionuclide brain imaging has been used in molecular imaging for tumor detection and therapy and has even exhibited the possibility of brain function studies. The SPECTbased reporter gene technique has been studied in patients [64]. The progress of gene expression, posttranscriptional events, and brain circuit function can be noninvasively visualized by repetitively using reporter genes. Most of the paired imaging probes of the studied reporter genes are already clinically approved, which causes reporter gene radionuclide imaging to be close to clinical usage. Cell tracking studies have experienced

great improvements. [18F]FHBG imaging was safe in tracking HSV1-tk reporter gene expressions present in chimeric antigen receptor-engineered cytotoxic T lymphocytes and enabled the longitudinal imaging of T cells that were stably transfected with a PET reporter gene in patients [58].

The difficulty when choosing a reporter is that host-compatible reporters have no immune rejection risks, while their endogenous expressions may interfere with the real signal. Radionuclide imaging cannot be used in long-term studies due to decay of the imaging probe. The reporter gene will be expressed by the undiluted target cells for the lifetime of the cells and even for expanding cells. Until the design of viral tools for noninvasive gene delivery can apply these reporters to biomedical radionuclide imaging of human brains [89], researchers must face safety, cost-effectiveness, and ethics challenges.

HSV1-tk and its mutant are the most popular reporter genes for PET imaging. The inability to pass through the blood–brain barrier makes it difficult for them to be used in brain studies [68]. Imaging probes (e.g., PET tracers and reporter probes) that can cross the BBB have been developed [61,71,73]. A central nervous system multiparameter PET optimization (CNS PET MPO) algorithm was developed by a group from Pfizer that provides predictions of the required physicochemical properties of clinically useful CNS PET agents [61,90]. Reporter gene PET imaging is combined with other modalities, such as MRI [74] and optical imaging [71].

### *4.4. Pharmacodynamic and Pharmacokinetic Assessment*

A wide spectrum of therapeutic viruses, genes, and cells have been developed in recent years for personalized treatment of cancers. Despite of many undergoing clinical trials and some now licensed for clinical use, the safety, accuracy, and efficiency of these therapies remain big concerns [91,92]. In vivo imaging tools are in urgent need for the longitudinal monitoring of the biological distribution and fate of these exogenous cells, genes, and viruses, as well as an evaluation of the therapeutic response and promotion of their clinical translation [91,93]. As one of the most promising approaches, reporter genes feature a high level of cellular specificity, without signal dilution, such as cellular proliferation or tumor metastasis. Among all the modalities, reporter genes expressing PET tracers have progressed quickly and are considered most valuable for clinical translation. Notably, the combination of PET and MRI reporter genes exhibits translational potential with superiority in both specificity and localization [94].

However, in practice, multiple factors need to be taken into accounts, including the background signal contaminations, the favorable dosage over radiation exposure, and the limited or no biological effect deriving from expression of the transgene. Additionally, the potential for nonhuman reporter genes to be immunogenic must also be taken into consideration when addressing ideal system design, since this may affect functionality and survival of transduced cells once injected into humans [2,14].

In addition to the performance improvement of reporter gene and imaging probe, the clinical usage of reporter gene imaging faces some major problems such as the stable transfer of reporter gene safely and efficient delivery of reporter gene into primary cells that cannot be cultured ex vivo for long period. Early collaborations between molecular imagers and cell gene therapists may solve these problems, which would afford a parallel approach starting with pre-clinical studies so that reporter gene imaging can be incorporated into the cell or gene therapy. Members of the molecular–genetic imaging industry such as CellSight Technologies have bridged the gap between reporter gene technologies and clinical trials [2,91].

#### *4.5. Neuron Imaging with Reporter Gene*

Reporter gene imaging for neuron imaging is a new rising direction with only a few studies reported. Wang's group imaged astrocytes in the whole brain with engineered AAVs and DWI in vivo [39]. They also use ferritin-encoding trans-synaptic [32] and adeno-associated virus [33] to detect neural connections ex vivo and longitudinal neural

connections in vivo, respectively. Shimojo et al. [57] used ecDHFR and TMP to visualize the neuronal circuit activities elicited by chemo-genetic manipulation in the mouse hippocampus. These studies provide substitutes for genetically encoded fluorescent probes, enabling in vivo gene targeted neuronal activity observation to be carried out in real time in larger animals. Hence, more mechanisms of neuron activity will be discovered in nonhuman primates or even humans in the future.

#### **5. Conclusions**

Reporter genes for use with clinical imaging modalities have greatly improved in recent years. For brain imaging studies, in addition to tumor detection and therapy, brain function-related studies cut a striking figure. Advances in gene techniques will bring great progress in brain imaging. The development of molecular functional MRI tools for animal research is accelerating, but the maximum benefits of this technology will eventually be achieved in human subjects. The abovementioned reporter gene technologies should be used and should not be limited to proof-of-concept experiments. Only in this way will it be possible to recognize the practical limitations of the emerging reporting mechanisms and identify important ways to improve them.

**Funding:** This research was funded by the National Natural Science Foundation of China grant number 81901907 and 82172112, the Beijing Institute of Technology Research Fund Program for Young Scholars, and the Fundamental Research Funds for the Central Universities grant number LY2022-22 and 2021CX11018.

**Conflicts of Interest:** The authors declare no conflict of interest.

### **Abbreviations**

#### **References**

## *Article* **Biomimetic Nanosponges Enable the Detoxification of** *Vibrio vulnificus* **Hemolysin**

**Shuaijun Zou 1,†, Qianqian Wang 1,†, Peipei Zhang 2 , Bo Wang 1 , Guoyan Liu 1 , Fuhai Zhang 1 , Jie Li 1 , Fan Wang 1 , Beilei Wang 1,\* and Liming Zhang 1,\***

**Abstract:** *Vibrio vulnificus* (*V. vulnificus*) infection-associated multiple antibiotic resistance has raised serious public health concerns. Recently, nanosponges (NSs) have been expected to provide innovative platforms for addressing antibacterial and drug-resistant challenges by targeting various pore-forming toxins (PFTs). In the present study, we constructed NSs to explore the effects and possible mechanism of recombinant *V. vulnificus* hemolysin (rVvhA)-induced injuries. In vitro, NSs significantly reversed rVvhA-induced apoptosis and necrosis, and improved toxin-induced intracellular reactive oxygen species (ROS) production, adenosine triphosphate (ATP) depletion, and apoptosis signaling pathway disruption. To explore the clinical translation potential of NSs, we established VvhA-induced septicemia and wound infection mouse models, respectively, and further found NSs could notably attenuate rVvhA-induced acute toxicity and septicemia-associated inflammation, as well as local tissue damage. In a conclusion, NSs showed excellent protective effects against rVvhA-induced toxicity, thus providing useful insights into addressing the rising threats of severe *V. vulnificus* infections.

**Keywords:** *Vibrio vulnificus*; nanosponge; VvhA; pore-forming toxins; biodetoxification

## **1. Introduction**

*Vibrio vulnificus* (*V. vulnificus*) is an alkalophilic, halophilic, rod-shaped, gram-negative bacterium of the *Vibrio* genus [1,2]. Exposure to *V. vulnificus*-contaminated seawater usually results in hemorrhagic necrotic skin lesions and secondary sepsis, while ingestion of contaminated raw or undercooked seafood mainly leads to primary septicemia [3–5]. Septicemia caused by *V. vulnificus* exerts high mortality (>50%), especially in patients with chronic liver diseases or immunocompromising conditions [6–9]. Currently, combination antibiotic therapy is the first choice for *V. vulnificus* infections and includes doxycycline, third-generation cephalosporin, fluoroquinolone, and trimethoprim-sulfamethoxazole, as well as aminoglycosides [10]; however, due to the excessive use of antibiotics in humans, agricultural and aquaculture systems, multiple antibiotic resistance has emerged in up to 50% of *V. vulnificus* strains in various countries, which raises serious public health and economic concerns [10,11]. Thus, the development of alternative agents is urgently needed.

*V. vulnificus* hemolysin (VvhA) is a pore-forming toxin (PFT) encoded by *vvhA*, which is a key virulence factor in the pathogenesis of *V. vulnificus* infections. Earlier studies [12,13] have shown that local and systemic administration of VvhA in animal models reproduces the same clinical and pathological manifestations as live *V. vulnificus*, and purified VvhA

**Citation:** Zou, S.; Wang, Q.; Zhang, P.; Wang, B.; Liu, G.; Zhang, F.; Li, J.; Wang, F.; Wang, B.; Zhang, L. Biomimetic Nanosponges Enable the Detoxification of *Vibrio vulnificus* Hemolysin. *Int. J. Mol. Sci.* **2022**, *23*, 6821. https://doi.org/10.3390/ ijms23126821

Academic Editor: Francesco Trotta

Received: 14 May 2022 Accepted: 17 June 2022 Published: 19 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

can induce lethal effects in vivo, including hypotension, tachycardia, and pulmonary damage; moreover, VvhA may also contribute to pathogen invasion, vasodilatation and septic shock [14–17]. These results indicate that VvhA should be a logical target for the development of alternative therapeutics to combat *V. vulnificus* infection. More importantly, targeting the VvhA strategy could effectively prevent drug resistance since it exerts less selective pressure on pathogens themselves; however, the cellular targeting sites of VvhA remain controversial and unclear [16]. For example, the cholesterol on cell membranes was believed to be the receptor for VvhA, while some other reports suggested that VvhA localized on both cholesterol and glycan moieties rich membrane domain and other membrane domains [18,19]; conversely, the molecular mechanism of cell damage after the interaction between VvhA and the host cell during *V. vulnificus* infection has also not been clarified yet. For example, some studies demonstrate that VvhA induced NF-κB-dependent mitochondrial cell death via lipid raft-mediated reactive oxygen species (ROS) production [20]; some other studies proposed that VvhA induced autophagy upregulation through the lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2α activation [18]. Collectively, structure-specific PFT-targeting strategies or some signal pathway inhibitors would be insufficient to thoroughly combat VvhA-induced injuries.

Recently, Hu et al. [21,22] proposed a broad-spectrum detoxification strategy, known as "nanosponges (NSs)", which focused on the whole membranes of affected host cells instead of targeting specific structures. Wrapped with red blood cell (RBC) membranes, NSs provide sufficient, intact, and natural targets for PFTs to bind with, and divert them away from the host cells; moreover, the natural membrane shells endow NSs with lower immunogenicity and prolonged circulation time in vivo [23]. Currently, the NS platform has been proven to be effective against injuries from gram-positive pathogen-derived toxins [24,25]; however, fewer studies have been conducted to investigate the protective effects and possible molecular mechanisms of NSs against gram-negative pathogen-derived PFTs.

In this study, we tried to construct NSs by fusing RBC vesicles onto poly (DL-lactide-coglycolide) (PLGA) nanoparticles (NPs), which are the biocompatible polymers approved by the US Food and Drug Administration [26–28]. In vitro, we chose human pulmonary artery endothelial cells (HPAECs) as effector cells, which are the most common target cells in *V. vulnificus*-related sepsis. Then, we examined the effects and possible mechanisms of NSs on toxin-induced intracellular oxidative stress, adenosine triphosphate (ATP) depletion, and the activation of apoptosis signaling pathways. In vivo, we respectively constructed VvhA-induced septicemia and wound infection mouse models to simulate clinical cases, then tested the protective effects of NSs against VvhA by recording the animal survival rates, inflammatory cascades as well as lung and tissue damage.

#### **2. Results**

#### *2.1. Characterization of NSs*

To characterize the hydrodynamic size and zeta potential, PLGA NPs and RBC-derived NSs were measured by dynamic light scattering (DLS). The results revealed that the diameters of NPs increased from 61.3 ± 1.1 to 77.2 ± 1.4 nm with a narrow size distribution, corresponding to the bilayered membrane coating on a polymeric core; moreover, the zetapotential of the PLGA NPs decreased from −35.8 ± 0.3 to −26.5 ± 0.6 mV, and the latter one corresponds to the potential of RBC vesicles (Figure 1A). These results demonstrated fusion between the NP cores and the RBC membranes. To characterize the nanostructures, the resulting NS sample was dried and examined by transmission electron microscopy (TEM). Compared to bare NPs, the resulting NSs exhibited a typical core-shell structure with PLGA cores enwrapped with thin membrane shells, verifying the formation of NSs (Figure 1B). To further characterize the protein contents retained on NSs, the resulting NS sample was examined by SDS-PAGE. The results showed that most membrane proteins were retained on NS shells, which were consistent with those of RBC ghosts and extruded RBC vesicles with a negligible loss during preparation (Figure 1C). Finally, NSs were suspended in

1 × phosphate-buffered saline (PBS) and 100% fetal bovine serum (FBS) and showed only negligible increases in particle size over 7 d at 4 ◦C, confirming the excellent stability of the prepared NSs (Figure 1D).

**Figure 1.** Characterization of NSs. (**A**) Hydrodynamic size, polydispersity index and zeta potential of PLGA NPs, RBC vesicles, and NSs. (**B**) TEM image showing the structure of PLGA NPs (lower) and NSs (upper). (Scale bar: 50 nm.) (**C**) Proteins in RBC ghosts, RBC vesicles, and NSs were examined by SDS-PAGE. (**D**) NSs were suspended in 1 × PBS or 100% FBS, and the hydrodynamic size was monitored for 7 d. **Figure 1.** Characterization of NSs. (**A**) Hydrodynamic size, polydispersity index and zeta potential of PLGA NPs, RBC vesicles, and NSs. (**B**) TEM image showing the structure of PLGA NPs (lower) and NSs (upper). (Scale bar: 50 nm.) (**C**) Proteins in RBC ghosts, RBC vesicles, and NSs were examined by SDS-PAGE. (**D**) NSs were suspended in 1 × PBS or 100% FBS, and the hydrodynamic size was monitored for 7 d.

#### *2.2. Inhibitory Effect of NSs on Cell Viability and Apoptosis Induced by Recombinant VvhA (rVvhA)*

After expression and purification of rVvhA (Figures S1 and S2) [29], we then used HPAECs as target cells to investigate the protective effect of NSs against VvhA-induced cytotoxicity. According to the dose-effect and time-effect relationship of the toxin (Figure 2A,B), we chose the optimal dose of rVvhA (500 ng/mL, the dose producing 50% inhibition) and exposure time (4 h) for subsequent detoxification study. rVvhA-induced cytotoxicity was significantly inhibited by NSs (10–40 µg/mL), and 40 µg/mL NSs almost completely reversed the cytotoxicity of rVvhA, while NSs exerted no effect on cell viability (Figure 2C,D). Similar detoxification effects of NSs (40 µg/mL) were also observed in the context of rVvhA (500 ng/mL)-induced cell apoptosis (Figure 2E,F).

*Int. J. Mol. Sci.* **2022**, *23*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/ijms

**Figure 2.** Inhibitory effects of NSs on rVvhA-induced cell death and apoptosis in HPAECs. (**A**) Doseresponse of cell viability in HPAECs treated with rVvhA for 4 h. (**B**) Time response of HPAECs treated with rVvhA (500 ng/mL) over a period of 8 h (0, 1, 2, 4, 6, 8 h). (**C**) HPAECs were treated with increasing concentrations of NSs (0, 1.25, 2.5, 5, 10, 20 and 40 μg/mL) for 4 h. (**D**) HPAECs were treated with increasing concentrations of NSs (0, 1.25, 2.5, 5, 10, 20 and 40 μg/mL) in the presence of rVvhA (500 ng/mL) for 4 h. (**E**,**F**) HPAECs were treated with PBS, NSs (40 μg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 μg/mL) for 4 h, respectively. The percentages of total apoptotic cells were measured and quantified by using Annexin V/PI staining and flow cytometry. (The data represent the means ± SE. *n* = 3. \* *p* <0.05). **Figure 2.** Inhibitory effects of NSs on rVvhA-induced cell death and apoptosis in HPAECs. (**A**) Doseresponse of cell viability in HPAECs treated with rVvhA for 4 h. (**B**) Time response of HPAECs treated with rVvhA (500 ng/mL) over a period of 8 h (0, 1, 2, 4, 6, 8 h). (**C**) HPAECs were treated with increasing concentrations of NSs (0, 1.25, 2.5, 5, 10, 20 and 40 µg/mL) for 4 h. (**D**) HPAECs were treated with increasing concentrations of NSs (0, 1.25, 2.5, 5, 10, 20 and 40 µg/mL) in the presence of rVvhA (500 ng/mL) for 4 h. (**E**,**F**) HPAECs were treated with PBS, NSs (40 µg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 µg/mL) for 4 h, respectively. The percentages of total apoptotic cells were measured and quantified by using Annexin V/PI staining and flow cytometry. (The data represent the means ± SE. *n* = 3. \* *p* < 0.05).

#### *2.3. NSs Inhibited ROS Production and ATP Depletion Induced by rVvhA*

It was reported that oxidative stress and intracellular ATP depletion were associated with cell necrosis and apoptosis [30,31]; thus, we measured intracellular ROS and ATP levels in this study. As shown in Figure 3A, rVvhA (500 ng/mL) induced a significant increase in intracellular ROS levels, which reached a peak at 1 h; however, NS treatment (40 µg/mL) completely attenuated rVvhA-induced ROS production. Concretely, fluorescence detection via flow cytometry revealed decreased fluorescence in NS-treated cells, and ROS-related fluorescence was quantitively weak under a fluorescence microscope after treatment (Figure 3B–D), indicating that NSs could prevent toxin-induced oxidative stress. Since ATP depletion is a terminal outcome of oxidative stress injuries in mitochondria, we further explored the effects of NSs on intracellular ATP depletion. Our results showed that rVvhA (500 ng/mL) caused a reduction in intracellular ATP in a time-dependent manner, and ATP was reduced by over 90% over a period of 12 h (Figure 3E,F); however, NSs also substantially prevented toxin-induced ATP depletion after 12 h (Figure 3G,H). Accordingly, we demonstrated that NSs may exert protective effects against rVvhA by improving oxidative stress and mitochondrial injury.

**Figure 3.** Inhibitory effects of NSs on rVvhA-induced ROS production and ATP reduction in HPAECs. (**A**) Time response of ROS production in HPAECs treated with rVvhA (500 ng/mL). (**B**) Flow cytometry results showing ROS production in HPAECs with various treatments. (**C**) ROS production (green) was visualized by fluorescence microscopy in HPAECs treated with PBS, NSs (40 μg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 μg/mL), respectively. (Original magnification × 200). (**D**) The quantification of ROS production in C. (**E**,**F**) Time response of ATP levels in rVvhA-stimulated cells over a period of 12 h (0, 1, 3, 6, 12 h) were examined by a luminometer and bioluminescence imaging. (**G**,**H**) Analysis of ATP levels for 12 h in HPAECs treated with PBS, NSs (40 μg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 μg/mL), respectively, as detected by a luminometer and bioluminescence imaging. (The data represent the means ± SE. *n* = 3. \* *p* < 0.05)*.* **Figure 3.** Inhibitory effects of NSs on rVvhA-induced ROS production and ATP reduction in HPAECs. (**A**) Time response of ROS production in HPAECs treated with rVvhA (500 ng/mL). (**B**) Flow cytometry results showing ROS production in HPAECs with various treatments. (**C**) ROS production (green) was visualized by fluorescence microscopy in HPAECs treated with PBS, NSs (40 µg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 µg/mL), respectively. (Original magnification × 200). (**D**) The quantification of ROS production in (**C**). (**E**,**F**) Time response of ATP levels in rVvhA-stimulated cells over a period of 12 h (0, 1, 3, 6, 12 h) were examined by a luminometer and bioluminescence imaging. (**G**,**H**) Analysis of ATP levels for 12 h in HPAECs treated with PBS, NSs (40 µg/mL), rVvhA (500 ng/mL), rVvhA (500 ng/mL) plus NSs (40 µg/mL), respectively, as detected by a luminometer and bioluminescence imaging. (The data represent the means ± SE. *n* = 3. \* *p* < 0.05).

#### *2.4. NSs Inhibit PKC/ERK/JNK and NF-κB Pathway Activation Induced by rVvhA*

According to previous studies, VvhA exposure can activate PKC/JNK/ERK phosphorylation, stimulate lipid raft-mediated ROS production, and ultimately cause NF-κBdependent cell apoptosis in intestinal epithelial cells [20,32]. We, thus, measured the protein expression of these signaling pathways in HPAECs to explore the intracellular changes and protective effects of NSs against VvhA. In our study, we confirmed that rVvhA could stimulate PKC phosphorylation and induce the phosphorylation of both JNK and ERK from 30 to 90 min (Figure 4A), and these events were responsible for the activation of the transcription factor NF-κB; however, NSs significantly blocked rVvhA-induced PKC/JNK/ERK phosphorylation (Figure 4B). Next, we examined the effect of rVvhA on the activation of the NF-κB pathway, which is a direct transcriptional target of the apoptotic signaling pathway. Our results showed that rVvhA (500 ng/mL) increased the phosphorylation level of NF-κB from 15 min to 60 min (Figure 4C); however, NSs notably attenuated toxin-induced NF-κB signaling pathway activation (Figure 4D). A similar inhibitory effect of NSs on NF-κB accumulation in the nucleus was also confirmed by immunofluorescence staining (Figure 4E). Finally, the expression of the main apoptosis factors, including Bcl-2, Bax, Caspase-9, and Caspase-3, was measured, and these factors play key roles in mitochondrial pathways. Our results revealed that rVvhA altered the Bcl-2/Bax ratio from 60 to 180 min, and these proteins are two NF-κB-dependent apoptosis-determining factors (Figure 4F). Consistently, rVvhA also stimulated caspase-9/-3 activation while promoting apoptotic cell death; however, NSs notably reversed VvhA-mediated changes in mitochondrial apoptotic indicators (Figure 4G).

**Figure 4.** NSs inhibited rVvhA-induced activation of the PKC/JNK/ERK and NF-κB pathways. (**A**) The phosphorylation of PKC/JNK/ERK in HPAECs treated with rVvhA (500 ng/mL) was examined by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min. (**B**) The phosphorylation of PKC/JNK/ERK in HPAECs treated with NSs (40 μg/mL) and rVvhA (500 ng/mL) for 60 min. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus Cont. (**C**) The phosphorylation of NF-κB in HPAECs treated with rVvhA (500 ng/mL) was examined by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min. (**D**) The phosphorylation of NF-κB in HPAECs treated with NSs (40 μg/mL) and rVvhA (500 ng/mL) for 60 min. The data represent the mean ± SE. *n* = 3. \* *p* < 0.05 versus Cont. (**E**) Nuclear accumulation of NF-κB was examined after 60 min by immunostaining with a Cy3-labeled NF-κB p65 antibody (Original magnification × 400). (**F**) HPAECs were incubated with rVvhA (500 ng/mL) for 180 min, and then Bcl-2 and Bax were analyzed by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min, # *p* < 0.05 versus 0 min. (**G**) The expression of Bcl-2 and Bax was measured in the presence of NSs. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus Cont, # *p* < 0.05 versus Cont. **Figure 4.** NSs inhibited rVvhA-induced activation of the PKC/JNK/ERK and NF-κB pathways. (**A**) The phosphorylation of PKC/JNK/ERK in HPAECs treated with rVvhA (500 ng/mL) was examined by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min. (**B**) The phosphorylation of PKC/JNK/ERK in HPAECs treated with NSs (40 µg/mL) and rVvhA (500 ng/mL) for 60 min. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus Cont. (**C**) The phosphorylation of NF-κB in HPAECs treated with rVvhA (500 ng/mL) was examined by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min. (**D**) The phosphorylation of NF-κB in HPAECs treated with NSs (40 µg/mL) and rVvhA (500 ng/mL) for 60 min. The data represent the mean ± SE. *n* = 3. \* *p* < 0.05 versus Cont. (**E**) Nuclear accumulation of NF-κB was examined after 60 min by immunostaining with a Cy3-labeled NF-κB p65 antibody (Original magnification × 400). (**F**) HPAECs were incubated with rVvhA (500 ng/mL) for 180 min, and then Bcl-2 and Bax were analyzed by Western blotting. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus 0 min, # *p* < 0.05 versus 0 min. (**G**) The expression of Bcl-2 and Bax was measured in the presence of NSs. The data represent the means ± SE. *n* = 3. \* *p* < 0.05 versus Cont, # *p* < 0.05 versus Cont.

#### *2.5. In Vivo Neutralization of rVvhA by NSs*

To examine the systemic detoxification effects of NSs against rVvhA in vivo, we first analyzed rVvhA-induced acute toxicity by injecting various amounts of rVvhA via the tail vein. Our results showed that the mortality of mice increased with increasing doses of rVvhA (Figure 5A). The survival curve shown in Figure 5B revealed that NS treatment dose-dependently improved the survival of mice that received lethal doses of rVvhA. When the dose of NSs exceeded 200 mg/kg, the survival rate was greater than 80%. Then, NSs (200 mg/kg) were used to examine the reduction in inflammatory cascade reactions caused by rVvhA (3.7 mg/kg, 0.75 × LD50, a rational dose to trigger inflammatory and lung damage without lethality). NSs dramatically reversed rVvhA-induced interleukin-6

(IL-6) and tumor necrosis factor-α (TNF-α) accumulation after 3 h (Figure 5C–F). NSs also apparently improved rVvhA-induced lung damage, such as typical hemorrhagic injuries, neutrophil aggregation, and alveolar structural changes (Figure 5G). *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 5 of 5

> **Figure 5.** Protective effects of NSs in vivo. (**A**) Different doses of rVvhA (3, 4, 5, 6, 7 mg/kg) were injected intravenously, and mouse mortality was recorded. (**B**) A lethal dose of rVvhA (6 mg/kg) was injected via the tail vein, and different doses of NSs (0, 50, 100, 150, 200, 250 mg/kg) were immediately administered for treatment. (**C**,**E**) Mice were exposed to sublethal doses of rVvhA (3.7 mg/kg), and inflammatory factors, including IL-6 and TNF-α, were examined at predetermined times (0, 3, 6, 12 h). (**D**,**F**) NSs (200 mg/kg) were used to neutralize rVvhA (3.7 mg/kg) for 3 h in vivo, and then IL-6 and TNF-α in the blood were measured. (**G**) rVvhA (3.7 mg/kg) was used to stimulate mice for 3 h with or without NSs (200 mg/kg), and the lungs were harvested for hematoxylin and eosin (H&E) staining and pathological observations. Untreated mice and mice injected with NSs were used as controls. (Original magnification × 400) (**H**) rVvhA (30 μg) was injected subcutaneously to imitate local infections, and NSs (50 mg/kg) were used for treatment. After 3 d, the injection sites were measured and photographed, and then the topical skin and muscle tissues were harvested for H&E staining and pathological observations. (Scale bar: 50 μm. The data represent the means ± SE. *n* = 6. \* *p* < 0.05)*.* **Figure 5.** Protective effects of NSs in vivo. (**A**) Different doses of rVvhA (3, 4, 5, 6, 7 mg/kg) were injected intravenously, and mouse mortality was recorded. (**B**) A lethal dose of rVvhA (6 mg/kg) was injected via the tail vein, and different doses of NSs (0, 50, 100, 150, 200, 250 mg/kg) were immediately administered for treatment. (**C**,**E**) Mice were exposed to sublethal doses of rVvhA (3.7 mg/kg), and inflammatory factors, including IL-6 and TNF-α, were examined at predetermined times (0, 3, 6, 12 h). (**D**,**F**) NSs (200 mg/kg) were used to neutralize rVvhA (3.7 mg/kg) for 3 h in vivo, and then IL-6 and TNF-α in the blood were measured. (**G**) rVvhA (3.7 mg/kg) was used to stimulate mice for 3 h with or without NSs (200 mg/kg), and the lungs were harvested for hematoxylin and eosin (H&E) staining and pathological observations. Untreated mice and mice injected with NSs were used as controls. (Original magnification × 400) (**H**) rVvhA (30 µg) was injected subcutaneously to imitate local infections, and NSs (50 mg/kg) were used for treatment. After 3 d, the injection sites were measured and photographed, and then the topical skin and muscle tissues were harvested for H&E staining and pathological observations. (Scale bar: 50 µm. The data represent the means ± SE. *n* = 6. \* *p* < 0.05).

> To examine the local detoxification effects of NSs against rVvhA in vivo, we further examined the effects of NSs (50 mg/kg) on a rVvhA-induced wound infection mouse model. Histological analyses showed that rVvhA induced obvious reddish ecchymosis and ulcerations on topical skin at 3 d, with typical edema, cell death and inflammation in the skin and muscles, while NS treatment significantly alleviated this tissue damage, confirming the local detoxification effects of NSs (Figure 5H).

#### **3. Discussion**

*V. vulnificus* is a highly fatal human pathogen that is increasingly being considered an emerging pathogen associated with public health concerns [33]; however, current antibiotic resistance combined with a lack of broad-spectrum virulence factor-targeting strategies highlights the need for innovative research into alternative strategies to combat diseases [10]. In this study, a stable and biocompatible biomimetic nanodetoxification system was developed to neutralize VvhA for the first time. Specifically, NSs showed significant inhibitory effects on not only VvhA-induced apoptosis/necrosis but also toxininduced septicemia and wound infections. In contrast to traditional strategies, NSs prevent the recognition and interaction between toxins and cells by absorbing and neutralizing VvhA toxins regardless of their specific structures, and further avoiding customization of inhibitors against VvhA-activating cell signaling molecules in treatment. Our work may offer significant potential for the treatment of severe *V. vulnificus* infections.

Multiple-organ failure caused by *V. vulnificus*-associated sepsis always begins with pulmonary dysfunction, which is characterized by increased vascular permeability due to pulmonary endothelial cell damage [34–37]. We, thus, used HPAECs as target cells to investigate the protective effect of NSs against VvhA-induced cytotoxicity. We first found that NSs could dramatically improve VvhA-induced apoptosis and necrosis in HPAECs. Since oxidative stress and intracellular ATP depletion are associated with necrosis and apoptosis [38–40], we further explored the effects of NSs on intracellular ROS production and ATP levels. Our results confirmed that NSs could prevent toxin-induced oxidative stress; moreover, we also found that NSs could apparently improve toxin-induced ATP depletion, which is a terminal outcome of oxidative stress injuries in mitochondria [38,40]. Accordingly, the above results revealed that NSs may exert protective effects against VvhA-induced apoptosis and necrosis by improving oxidative stress and mitochondrial injury. Then, we further explored the effects of NSs on various cell signaling pathways related to oxidative stress and mitochondrial injury. We confirmed that VvhA could induce NF-κB-dependent cell apoptosis via ROS production through the distinct activation of PKC/JNK/ERK in HPAECs. Additionally, NS treatment significantly blocked the activation of the above signaling pathways, as well as apoptosis-related proteins, confirming the protective mechanism of NSs on molecular levels. In previous studies [21,24,41–43], NSs were indicated to bind and neutralize toxins via lipid membrane interactions with a high natural affinity and prevent host-toxin interactions. Thus, we hypothesize that the protective mechanism of NSs against VvhA-induced cell apoptosis and necrosis may be associated with toxin sequestration and blocking toxin-host cell interactions, thus radically avoiding the downstream activation of toxin-induced cell signaling disruptions, as well as further cellular injuries. Therefore, for VvhA-induced sepsis, especially pulmonary dysfunction, the sequestration of VvhA by NSs can exert broadly protective effects without consideration of customization of inhibitors against disease-related cell signaling molecules in clinical trials.