**Abstract:** The effect of low-dose photodynamic therapy on in vivo wound healing was investigated using optical coherence tomography. This work aims to develop an approach to quantitative assessment of the wound's state during wound healing including the effect of low-dose photodynamic therapy using topical application of two different photosensitizers, 5-aminolevulinic acid and methylene blue, and two laser doses of 1 J/cm2 and 4 J/cm2 . It was concluded that the laser dose of 4 J/cm2 was better compared to 1 J/cm2 and allowed the wound healing process to accelerate.

**Keywords:** low dose photodynamic therapy; wound healing; 5-aminolevulinic acid; methylene blue; optical coherence tomography

### **1. Introduction**

According to the World Health Organization (WHO), burn wounds result in approximately 180,000 deaths every year and nearly 11 million injuries that require medical treatment worldwide [1]. Cutaneous wounds are widespread and differentiated into acute and chronic wounds [2].

Wound healing is a complex physiological process at the cellular and molecular levels including the extracellular matrix synthesis, the replacement of type III collagen with type I collagen, and scar tissue formation [3–6]. These processes are divided into four overlapping stages: coagulation (hemostasis), inflammation, proliferation, and remodeling [7,8]. Some underlying diseases affect the wound healing process including peripheral arterial and venous disease or diabetes; acute wounds may have impaired healing, which can lead to a chronic stage [9–11]. In developed countries, 1–6% of the population suffers from chronic wounds [12–14].

It is known that a low dose photo process with photoactive compounds promotes the healing of skin diseases and leads to results in rejuvenation and wound healing [15,16]. Low-dose photodynamic therapy (LDPDT) is widely used to treat skin diseases and wound healing where it reduces the treatment time, accelerates tissue repair, and promotes healing [17,18]. The method is based on using a photosensitizer (PS), which accumulates in tissues, followed by irradiation of the tissue with a light source with an appropriate wavelength. The latter causes the formation of reactive oxygen species (ROS) [19,20]. Low concentrations of ROS can trigger cell repair processes including proliferation and offer promising treatments to accelerate healing. Different PSs have been studied in the wound healing process, which has a relevant role in ensuring PDT effectiveness in skin wound healing [21] such as 5-aminolevulinic acid (5-ALA) and methylene blue (MB) [22–25]. MB is a popular PS among the phenothiazinium derivatives that have attracted the attention of different research groups working and achieving good results in wound healing [25–27].

**Citation:** Zuhayri, H.; Nikolaev, V.V.; Lepekhina, T.B.; Sandykova, E.A.; Krivova, N.A.; Kistenev, Y.V. The In Vivo Quantitative Assessment of the Effectiveness of Low-Dose Photodynamic Therapy on Wound Healing Using Optical Coherence Tomography. *Pharmaceutics* **2022**, *14*, 399. https://doi.org/10.3390/ pharmaceutics14020399

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 18 December 2021 Accepted: 9 February 2022 Published: 11 February 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/).

Recently, MB was shown to have an antioxidant role [28]. Additionally, 5-ALA is among the most effective photosensitizers and is widely used to present a better achievement concerning wound healing [17,22,29].

The Arndt–Schultz Law is an appropriate model to demonstrate that low levels of light have a better effect in wound healing than higher levels, which may have an inhibitory or cytotoxic effect [30,31]. Hawkins and Abrahamse studied the behavior in vitro of human skin fibroblasts using different irradiation doses of 0.5, 2.5, 5, 10, and 16 J/cm2 . They demonstrated that higher laser doses (10 and 16 J/cm2 ) resulted in increased cellular damage as well as decreased cell viability and proliferation [32]. Results for different energy doses were described for 4 J/cm2 [33] and for 1 J/cm2 and 2 J/cm2 [34]. Basso et al. demonstrated that irradiation of cultured human gingival fibroblasts with energy doses of 0.5 and 3 J/cm2 resulted in a significant increase in cellular metabolism compared with the non-irradiated control group and the cells irradiated with higher energy doses of 5 and 7 J/cm2 [35]. The most significant biological effects were seen with predominant dose values (i.e., up to 5 J/cm2 ), which were within the Arndt–Schultz curve [36].

Traditionally, wounds have been observed invasively with a histochemical assessment of the biopsies [3,8]. Visual observation is a common tool for wound assessment. Additionally, clinical wound evaluation is a widely used and the least expensive method of assessing wound depth. This method relies on a subjective evaluation of the external features of the wound such as wound appearance, capillary refill, and burn wound sensibility to touch and pinprick, providing diagnostic accuracy at the level of 60–75% [37]. These methods are not quantitative and can lead to additional tissue damage and impair healing. Accordingly, the development of noninvasive and accurate methods of wound analysis is relevant.

Optical coherence tomography (OCT) is a noninvasive 3D imaging method of biological tissues with a spatial resolution of 5–10 µm and a penetration depth of 1–2 mm [38,39]. Epidermal thickness is a critical parameter for assessing epithelialization during wound healing [40,41].

OCT could detect essential morphological changes during wound healing (e.g., epidermis, dermis, adipose tissue, and granulation) that was based primarily on their backscattering characteristics [42–44]. The use of polarization-sensitive OCT revealed higher birefringence in scars compared to healthy skin [45]. OCT-based angiography provides in vivo, three-dimensional vascular information by using flowing red blood cells as intrinsic contrast agents, allowing visualization of functional vessel networks within microcirculatory tissue beds non-invasively, without needing dye injection [46].

This work aims to develop a method for quantitative in vivo evaluation of wounds using OCT during the wound healing process including a quantitative assessment of the effect of LDPDT using the topical application of two different photosensitizers (5-ALA and MB) and two laser doses of 1 J/cm2 and 4 J/cm2 .

#### **2. Materials and Methods**

#### *2.1. Wound Model Protocol*

This study used 15 male laboratory CD1 mice, weighing 25–30 g and aged 6–7 weeks, obtained from the Department of Experimental Biological Models of the Research Institute of Pharmacology, TSC SB RAMS. Before the experiment, the mice were kept seven days in the standard conditions of a conventional vivarium with free access to water and food, and a 12/12 light regime, in a ventilated room at a temperature of 20 ± 2 ◦C and a humidity of 60%. The experimental protocol of this research was approved by the Bioethical Committee of Tomsk State University (Protocol No. 4, 10.02.2021), registration No. 6.

The mice were anesthetized by isoflurane using the Ugo Basile gas anesthesia system, where the mice were put in a glass chamber connected to isoflurane (Figure 1). The wound area was prepared through depilation using Veet cream (made in France), rinsed with saline solution, and sterilized using chlorhexidine 20%. A full-thickness cutaneous wound (diameter 5 mm) was formed by cutting out a whole layer skin flap with scissors on both of the hind paws of each animal under isoflurane anesthesia. The experiment was performed

in a time-lapsed schedule for the wound aging on days 1, 3, 7, and 14. Additionally, the day of wound formation was defined as day 0. performed in a time-lapsed schedule for the wound aging on days 1, 3, 7, and 14. Additionally, the day of wound formation was defined as day 0.

wound area was prepared through depilation using Veet cream (made in France), rinsed with saline solution, and sterilized using chlorhexidine 20%. A full-thickness cutaneous wound (diameter 5 mm) was formed by cutting out a whole layer skin flap with scissors on both of the hind paws of each animal under isoflurane anesthesia. The experiment was

*Pharmaceutics* **2022**, *14*, x 3 of 13

**Figure 1.** Anesthetized mice by isoflurane using the Ugo Basile gas anesthesia system. **Figure 1.** Anesthetized mice by isoflurane using the Ugo Basile gas anesthesia system.

#### *2.2. Low Dose Photodynamic Therapy Protocol*

*2.2. Low Dose Photodynamic Therapy Protocol*  Both of the photosensitizers 5-ALA 20% and MB 0.01% in saline solution were topically administered directly on the wound; after 30 min, the irradiation was started by an AlGalnP laser (λ = 630 nm, P = 5 mW) with two doses: 1 J/cm2 and 4 J/cm2, and the procedure was carried out under the influence of isoflurane. 5-ALA was applied on the wounds on the right hind paws, MB was applied on the left ones. The animals were divided according to the laser dose and photosensitizer into five groups: the control group, Both of the photosensitizers 5-ALA 20% and MB 0.01% in saline solution were topically administered directly on the wound; after 30 min, the irradiation was started by an AlGalnP laser (λ = 630 nm, P = 5 mW) with two doses: 1 J/cm2 and 4 J/cm2 , and the procedure was carried out under the influence of isoflurane. 5-ALA was applied on the wounds on the right hind paws, MB was applied on the left ones. The animals were divided according to the laser dose and photosensitizer into five groups: the control group, the LDPDT–5-ALA 1 J/cm2 , LDPDT–MB 1 J/cm2 , LDPDT–5-ALA 4 J/cm2 , and LDPDT–MB 4 J/cm2 . LDPDT procedure was repeated once immediately after wound formation.

#### the LDPDT–5-ALA 1 J/cm2, LDPDT–MB 1 J/cm2, LDPDT–5-ALA 4 J/cm2, and LDPDT–MB *2.3. Optical Coherence Tomography Protocol*

4 J/cm2. LDPDT procedure was repeated once immediately after wound formation. *2.3. Optical Coherence Tomography Protocol*  The experiments were carried out using optical coherence tomography (OCT) on the GANYMEDE−II system (Thorlabs, USA) with the basic scanning module OCTG-900. It is possible to obtain information on the optical characteristics, morphology, and elastic properties of biological tissues using OCT. The GANYMEDE-II system uses a superluminescent diode with an operating wavelength of 930 ± 50 nm. The superluminescent diode allows one to reach a signal penetration depth up to 2.9 mm with an axial resolu-The experiments were carried out using optical coherence tomography (OCT) on the GANYMEDE−II system (Thorlabs, USA) with the basic scanning module OCTG-900. It is possible to obtain information on the optical characteristics, morphology, and elastic properties of biological tissues using OCT. The GANYMEDE-II system uses a superluminescent diode with an operating wavelength of 930 ± 50 nm. The superluminescent diode allows one to reach a signal penetration depth up to 2.9 mm with an axial resolution of up to 6.0 microns (air/tissue). The width of the spectral band was 100 nm. Figure 2 shows an example of placing a mouse on the substrate of OCT. As a result, B-scans were obtained—two-dimensional images. Data processing was carried out using ThorImageOCT 5.0.1., with the following parameters: size 2469 × 675 pixel, FOV 4.66 × 1.94 mm, and pixel size 1.89 × 2.88 µm, with 20 frames. The experiment was repeated with a 30◦ rotation around the previous position.

tion of up to 6.0 microns (air/tissue). The width of the spectral band was 100 nm. Figure 2 shows an example of placing a mouse on the substrate of OCT. As a result, B-scans were obtained—two-dimensional images. Data processing was carried out using ThorImageOCT 5.0.1., with the following parameters: size 2469 × 675 pixel, FOV 4.66 × 1.94 mm, and pixel size 1.89 × 2.88 µm, with 20 frames. The experiment was repeated with a 30°

rotation around the previous position.

**Figure 2.** The mouse positioning during OCT imaging. **Figure 2.** The mouse positioning during OCT imaging.

#### *2.4. Statistical Analysis*

*2.4. Statistical Analysis*  The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically sig-The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically significant differences among groups under study. Statistical power was used at 0.95 and 0.99. The *p*-values were calculated for all groups on all days.

#### nificant differences among groups under study. Statistical power was used at 0.95 and **3. Results**

#### 0.99. The *p*-values were calculated for all groups on all days. *3.1. OCT Imaging*

**3. Results**  *3.1. OCT Imaging*  Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was lo-Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was located at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4. The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm.

cated at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4. Photos of the observation area and B-scans of the wound at different time points on days 1, 3, 7, and 14 for the control (without LDPDT) are shown in Figure 5.

The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm. The wound healed typically without pathologies, and the injury was close to healing on day 14. The difference in signal intensities at different stages of wound healing on different days is shown in Figure 6. On day 1 after the wound forming procedure, the signal had a low intensity, while the signal of the dermis started decreasing from 0.3 µm, so the signal from 0 to 0.3 corresponded to the formed wound scab. The signal intensity increased on days 3 and 7. On day 14, the signal intensity values were close to healthy skin.

OCT images for the LDPDT-5-ALA groups are shown for a laser dose of 1 J/cm2 in Figure 7 and 4 J/cm2 in Figure 8. The intensity signal for LDPDT-5-ALA 4 J/cm2 had the same behavior as the control. On day 1 for LDPDT-5-ALA 1 J/cm2 , the attenuation signal corresponding to the dermis started from ~0.3 mm. Similar to the control, the signal

**Figure 3.** (**a**) Visual observation for healthy skin, (**b**) OCT imaging.

(**a**) (**b**)

(**a**)

(**b**)

intensity increased on days 3 and 7, and on day 14, the signal intensity values were close to healthy skin, as shown in Figure 9a. In the same way for LDPDT-5-ALA 4 J/cm2 , the intensity signal started decreasing from ~0.2 mm on day 1. On days 3 and 7, the signal intensity values increased to close to the value of healthy skin on day 14 more than the LDPDT-5-ALA 1 J/cm2 group, as shown in Figure 9b. Figure 3a shows a photo of healthy skin, and an OCT B-scan for the area, marked with a red arrow, is shown in Figure 3b. The B-scan data were normalized so that the stratum corneum, corresponding to the region with the highest pixel intensity, was located at the top of the image. After normalization, the signal intensity was calculated at different tissue depths from 0 to 0.8 mm (A-scan) and visualized as shown in Figure 4.

The OCT data were exported using the ThorImageOCT 5.0.1 program to files. txt. Statistical analysis and data processing were carried out in Python 3.6 using libraries (numpy, scipy, matplotlib). All calculated parameters were expressed as the mean ± standard deviation. The Pearson test was applied to assess the level of statistically significant differences among groups under study. Statistical power was used at 0.95 and

Measurements were similarly carried out for the MB photosensitizer with two laser doses of 1 J/cm2 (Figure 10) and 4 J/cm2 (Figure 11). The intensity signal for LDPDT-MB on day 14 was close to the values for healthy skin for different exposure doses, as shown in Figure 12. The maximum intensity values were at a depth of 0 to 2–4 µm, which corresponded to the stratum corneum, and then the signal intensity gradually decreased to ~20 µm, which corresponded to the epidermis. The dermis starts from 30–40 µm, which was accompanied by a decrease in intensity to the minimum values at a depth of 0.8 mm.

**Figure 3. Figure 3.** (**a** (**a**) Visual observation for healthy skin, ( ) Visual observation for healthy skin, ( **b**) OCT imaging. **b**) OCT imaging.

*Pharmaceutics* **2022**, *14*, x 4 of 13

**Figure 2.** The mouse positioning during OCT imaging.

0.99. The *p*-values were calculated for all groups on all days.

*2.4. Statistical Analysis*

**3. Results**

*3.1. OCT Imaging*

**Figure 4.** Dependence of signal intensity on depth for healthy skin: S1 start point of the stratum corneum; S2—the endpoint of epidermis; S3—start point of the dermis. **Figure 4.** Dependence of signal intensity on depth for healthy skin: S1 start point of the stratum corneum; S2—the endpoint of epidermis; S3—start point of the dermis.

days 1, 3, 7, and 14 for the control (without LDPDT) are shown in Figure 5.

skin.

 **Figure 5.** (**a**) Visual observation and (**b**) the corresponding B-scans for the control group.

The wound healed typically without pathologies, and the injury was close to healing

on day 14. The difference in signal intensities at different stages of wound healing on different days is shown in Figure 6. On day 1 after the wound forming procedure, the signal had a low intensity, while the signal of the dermis started decreasing from 0.3 µm, so the signal from 0 to 0.3 corresponded to the formed wound scab. The signal intensity increased on days 3 and 7. On day 14, the signal intensity values were close to healthy

Photos of the observation area and B-scans of the wound at different time points on

**Figure 5.** (**a**) Visual observation and (**b**) the corresponding B-scans for the control group.

**Figure 6.** Dependence of signal intensity on depth for the control group during wound healing.

**Figure 7.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-5-ALA 1 J/cm2 group.

**Figure 8.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-5-ALA 4 J/cm2 group.

**Figure 9.** Dependence of signal intensity on depth for the (**a**) LDPDT-5-ALA 1 J/cm2 group and (**b**) LDPDT-5-ALA 4 J/cm2 group.

**Figure 10.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-MB 1 J/cm2 group.

**Figure 11.** (**a**) Visual observation and (**b**) the corresponding B-scans for the LDPDT-MB 4 J/cm2 group.

**Figure 12.** Dependence of signal intensity on depth for the (**a**) LDPDT-MB 1 J/cm2 group and (**b**) LDPDT-MB 4 J/cm2 group.

#### *3.2. Quantitative Comparison of the Spatial Proximity of the OCT Signal Intensity*

For a quantitative comparison of spatial profiles, we used the curve proximity factor (CPF) *S*, similar to the Pearson's correlation coefficient, to compare healthy skin and wound curves in all days to all groups [47]:

$$S = \frac{\sum\_{i} |X\_{i} - Y\_{i}|}{\frac{1}{2} \sum\_{i} |X\_{i} + Y\_{i}|},\tag{1}$$

where *Xi* , *Yi* is the intensity of the OCT signal for a definite depth from the wound and healthy skin, respectively. The higher the CPF value, the closer the wound state to healthy skin. The 0.01 and 0.05 significance levels were used to assess the statistical differences between the wound and healthy skin groups. The CPF values for all groups are shown in Table 1.

**Table 1.** CPF values for the studied groups (mean ± standard deviation).

\* *p* > 0.01,\*\* *p* > 0.05.

The CPF (1) was also used to estimate the effectiveness of different laser doses for each photosensitizer on day 14. The CPF values calculated for the OCT signal intensity curves corresponding to 4 J/cm2 and 1 J/cm2 for 5-ALA (Figure 9) and MB (Figure 12) are shown in Table 2. These quantitative estimations demonstrated a "proximity" between the curves corresponding to 4 J/cm2 laser dose and the curves corresponding to the 1 J/cm2 laser dose for the same photosensitizer.

**Table 2.** CPF values for the studied groups (mean ± standard deviation).

## **4. Discussion**

The proposed method of wound state quantitative evaluation is based on the OCT visualization of tissue structure transformation. The averaged scatter A-line intensity profile obtained from the horizontal rectangle in the OCT B-scan image of healthy skin is shown in Figure 4. Three areas are highlighted in the figure, representing changes in the attenuation coefficient. The red (S1), green (S2), and blue (S3) lines correspond to the beginning of the stratum corneum, the end of the epidermis, and the beginning of the dermis, respectively. After inflicting a wound in the first days, there are no surface layers of the skin (horny, epidermis); instead, a scab forms on the surface. These changes in the skin are visible on A-scans. Over time, the skin recovers, and on A-scans, we can see the appearance of areas characteristic of the epidermis's end and the dermis's beginning. These changes are reflected in the OCT signal attenuation curve (see Figures 6, 8, 9 and 12).

For wound state quantitative estimation, we used the curve proximity factor, introduced by us earlier [47]. According to Table 1, in the control group, the CPF mean value on day 1 was about 0.053, and decreased to 0.021 on day 14, while in the LDPDT groups, the mean values of this coefficient on day 14 ranged from 0.017 to 0.021. The Pearson test demonstrated that for LDPDT groups on day 14, *p*-value did not exceed 0.01, while for the control group, this value was equal to 0.03. Therefore, for all LPDT groups, the wound state had no statistically significant difference compared to healthy skin for the used statistical power levels.

The CPF value for LDPDT groups for the 4 J/cm2 laser dose was smaller than the LDPDT groups for 1 J/cm2 . We also calculated the CPF values for the OCT signal intensity curves corresponding to 4 J/cm2 and 1 J/cm2 for 5-ALA (the first column in Table 1) and MB (the second column in Table 2) and conducted a Pearson test to check the statistical significance of these differences. *p*-value was shown to be larger for 5-ALA.

Therefore, after comparing the CPF parameter for five groups: control, LDPDT 5-ALA 4 J/cm2 , LDPDT 5-ALA 1 J/cm2 , LDPDT-MB 4 J/cm2, and LDPDT-MB 1 J/cm2 , it was concluded that the laser dose of 4 J/cm2 for LDPDT 5-ALA was definitely better compared to 1 J/cm2 and probably better for LDPDT MB. It should be noted that the conclusion depends on the volume and quality of the dataset.

A possible reason for the 4 J/cm2 dose preference relative to the 1 J/cm2 dose is as follows. According to previous works [33–35], when low-level laser light is applied and a dose is too low, no tissue response will occur. If too a high dose is applied, it can inhibit a tissue response. It has been seen in studies of wound healing where too low a dose did not have an impact, and too high a dose (above 5 J/cm2 ) prolonged wound healing while the optimal dose resulted in faster healing. In this interval, according to the Arndt–Schultz curve, the larger dose causes a stronger biological effect.

In any case, LDPDT allows for accelerating of the wound healing process, which is consistent with the literature data [32–34,48,49].

#### **5. Conclusions**

In this paper, a study of quantitative in vivo evaluation of wounds using OCT during the wound healing process was carried out. 5-ALA and MB were used as photosensitizers for LDPDT, with two laser doses of 1 and 4 J/cm2 .

An approach to quantitative estimation of wound state based on the CPF, Equation (1) [47] was proposed. The method was used to quantify the effectiveness of LDPDT to accelerate the wound healing process. CPF parameter estimation allowed us to compare LDPDT regimes quantitatively and to obtain objective arguments about the superiority of one regime over another.

Therefore, the proposed CPF parameter, estimated from OCT data, has demonstrated its feasibility for the quantitative estimation of the human wound state during healing. This approach is noninvasive, simple in implementation, and suitable for continuous monitoring throughout the wound healing process and sufficient resolution to assess both anatomy and pathology. It makes it a promising technique for applications in wound healing and the evaluation of novel therapeutics.

Another approach, which was proven to be effective in monitoring wound healing is two-photon microscopy. Previously, our group analyzed the two-photon microscopy images of the wound healing process and succeeded in quantitatively assessing the state of the wound and studying the effect of low-dose photodynamic therapy using the techniques of two-photon microscopy. The results of this study are completely consistent with the results obtained earlier [50].

**Author Contributions:** Conceptualization, Y.V.K.; Methodology, H.Z. and E.A.S.; Software, V.V.N.; Validation, H.Z. and V.V.N.; Investigation, H.Z., V.V.N., T.B.L. and N.A.K.; Writing—original draft preparation, E.A.S.; Writing—review and editing, Y.V.K.; Visualization, H.Z.; Supervision, Y.V.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by a grant under the Decree of the Government of the Russian Federation No. 220 of 9 April 2010 (Agreement No. 075-15-2021-615 of 4 June 2021).

**Institutional Review Board Statement:** The study was approved by the Ethics Committee of Tomsk State University (Protocol No.4, 10.02.2021), registration No. 6.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

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

#### **References**

## *Article* **In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on** *Candida* **spp.**

**Vanesa Pérez-Laguna 1,\* , Yolanda Barrena-López 2 , Yolanda Gilaberte 3,4,† and Antonio Rezusta 2,3,5,†**

**Citation:** Pérez-Laguna, V.; Barrena-López, Y.; Gilaberte, Y.; Rezusta, A. In Vitro Effect of Photodynamic Therapy with Different Lights and Combined or Uncombined with Chlorhexidine on *Candida* spp. *Pharmaceutics* **2021**, *13*, 1176. https://doi.org/10.3390/ pharmaceutics13081176

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis and Gerhard Litscher

Received: 18 July 2021 Accepted: 28 July 2021 Published: 30 July 2021

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

**Copyright:** © 2021 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/).

**Abstract:** Candidiasis is very common and complicated to treat in some cases due to increased resistance to antifungals. Antimicrobial photodynamic therapy (aPDT) is a promising alternative treatment. It is based on the principle that light of a specific wavelength activates a photosensitizer molecule resulting in the generation of reactive oxygen species that are able to kill pathogens. The aim here is the in vitro photoinactivation of three strains of *Candida* spp., *Candida albicans* ATCC 10231, *Candida parapsilosis* ATCC 22019 and *Candida krusei* ATCC 6258, using aPDT with different sources of irradiation and the photosensitizer methylene blue (MB), alone or in combination with chlorhexidine (CHX). Irradiation was carried out at a fluence of 18 J/cm2 with a light-emitting diode (LED) lamp emitting in red (625 nm) or a white metal halide lamp (WMH) that emits at broad-spectrum white light (420–700 nm). After the photodynamic treatment, the antimicrobial effect is evaluated by counting colony forming units (CFU). MB-aPDT produces a 6 log10 reduction in the number of CFU/100 µL of *Candida* spp., and the combination with CHX enhances the effect of photoinactivation (effect achieved with lower concentration of MB). Both lamps have similar efficiencies, but the WMH lamp is slightly more efficient. This work opens the doors to a possible clinical application of the combination for resistant or persistent forms of Candida infections.

**Keywords:** candidiasis; *C. albicans*; antimicrobial photodynamic therapy; methylene blue

#### **1. Introduction**

*Candida* spp. are commensal fungal species commonly colonizing human mucosal and skin surfaces, but they may become pathogenic in some particular scenarios such as treatment with antibiotics, immunocompromised patients, etc., producing in these cases infections that range from superficial to severe skin and mucosal lesions, to even systemic invasion at its worst degree [1]. For example, oral candidiasis is the most common opportunistic infection affecting the human oral cavity. It is caused by an overgrowth of *Candida* spp., being the most prevalent *Candida albicans* [2,3].

Due to the recurrence of *Candida* spp. infections, high systemic antifungal therapy have been widely used, thereby antifungal resistances are increasing. Moreover, patientdependent, interactions with other medical regimens and organ toxicity can happen [4].

Therefore, it is necessary to develop new treatments such as antimicrobial photodynamic therapy (aPDT). It is based on the use of photosensitizing molecules that are excited with visible light of the appropriate wavelength and reacts with the oxygen, generating reactive species of oxygen to destroy the target pathogen [5–7].

Superficial wound infections are potentially suitable for treatment by aPDT because of the ready accessibility of these wounds for both topical delivery of the photosensitizer and light, and because of the exposure to oxygen [6,8,9].

Different aPDT studies have demonstrated that *Candida* spp. can be effectively photoinactivated in vitro and in vivo [5,10–13].

Future directions of aPDT include the combination with antimicrobials in order to enhance the microbial inactivation and prevent the regrowth when the light from aPDT is turned off and the photoinactivation ends. This original approach has already shown significant potential. It could help to implement the use of aPDT and reduce the amount of antimicrobials used and, thus, the multidrug resistance problem [14–16].

Chlorhexidine (CHX) is an antiseptic drug, mainly available in over-the-counter products as routine hand hygiene in healthcare personnel, to clean and prepare the skin before surgery, and before injections in order to help reduce the amount of microorganisms that potentially can cause skin infections [17–20]. CHX gluconate is also available as a prescription mouthwash to treat gingivitis and as a prescription oral chip to treat periodontal disease [21–23] and recently against COVID-19 in dentistry [24].

Here, we investigate the aPDT and the CHX uncombined or in combination against *Candida* spp. As a photosensitizing molecule, we use methylene blue (MB), the main member of the phenothiazine family, well known for its ability to produce singlet oxygen when it is irradiated by red light and react with molecular oxygen [6,8]. As a source of irradiation, we use a light-emitting diode (LED) lamp emitting in red or a white metal halide lamp (WMH) that emits at broad-spectrum white light which is comparable to the emission spectrum of daylight.

The aim is to compare the antimicrobial effect of MB-aPDT when a specific irradiation source or a non-specific broad-spectrum source is used to excite different concentrations of MB. Furthermore, the effects of the combination of aPDT with CHX are evaluated.

#### **2. Materials and Methods**

The procedure used tried to follow the materials and methods of our previous works and was adapted as follows [25–27]:

#### *2.1. Chemicals, Media, Strains and Light Sources*

fluence corresponds to a 42.86 min (≈43 min) irradiation time for the samples using the red-LED lamp and 3 min and 25 sec for the samples irradiated with WMH lamp.

#### *2.2. In Vitro Photodynamic Treatment of Yeast Suspension*

*C. albicans*, *C. parapsilosis* or *C. krusei* seeded on Sabouraud dextrose agar were cultured aerobically overnight at 35 ◦C. The inoculum was prepared in distilled water and adjusted to 5 ± 0.03 on the McFarland scale (concentrations in the range of >1 × 106 colony-forming units (CFU) per 100 µL and was deposited into 96-well microtiter plates. Two-fold serial dilutions concentrations from 640 µg/mL to 0.03 µg/mL of the MB were added, in absence or presence of 10 µg/mL of CHX (MB+/CHX−/light+) (MB+/CHX+/light+). The final volume in each well was 100 µL. Irradiation proceeded with no preincubation period; the suspensions were immediately subjected to irradiation with fluence of 18 J/cm2 using the red-LED lamp or the broad spectrum-WMH lamp. Control samples were subjected to identical treatment, in the absence or presence of the photosensitizer, and were either kept in darkness or irradiated to evaluate the effect of each parameter: negative or initial control (MB−/CHX−/light−), irradiation control (MB−/CHX−/light+), control of photosensitizer in darkness (MB+/CHX−/light−) and antiseptic controls (MB−/CHX+/light−) (MB−/CHX+/light+). After completing the aPDT protocol, samples and controls were assessed in serial dilutions of each suspension and were cultured on blood agar and incubated overnight at 35 ◦C. The dilutions were made and aliquots were cultured to have blood agar plates with a number of CFUs in the range of 0 to 200 per plate in order to be able to count them reliably.

#### *2.3. Efficacy*

The efficacy of aPDT treatment was assessed by counting the number of CFU/100 µL using a Flash and Go automatic colony counter (IUL S.A., Barcelona, Spain). A reduction of 6 log10 in the number of CFU/100 µL was considered indicative of fungicidal activity. The minimum concentration of MB that reduced yeast survival by 3 log10 was also evaluated. All experiments were carried out at least five times. The results are expressed as mean and standard deviation.

#### **3. Results**

## *3.1. Photoinactivation of Yeasts by MB-aPDT (MB+/CHX*−*/Light+)*

MB-aPDT effectively inactivated *Candida* spp. achieving a reduction of 6 log10 in the number of CFU/100 µL in all the studied strains (Figure 1). The minimum concentration of MB required to achieve this effect was 320 µg/mL in all cases except in those irradiated with a WMH lamp in *C. parapsilosis* that required 80 µg/mL and in *C. krusei* between 320–640 µg/mL (Table 1). Analyzing in more detail the sensitivity of each strain to MBaPDT, *C. krusei* is the most resistant and *C. parapsilosis* and *C. albicans* show a very similar ratio of response, although *C. parapsilosis* is slightly more sensitive to white light than *C. albicans* (Figure 1 and Supplementary Material Figure S2I).

**Figure 1.** Photoinactivation by antimicrobial photodynamic therapy with methylene blue alone or in combination with chlorhexidine of *C. albicans* (**A**,**D**) *C. parapsilosis* (**B**,**E**) and *C. krusei* (**C**,**F**) using the 625 nm lamp-LED (**A**–**C**) or the WMH lamp (**D**–**F**). The error bars represent the standard deviation calculated for five measurements. C0, initial inoculum control; **Figure 1.** Photoinactivation by antimicrobial photodynamic therapy with methylene blue alone or in combination with chlorhexidine of *C. albicans* (**A**,**D**) *C. parapsilosis* (**B**,**E**) and *C. krusei* (**C**,**F**) using the 625 nm lamp-LED (**A**–**C**) or the WMH lamp (**D**–**F**). The error bars represent the standard deviation calculated for five measurements. C0 , initial inoculum control; CHX, chlorhexidine; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.

CHX, chlorhexidine; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.

**Table 1.** Minimum concentrations of methylene blue (µg/mL) required to reduce the number of *Candida* spp. 3 or 6 log10 in the number of colony forming units using the 625 nm-LED lamp or the WMH lamp.

aPDT: antimicrobial photodynamic therapy; CHX: chlorhexidine; CFU: colony forming units; LED: light-emitting diodes; MB: methylene blue; WMH: white metal halide.

#### *3.2. Fungicidal Effect of MB-aPDT Combined with CHX (MB+/CHX+/Light+)*

The antimicrobial effect of MB-aPDT on *Candida* spp. was maintained in the presence of CHX, as evidenced by the 6 log10 reduction in the number of CFU/100 µL in all experiments. Moreover, the combination of MB-aPDT using the WMH lamp + CHX achieves this degree of reduction on *C. albicans* decreasing 4-fold the required photosensitizer concentration (the necessary concentration is 1/4) (Figure 1 and Table 1).

To achieve a 3 log10 reduction in the number of CFU/100 µL when MB-aPDT is used in combination with CHX, the required concentration of MB needed is at least half compared to the concentration needed using MB-aPDT alone. The greatest reduction of the photosensitizer concentration (8-fold) is achieved against *C. albicans* using the red-LED lamp. On the other hand, the greatest reduction against *C. parapsilosis* occurs with the WMH irradiation (1/4–1/8 of the initial photosensitizer concentration) (Figure 1 and Table 1).

## *3.3. Control of Inoculum and Toxic Effects of MB (MB+/CHX*−*/Light*−*), CHX (MB*−*/CHX+/Light*−*) and Irradiation (MB*−*/CHX*−*/Light+)*

No reduction in the number of CFU/100 µL from the initial inoculums (MB−/CHX−/ light−) was observed.

Samples with the different MB concentrations evaluated under the same conditions used in irradiation but keeping it in darkness (MB+/CHX−/light−) (dark MB in Figure 1) show significant reductions at the highest concentrations tested as follows: reductions of up to a maximum of 3.5 log10 in *C. albicans*, 4 log10 in *C. parapsilosis* and 4.5 log10 in *C. krusei* were achieved by 640 µg/mL of MB. In all experiments, the effects of keeping the microbial suspension with the different MB concentrations in dark (light−) for 43 min or 3 min and 25 sec (using the time of the irradiation with the red-LED or the WMH lamp respectively) is similar, except for *C. krusei* (reduction of 4.5 log10 after 43 min vs. 2.5 log10 after 3 min and 25 sec) (Figure 1).

The irradiation with the red-LED lamp or with WMH lamp in the absence of photosensitizer and antimicrobial (MB−/CHX−/light+) did no significantly reduce the number of yeasts (reduction ≤0.3 log10, Figure 1).

In the absence of photosensitizer and irradiation, the tested concentration of CHX (10 µg/mL) (MB−/CHX+/light−) (dark MB-CHX with the value of 0 MB concentration in Figure 1) failed to effectively inactivate the yeast. A maximum reduction of 1 log10 was observed against *C. parapsilosis.*

The cumulative effect of CHX and irradiation (MB−/CHX+/light+) (Figure 1) reaches a maximum reduction in the number of CFU/100 µL of 1.3 log10 against *C. parapsilosis* being the most sensitive strain to this effect.

#### **4. Discussion**

MB-aPDT is effective in eradicating *Candida* spp. (>6 log10 reduction in the number of CFU/100 µL of *C. albicans*, *C. parapsilosis* or *C. krusei*) and the combination with CHX enhances the photoinactivation, i.e., the effect is achieved with lower aPDT-dose (Figure 1 and Table 1).

Regarding the comparison of MB-aPDT results obtained with those reported by other authors, many variables should be considered. Table 2 summarizes different studies against *Candida* spp. in suspension, specifying the methodology and results. Daliri et al. reported a reduction of 3.43 log10 of *C. albicans* using 200 µg/mL of MB which is notably lower than the one reached in the present study (MB concentration range of 80–160 µg/mL is able to inhibit 4 log10). They used a bigger number of CFU in the inoculum and this could affect but the mismatch may be because they use a laser irradiation source [28]. Application times are usually short when lasers are used and it does not always guarantee adequate oxygenation [6]. Valkov et al. report the absence of effect of MB-aPDT using very low MB concentration (<2 µg/mL) [13]. Ferreira et al. and de Oliveira-Silva et al. achieved different reductions of *C. albicans*, 0.5 log10 and 6 log10 respectively, with 32 µg/mL of MB and a fluence of 30 J/cm2 for 3–4 min. This shows the variability between experiments [29,30]. The results of Ferreira et al. are closer to those of this work (32 µg/mL of MB does not produce complete photoinactivation) (Table 2).

Focusing on *C. parapsilosis*, Güzel Tunçcan et al. achieved a reduction of 4 log10 with 25 µg/mL of MB. The comparison with our data and the possible explanation is very difficult because the methodology used is dissimilar [31]. Cern ˇ áková et al. demonstrated that using 9.6 µg/mL of MB inhibited between 1.13–1.27 log10 of *C. parapsilosis* in suspension, similar results to this work (this concentration does not generate complete photoinactivation) [32]. Finally, Ahmed et al. used 100 µg/mL of MB and achieved reductions of 0.58–0.85 log10 at fluences of 90–180 J/cm2 respectively [33]. Again, the difference may be due to the fact that they used a laser irradiation source and therefore it would be less effective (Table 2).

Against *C. krusei*, concentrations of 16 µg/mL [34] or150 µg/mL of MB [35] even at high fluences only achieves a maximum reduction of 0.65 log10. More similar result to ours was obtained by Souza et al. using 100 µg/mL with a reduction of 1.54 log10 using a fluence of 28 J/cm2 [36]. All MB-aPDT studies together lead us to conclude that *C. krusei* is the most resistant *Candida* spp. to MB-aPDT as well as it is more resistant to antifungals in general mainly due to the characteristics of its membrane [37] (Table 2).

Regarding the MB-aPDT combination with CHX, it stands out that >6 log10 reduction in the number of CFU/100 µL of *C. albicans* was achieved reducing the concentration of photosensitizer needed from 320 to 80 µg/mL when WMH lamp was used (Figure 1, Table 1 and Supplementary Figure S2). Furthermore, the addition of CHX halved the concentration of MB required to reach a reduction of 3 log10 in *C. albicans* and *C parapsilosis*, and slightly less than half against *C. krusei*. Therefore, a synergistic effect is seen between MB at concentrations unable to achieve complete photoinactivation and CHX. These results are relevant because the presence of CHX could help to avoid the microbial regrowth of those microorganisms not completely destroyed when PDT is finished. This is one of the disadvantages of using aPDT for infections in the clinic, the risk of microbial regrowth after its application. The combination with antimicrobials could play a crucial role to overcome this limitation of aPDT in this context [14,15].

To our knowledge, there are not studies combining aPDT plus CHX in vivo against *Candida* spp. Recently, the effectiveness of MB-aPDT combined with CHX and zinc oxide ointment has been studied on wound healing process after rumenostomy. This study in cattle ratifies the use of aPDT and suggests that it could be performed for other surgical procedures as a complementary approach or an alternative for topical administration of antibiotics [38]. The combination of CHX plus aPDT has been tried against other microorganisms such as *Porphyromonas gingivalis* biofilm on a titanium surface in a dental framework. The application of CHX and subsequent aPDT using toluidine blue O was

shown to be an efficient method to reduce *P. gingivalis* in titanium surfaces [39]. Regarding other studies of antimicrobials plus aPDT against *Candida* spp., Giroldo et al. demonstrated that yeasts, both in suspension and in biofilms, were much more susceptible to antifungal treatments after MB-aPDT, explained by the increase of membrane permeability caused by aPDT [40]. Regarding the in vitro combination of MB-aPDT with fluconazole against resistant strains of *C. albicans, C. glabrata* and *C. krusei*, a synergistic effect was found in fluconazole resistant strains of *C. albicans* and *C. glabrata*, but not against *C. krusei*. [34]. These results do not agree with those found by Snell et al. They showed that fluconazole did not increase the aPDT inactivation of *C. albicans* using MB or another photosensitizer of the protoporphyrin family. However, miconazole did enhance the fungicidal activity of aPDT [41]. Moreover, to our knowledge, there are no studies using aPDT in combination with antimicrobials that report antagonistic effects, which support the use of aPDT in combination with CXH due to the possible advantages [15].

Considering clinical practice, MB-aPDT (660 nm and 7.5 J/cm2 ) has been tried in HIV patients diagnosed with oral candidiasis comparing it with an antifungal commonly used in candidiasis. After 30 days, the antimicrobial was effective, but there were recurrences except when 450 µg/mL of MB was used [42].

All together indicates that aPDT or antimicrobial alone may not be entirely effective against *Candida* spp. that is characterized for causing highly recurrent infection especially in predisposed or immunosuppressed patients. On the other hand, combined treatments such as aPDT plus antimicrobials may prevent recurrent infections and avoid resistance. In addition, the combination in terms of clinical application would decrease the intensity of blue staining caused when the MB is applied on the skin or mucous membranes, making the aPDT procedure more cosmetically appealing.

Regarding the concentration of 10 µg/mL CHX used for this study, it was chosen by taking into account other protocols for the combination of antimicrobials plus aPDT and considering that by itself produces no effect under experimental conditions [25,27], Figure 1 C0-CHX. Other studies using other conditions report very different results: e.g., Azizi et al. using 1000 µg/mL of CHX achieved a reduction of 0.71 log10 [43]; Do Vale et al. calculated that the minimum inhibitory concentration for *C. albicans* was 3.74 µg/mL using an exposure time of 12–48 h [44]; Ellepola et al. proved that with 50 µg/mL of CHX applied for 30 min achieved 0.38 and 0.5 log10 of *C. albicans* and *C. krusei* reduction respectively [45].

Regarding the use of the red-LED lamp or the WHM lamp as a source of irradiation for aPDT, the second proved to be more effective in photoinactivating *Candida* spp. with the exception of against *C. krusei* (Figure 1, Table 1 and Supplementary Material Figure S2II). The use of LED lamp emitting in red matching the absorption spectra peak of the MB tends to be more efficient in the sense of not wasting irradiation energy and therefore red emission sources are usually the ones chosen for MB-aPDT studies (e.g., shown in Table 2). In addition, red LEDs lamps have added advantages at the time of transferring the use of aPDT to clinical application because they are available in all the PDT clinical units; in addition, these lamps stimulate cellular repair mechanisms in fibroblasts [46] and are already used to treat acne vulgaris, herpes simplex virus infection, shingles, or severe wound healing [6,47].

It is also worth noting the time factor to facilitate the use in the clinic since the WMH lamp needs 3 min and 25 seconds to photoinactivate *Candida* spp. compared to 43 min for the LED lamp, due to the greater irradiance of the former compare to the latter (90 mW/cm2 vs. 7 mW/cm2 respectively). Furthermore, the experiments were performed without preincubation of the photosensitizer MB with *Candida* spp. prior to irradiation. Andrade et al. and Soria-Lozano et al. demonstrated that a pre-incubation time did not produce greater inactivation of the microorganism, so it is not necessary to add more time to the aPDT procedure [12,48].

On the other hand, a broad-spectrum WMH lamp could be a model of daylight, i.e., which could be used as a source of irradiation for aPDT instead of this lamp. The advantages are that the treatment could be carried out at home and it would require less equipment and personnel (cheaper). However, it also has disadvantages, such as the imprecision in the quantification of the dose of light or duration of exposure, considering that intensity of daylight depends on the season of the year, weather conditions, or geographic location [9,49–51]. Another limitation for the use of daylight is the limitation to treat Candida infections not accessible for this light, such as the mouth or the genitalia, which otherwise are the most frequent. Nevertheless, the WMH lamp achieves better results than the LED lamp in this work, demonstrating its efficacy.

Overall, our study aims to open the way for the application of this alternative therapy, MB-aPDT alone or better in combination with CHX, either using lamps with a specific or broad- emission spectrum or even daylight as an irradiation source, to deal with cutaneous and mucosal candidiasis. However, it should be borne in mind that the present findings were obtained following in vitro irradiation of *Candida* spp., therefore clinical studies are required to confirm these results.

#### **5. Conclusions**

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pharmaceutics13081176/s1, Figure S1: Photos and emission spectra of the lamps used in the photoinactivation experiments. A: graph of the emission spectrum of the red-LED lamp and B: emission spectrum of the white metal halide lamp, Figure S2: Photoinactivation of *Candida* spp. using MB-aPDT. I: Comparison of the response of yeast when they are irradiated with the red-LED lamp (left A) or with the WMH lamp (right B). II: Comparison of the response of each strain to irradiation with the two lamps (A: *C. albicans;* B: *C. parapsilosis*; C: *C. krusei*). The error bars represent the standard deviation calculated for five measurements. C0 , initial inoculum control; LEDs, light-emitting diodes; MB, methylene blue; WMH, white metal halide.

**Author Contributions:** Conceptualization, V.P.-L., Y.G. and A.R.; methodology, validation and figures, V.P.-L. and Y.B.-L.; analysis, V.P.-L.; resources and funding acquisition, V.P.-L., Y.G. and A.R.; writing—original draft preparation, V.P.-L.; writing—review and editing, V.P.-L., Y.B.-L., Y.G. and A.R.; supervision, V.P.-L. and A.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grant CTQ2013-48767-C3-2-R from the Spanish Ministerio de Economía y Competitividad. Antonio Rezusta and Yolanda Gilaberte were funded by the Aragón Government: B10\_17R Infectious Diseases of Difficult Diagnosis and Treatment research group and B18\_17D Dermatology and Photobiology research group, respectively as recognized by the Government of Aragon.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank the IIS Aragon for the GIIS-023.

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

#### **References**

## *Article* **Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model**

**Mohammad Sadraeian 1,2,\* , Fabio Francisco Pinto Junior 1 , Marcela Miranda 1 , Juliana Galinskas 3 , Rafaela Sachetto Fernandes 1 , Edgar Ferreira da Cruz 3 , Libing Fu 2 , Le Zhang 2 , Ricardo Sobhie Diaz 3 , Gustavo Cabral-Miranda 4,5 and Francisco Eduardo Gontijo Guimarães 1,\* ,†**

**Abstract:** Different light-based strategies have been investigated to inactivate viruses. Herein, we developed an HIV-based pseudotyped model of SARS-CoV-2 (SC2) to study the mechanisms of virus inactivation by using two different strategies; photoinactivation (PI) by UV-C light and photodynamic inactivation (PDI) by Photodithazine photosensitizer (PDZ). We used two pseudoviral particles harboring the Luciferase-IRES-ZsGreen reporter gene with either a SC2 spike on the membrane or without a spike as a naked control pseudovirus. The mechanism of viral inactivation by UV-C and PDZ-based PDI were studied via biochemical characterizations and quantitative PCR on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDZ treatments could destroy single stranded RNA (ssRNA) and the spike protein of the virus, with different ratios. However, the virus was still capable of binding and entering into the HEK 293T cells expressing angiotensin-converting enzyme 2 (ACE-2). A dose-dependent manner of UV-C irradiation mostly damages the ssRNA, while PDZ-based PDI mostly destroys the spike and viral membrane in concentration and dosedependent manners. We observed that the cells infected by the virus and treated with either UV-C or PDZ-based PDI could not express the luciferase reporter gene, signifying the viral inactivation, despite the presence of RNA and DNA intact genes.

**Keywords:** viral inactivation; photodynamic inactivation; SARS-CoV-2 pseudovirus; enveloped virus; UV-C light; photosensitizer

### **1. Introduction**

Novel coronavirus disease (COVID-19), caused by the SC2 virus, was first detected in December 2019 in the Hubei province of China, and has since sparked a global health crisis, with 5.1 million deaths reported by the World Health Organization (WHO) as of November 20 in 2021 [1]. This pandemic situation demands urgent attention toward finding novel strategies that might contribute to the prevention of viral spread via the inactivation of virions on surfaces, aerosols, and the human body.

**Citation:** Sadraeian, M.; Junior, F.F.P.; Miranda, M.; Galinskas, J.; Fernandes, R.S.; da Cruz, E.F.; Fu, L.; Zhang, L.; Diaz, R.S.; Cabral-Miranda, G.; et al. Study of Viral Photoinactivation by UV-C Light and Photosensitizer Using a Pseudotyped Model. *Pharmaceutics* **2022**, *14*, 683. https://doi.org/10.3390/ pharmaceutics14030683

Academic Editor: Maria Nowakowska

Received: 26 February 2022 Accepted: 15 March 2022 Published: 21 March 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/).

The UV-C light has been used in healthcare facilities for environmental disinfection (air, liquid, and solid surfaces) [2]. The efficacy of this inactivation may depend not only on the wavelength but also on factors such as the pathogens (e.g., bacterial or viral species), light output, and environmental conditions [3]. UV-C light at 254 nm radiation enables the deposit of the energetic photons during interaction with the coronavirus, damaging the viral genome, and, consequently, the virus replication and proliferation can theoretically be abrupted [4]. In the case of RNA viruses like SC2, UV irradiation forms several RNA photoproducts, primarily from adjacent pyrimidine nucleotides, such as uracil dimers, as well as RNA–protein cross-links [3]. The formation of the uracil dimer potentially leads to frameshift or point mutations in the genome, known as UV-signature mutations of virus [5]. Hence, we should remain vigilant about the long-term effects of irradiation-mediated strategies for viral inactivation. There are several studies on the effects of UV-C for LD90 viral inactivation based on the time and dose of irradiation [2,6,7]; however, the mechanism of action of how UV-C inactivates viruses is still unclear [7,8].

Photodynamic therapy (PDT) is another light-based strategy that has been proposed to treat infections by damaging microorganisms, fungi, parasites, and viral particles. PDT is based on the use of photo-sensitive agents named photosensitizers (PS) which, in light-excited conditions and the presence of molecular oxygen, produce reactive oxygen species (ROS) [9–15]. PDT may damage cells via ROS generation, causing necrosis and apoptosis without harming the neighboring tissues. The advantages of utilizing photosensitizers for photodynamic inactivation (PDI) include its short-term toxicity, the absence of cell genome alterations, and avoiding the development of viral-induced resistance. Hence, the antiviral potential therapeutic effects of PDT and PDI on SC2 have been investigated with promising results [16,17]. Photoditazine photosensitizer (PDZ) is a porphyrin derivative with a chlorine core which allows it a high absorption in the red light spectrum with λmax of between 650–670 nm, as an advantage compared to the first generation of photosensitizers which are porphyrin core-based and which absorbs wavelengths too short for superior tissue penetration [18]. Understanding the mechanism of viral photoinactivation is important in finding and optimizing light-based strategies to battle viral infection. There are several reports on the mechanisms of viral photoinactivation with limited experiments on virion damage and viral propagation [2,19,20] due to the restriction of working with highly pathogenic viruses like HIV and SC2 viruses. Addressing these containment issues, the setting up of pseudotyped models in BSL2 labs can speed up studying the viral–cell mechanism and neutralizing assay towards in vivo studies [21,22]. Herein, we introduced the application of a pseudotyped model for studying the viral mechanism on four levels; virion damage; viral-cell entry; DNA integration; and expression of reporter genes. In this study, we followed the effects of UV-C irradiation and PDI on viral spike proteins and ssRNA in a HIV-based pseudotyped model of SC2 containing the Luciferase-IRES-ZsGreen reporter gene. Finally, we aimed to study the pseudovirus during cell internalization, genome integration, and reporter gene expression, after undergoing treatments by UV-C and PDZ photosensitizer under different concentrations and conditions (Figure 1).

**Figure 1.** Schematic picture of the mechanism of SARS-CoV-2 pseudovirus infectivity. Unlike SC2 ssRNA virus, which has viral reproduction independent of the host genome, this counterpart pseudovirus carries on reporter ssRNA with LTR, which causes integration into the genome. In this study, the pseudovirus has been treated with either UV-C irradiation or photodynamic inactivation (PDI) by Photodithazine photosensitizer. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. The figure was created with BioRender software. **2. Materials and Methods Figure 1.** Schematic picture of the mechanism of SARS-CoV-2 pseudovirus infectivity. Unlike SC2 ssRNA virus, which has viral reproduction independent of the host genome, this counterpart pseudovirus carries on reporter ssRNA with LTR, which causes integration into the genome. In this study, the pseudovirus has been treated with either UV-C irradiation or photodynamic inactivation (PDI) by Photodithazine photosensitizer. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on four levels; free-cell viral damage; viral cell entry; DNA integration; and expression of reporter genes. The figure was created with BioRender software.

#### *2.1. Chemical Reagents*  **2. Materials and Methods**

#### *2.1. Chemical Reagents*

All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise stated. All reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA), unless otherwise stated.

#### *2.2. Cells and Viruses 2.2. Cells and Viruses*

*2.3. Plasmids*

The HEK 293T cells expressing ACE-2 receptor were gifted from BEI Resources as catalog number NR-52511. ACE-2 enzyme is a critical receptor for virus entrance into the host cell. The HEK 293T cells were used as control cells for assays, and for pseudovirus generation. The cells were maintained at 37 °C in 5% CO2 in DMEM medium-high glucose (DMEM-HG) with 10% fetal bovine serum (Gibco Invitrogen, Grand Island, NY, USA). HEK 293T is a derivative human cell line isolated from human embryonic kidneys (HEK) and expresses a mutant version of the SV40 large T antigen. The HEK 293T cells expressing ACE-2 receptor were gifted from BEI Resources as catalog number NR-52511. ACE-2 enzyme is a critical receptor for virus entrance into the host cell. The HEK 293T cells were used as control cells for assays, and for pseudovirus generation. The cells were maintained at 37 ◦C in 5% CO2 in DMEM medium-high glucose (DMEM-HG) with 10% fetal bovine serum (Gibco Invitrogen, Grand Island, NY, USA). HEK 293T is a derivative human cell line isolated from human embryonic kidneys (HEK) and expresses a mutant version of the SV40 large T antigen.

#### *2.3. Plasmids*

Vector backbone pMD2.G and Vector backbone psPAX2 were gifts from Didier Trono (Addgene plasmid # 12259 and # 12260, respectively). The other plasmids were donated by BEI Resources and their sequences are available at (https://www.beiresources.org/ (accessed on 11 February 2022)) with the following catalog numbers [23]:

HDM-IDTSpike-fixK (BEI catalog number NR-52514): Plasmid expressing under a CMV promoter the Spike viral entry from SARS-CoV-2 strain Wuhan-Hu-1 (Genbank NC\_045512);

pHAGE-CMV-Luc2-IRES-ZsGreen-W (BEI catalog number NR-52516): Lentiviral backbone plasmid that uses a CMV promoter to express luciferase followed by an IRES and ZsGreen;

HDM-Hgpm2 (BEI catalog number NR-52517): lentiviral helper plasmid expressing HIV Gag-Pol under a CMV promoter;

HDM-tat1b (NR-52518): Lentiviral helper plasmid expressing HIV Tat under a CMV promoter.

pRC-CMV-Rev1b (NR-52519): Lentiviral helper plasmid expressing HIV Rev under a CMV promoter.

#### *2.4. UV-Vis Spectroscopy*

UV-Vis spectroscopy was used to determine plasmid and protein concentrations by using Nanodrop 1000 UV-Visible spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [24].

#### *2.5. Dynamic Light Scattering*

Hydrodynamic radii, electrophoretic mobility, and polydispersity of SC2 Spikepseudovirus were measured before and after photo inactivation. For UV-C inactivation and PDI inactivation, we followed the inactivation protocols as explained in Sections 2.7 and 2.8. Then, samples with 70 µL volume at 1 mg/mL in UV-transparent 96-well plates were measured using a DLS Wyatt Möbius (Wyatt Technologies, Dernbach, Germany) with incident light at 532 nm, at an angle of 163.5◦ . Samples were equilibrated at 25 ± 0.1 ◦C for 600 s before the measurements, and this temperature was held constant throughout the experiments. All samples were measured in triplicate with 10 acquisitions and a 5 s acquisition time. The change in the cumulant-fitted hydrodynamic radius in nanometers was monitored during the storage period. Results were calculated using the Dynamics 7.1.7 software (Wyatt Technologies, Santa Barbara, CA, USA).

#### *2.6. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control*

SC2 Spike pseudotyped lentiviruses were generated by transfecting 293T cells, adjusted with the protocol explained by Thermo Fisher Scientific (Waltham, MA, USA). Briefly, seed 293T cells to be 95–99% confluent at transfection. At 16–24 h after seeding, the cells were transfected with the plasmids required for lentiviral production by using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions and using the following plasmid with 1 mL total volume per well of a six-well plate. The 293T cells were transfected with a lentiviral backbone plasmid encoding Firefly luciferase and ZsGreen reporter proteins, a plasmid expressing SC2 Spike, and plasmids expressing HIV-1 *gag*, *pol*, and *tat* proteins, to assemble the membrane of viral particles. The same protocol was followed to generate naked control pseudovirus without adding the viral entry plasmid encoding SC2 Spike. At 8 h post-transfection, the packaging medium was removed and replaced. At 24 h post-transfection, the entire volume of cell supernatant was harvested and stored at 4 ◦C. Then, 1 mL of fresh medium was replaced. At 52 h post-transfection, the entire volume of the cell supernatant was harvested. The pseudovirus product was aliquoted in small volumes of 400 µL and stored at −80 ◦C prior to use and underwent a single freeze-thaw.

#### *2.7. Viral Inactivation Using UV-C Irradiation*

A total of 40 µL of pseudovirus were diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm (HNS G5, OSRAM Germicidal Puritec, Munich, Germany) placed 1 cm above the plate to allow a uniform irradiance over the plate (10 ± 2 mW/cm2 ). Light was delivered by 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2 , respectively. Controls were not submitted to irradiation. After irradiation, aliquots of 80 µL were placed into the plates containing the previously seeded 293T/ACE2 cells and incubated for 8 h at 37 ◦C with 5% CO2 for viral adsorption. Then, 120 µL of DMEM-HG medium containing 12% fetal bovine serum was added, and the plate was incubated for 48 h at 37 ◦C with 5% CO2. Afterward, the cells were placed into a lysis buffer solution to proceed with either Firefly luciferase assay or proviral DNA assay. Results were normalized in relation to controls for the calculation of viral inhibition rates of each sample.

#### *2.8. Photosensitizer-Based Photodynamic Inactivation*

A total of 40 µL of pseudovirus were diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate. The Photodithazine photosensitizer (PDZ) (Photodithazine® Company, Moscow, Russia) with a serial dilution of 10, 50, and 250 µg/mL was added, and incubated in the dark at RT (22 ◦C) for 15 min, then were irradiated using a homemade LED device at 670 nm (red light). All irradiations were performed with an irradiance rate of 30 mW/cm2 in a time-dependent manner of 1, 10, and 20 min which equal the light doses of 1.8, 18, and 36 J/cm2 , respectively. Afterwards, the treated ssRNA viruses were either harvested for the viral RNA load and DLS characterization or were incubated with the 293T/ACE-2 cells, as described in Section 2.7. After that, the cells were harvested for previral DNA load and luciferase activity measurement.

### *2.9. Quantification of Viral RNA and Proviral DNA*

The total ssRNA pseudovirus, before and after irradiation, was extracted and purified using the RNeasy Lipid Tissue Mini Kit, according to the manufacturer's (QIAGEN, Hilden, Germany) protocol.

The pseudovirus was treated with either 36 s UV-C irradiation or 10 min PDI in the presence of 10 µg/mL PDZ. The viral RNA load refers to the virus genome of freecell pseudovirus, before and after treatment. Viral load measurement was carried out using one-step reverse transcriptase (RT) and real-time PCR in a single buffer system using the Abbott Real Time on the automated m2000, over the dynamic range of detection of 40 to 10,000,000 copies/mL (Abbott, IL, USA) [25]. The protocol was followed as described by Kumar et al. for the TaqMan One-Step RT and PCR Master Mix Reagents Kit (Thermo Fisher Scientific, Waltham, MA, USA) with primers and probes for long terminal repeat (LTR) region of 640 bp, with two identical regions located at both ends of the either proviral DNA or RNA viral of SC2 pseudovirus. Briefly, a volume of 5 µL RNA sample and 20 µL Master Mix were used for a one-step RT-qPCR reaction with 20 µM forward primer (5-GCCTCAATAAAGCTTGCCTTGA-3); 20 µM reverse primer (5- GGGCGCCACTGCTAGAGA-3); 10 µM Taqman probe (5-FAM-CCAGAGTCACACAACAG ACGGGCACA-TAMRA-3); and an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA), as reported previously [25–27].

Quantification of Proviral DNA were completed with the TaqMan Real-Time PCR Assay. The cells were infected with the treated virus (Sections 2.7 and 2.8). The cells were harvested three days after infection, centrifuged, and separated the pellets. The number of infected cells containing proviral DNA of pseudovirus was measured using qPCR. The quantification was executed based on the previously published protocol [27,28] for amplification of proviral DNA of pseudovirus (region LTR) with the primers described in Section 2.9.

#### *2.10. Flow Cytometry*

Direct fluorescence detections were applied using flow cytometry (Becton-Dickson Accuri C6, Mountain View, CA, USA) to analyze the expression of ZsGreen in the 293T/ACE-2 cells incubated with treated pseudovirus, as explained before (Sections 2.7 and 2.8). After 48 h, the ZsGreen-positive cells were harvested, fixed by 2% paraformaldehyde (PFA), quantified by blue laser (20 mW) irradiation at 488 nm and analyzed in the channel FL1: 533/30. The acquired data were analyzed by Flow-Jo software version 7.5 (Tree Star Inc., Ashland, OR, USA).

## *2.11. Luciferase Assay*

The infected cells which were harvested before (Sections 2.7 and 2.8) were lysed with 20 µL of Luciferase Cell Culture Lysis Reagent (Promega, Madison, WI, USA), then mixed with 100 µL of Luciferase Assay Reagent (Promega, Madison, WI, USA), and the light emission was measured.

#### *2.12. Titration of Pseudovirus*

The pseudovirus particles were titrated using a method similar to SC2 pseudovirus generation. Virus titers were determined by measuring relative luciferase units (RLUs). The HEK293T cells expressing human ACE-2 (293T-ACE-2) were produced to test the correlation between ACE-2 expression and SC2 pseudovirus susceptibility. Particles were generated in two forms; with a SC2 spike and a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. In a mirror plate, the percentage of cell viability was measured during the viral infection with a serial dilution of the virus starting at 50 µL pseudovirus in a total volume of 100 µL (0.5) for the spike pseudovirus. After 8 h of pseudovirus incubation, the media were replaced with 150 µL fresh media. After 48 h incubation, the wells containing 50 µL pseudovirus were studied for cell confluency. Afterwards, the titers of pseudotyped particles were quantified by a Luciferase assay expressed in RLU, to determine the number of transducing particles per mL.

#### *2.13. Confocal Microscopy*

One day before UV-C or PDI treatment, 2 × 104 cells per well of 293T/ACE-2 cells were seeded on a multiple-chamber slide (Nalge-Nunc International, Naperville, Ill, USA). The next day, cells were incubated with treated pseudovirus, as explained before (Sections 2.7 and 2.8). After 48 h, the ZsGreen-positive cells were washed four times with PBS, fixed by 2% PFA. Images were obtained with an inverted LSM 780 multiphoton laser scanning confocal microscope (Zeiss, Jena, Germany), a 63 × 1.2 water immersion objective to couple with the bottom side of the cover slip, and the Zeiss LSM software was used to treat the images. The wavelength of Argon ion laser at 488 nm was used to excite the expressed ZsGreen protein compared to cell autofluorescence. The molecular localization of ZsGreen was analyzed for each image pixel in spectral and channel modes in the ranges 492–700 nm and 492–537 nm, respectively. The cells' autofluorescence were analyzed from 585 to 692 nm.

Each pixel was associated with an emission spectrum which allowed the spatial separation of the expressed ZsGreen fluorescence (bright blue-greenish color) and the cell auto-fluorescence (yellow-orange color). Considering the spectral of the cell autofluorescence (maximum at 575 nm) is almost constant, the expression of the ZsGreen by the active pseudovirus internalization would be promptly signaled by the spectral superposition of the protein emission at around 515 nm.

#### *2.14. Statistical Analyses*

Statistical analyses were performed using the GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data are shown as mean and SEM of the indicated number of replicate values. If no error bar appears present, the error bars are smaller than, and obscured by, the symbol. The method for statistical comparison used was unpaired twotailed Student's *t*-test, unless specifically indicated otherwise. was unpaired two-tailed Student's *t*-test, unless specifically indicated otherwise. **3. Results and Discussion**

indicated number of replicate values. If no error bar appears present, the error bars are smaller than, and obscured by, the symbol. The method for statistical comparison used

#### **3. Results and Discussion** *3.1. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control*

#### *3.1. Generation of Pseudovirus with SARS-CoV-2 Spike and Naked Control* The spike-pseudotyped lentiviral particles were generated, which can infect 293T

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The spike-pseudotyped lentiviral particles were generated, which can infect 293T cells expressing the human ACE-2 receptor. In parallel, the naked control pseudovirus was generated, which harbors a backbone plasmid-encoding luciferase-IRES-ZsGreen reporter, but without the SC2 Spike on the membrane (Figure 1). cells expressing the human ACE-2 receptor. In parallel, the naked control pseudovirus was generated, which harbors a backbone plasmid-encoding luciferase-IRES-ZsGreen reporter, but without the SC2 Spike on the membrane (Figure 1). *3.2. Titration of Pseudovirus*

#### *3.2. Titration of Pseudovirus* The pseudovirus particles were titrated in two forms; particles with a SC2 spike and

The pseudovirus particles were titrated in two forms; particles with a SC2 spike and a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. After 48 h incubation, the cell confluence reached 100% for all wells in a mirror plate containing non-transduced cells, however, the wells containing 50 µL pseudovirus showed 90% cell confluence (Figure 2A). The titers of pseudotyped particles were quantified by a Luciferase assay. Titers of >105 RLUs per mL were measured in a 96-well plate (Figure 2B). Unsurprisingly, the ACE2-expressing cells incubated with naked pseudovirus without a spike did not show the expression of luciferase (Figure 2C). The other negative control was the incubation of 293T non-ACE2 control cells with Spikepseudovirus, which did not show the luciferase expression, as expected (Figure 2D). In previous reports, researchers used polybrene to facilitate the lentiviral infection through minimizing charge-repulsion between the virus and cells [29], but we found this SC2 pseudovirus no need to polybrene for binding to the ACE-2 receptor. a negative control without a viral entry protein. Both pseudo-typed particles harbored a Luciferase-IRES-ZsGreen backbone. After 48 h incubation, the cell confluence reached 100% for all wells in a mirror plate containing non-transduced cells, however, the wells containing 50 μL pseudovirus showed 90% cell confluence (Figure 2A). The titers of pseudotyped particles were quantified by a Luciferase assay. Titers of >105 RLUs per mL were measured in a 96-well plate (Figure 2B). Unsurprisingly, the ACE2-expressing cells incubated with naked pseudovirus without a spike did not show the expression of luciferase (Figure 2C). The other negative control was the incubation of 293T non-ACE2 control cells with Spike-pseudovirus, which did not show the luciferase expression, as expected (Figure 2D). In previous reports, researchers used polybrene to facilitate the lentiviral infection through minimizing charge-repulsion between the virus and cells [29], but we found this SC2 pseudovirus no need to polybrene for binding to the ACE-2 receptor.

**Figure 2.** Titration of SARS-CoV-2 spike-pseudovirus particles in 293T cells expressing ACE-2. (**A**) Study of the percentage of cell viability during the viral infection respecting a serial dilution starting at 1:2 (0.5); (**B**,**C**) The graph shows the titers of the expression of Luciferase reporter as determined by measuring relative luciferase units (RLUs). The RLU data are the average of three-fold serial dilution of virus starting at 50 μL virus in a total volume of 100 μL (0.5) for the Spike-pseudovirus (**B**); naked pseudovirus without spike (**C**); or the Spike-pseudovirus with 293T cells without ACE2 receptor (**D**). After 8 h of pseudovirus incubation, the media were replaced with 150 μL fresh media. *3.3. Viral Inactivation using UV-C Irradiation or Photosensitizer-Based PDI*  **Figure 2.** Titration of SARS-CoV-2 spike-pseudovirus particles in 293T cells expressing ACE-2. (**A**) Study of the percentage of cell viability during the viral infection respecting a serial dilution starting at 1:2 (0.5); (**B**,**C**) The graph shows the titers of the expression of Luciferase reporter as determined by measuring relative luciferase units (RLUs). The RLU data are the average of threefold serial dilution of virus starting at 50 µL virus in a total volume of 100 µL (0.5) for the Spikepseudovirus (**B**); naked pseudovirus without spike (**C**); or the Spike-pseudovirus with 293T cells without ACE2 receptor (**D**). After 8 h of pseudovirus incubation, the media were replaced with 150 µL fresh media.

#### A volume of 40 μL of pseudovirus was diluted in 60 μL of DMEM-HG without *3.3. Viral Inactivation Using UV-C Irradiation or Photosensitizer-Based PDI*

supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm for 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2, respectively. Figure 3A represents the effect of UV-C irradiation on the photo-inactivation of ssRNA pseudovirus. The results showed that UV-irradiation may inactivate 74%, 93%, and A volume of 40 µL of pseudovirus was diluted in 60 µL of DMEM-HG without supplementation in each well of a 96-well plate, which were exposed to the UV-C lamp 254 nm for 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2 , respectively. Figure 3A represents the effect of UV-C irradiation on the photo-inactivation of ssRNA pseudovirus. The results showed that UV-irradiation may inactivate 74%, 93%, and 99.99% of SC2 Spike-pseudovirus particles during 1, 6, and 36 s irradiation, respectively. These

results are comparable with the published results on SC2 elsewhere [20], due to the discrepancies of the cell-entry mechanism among virus and pseudovirus. Furthermore, a time-dependent manner of PDI was performed to find the maximum viral inactivation with the minimum time and concentration of Photogem PS (PDZ). As Figure 3B demonstrates, the viral inactivation depended on both time and the PS concentration. We observed that 99.8% of the pseudovirus were inactivated in the presence of 50 µg/mL PDZ with 10 min irradiation. Hence, we selected this time and concentration for further studies. These results indicate that both UV-C irradiation and PDI, as two distinct strategies, are highly effective in inactivating pseudovirus replication, while there could be some differences in the mechanism of infectivity between UV-C irradiation and PDI. Hence, we extended our studies focusing on the viral RNA and proviral DNA loads, as described in Section 3.4. discrepancies of the cell-entry mechanism among virus and pseudovirus. Furthermore, a time-dependent manner of PDI was performed to find the maximum viral inactivation with the minimum time and concentration of Photogem PS (PDZ). As Figure 3B demonstrates, the viral inactivation depended on both time and the PS concentration. We observed that 99.8% of the pseudovirus were inactivated in the presence of 50 μg/mL PDZ with 10 min irradiation. Hence, we selected this time and concentration for further studies. These results indicate that both UV-C irradiation and PDI, as two distinct strategies, are highly effective in inactivating pseudovirus replication, while there could be some differences in the mechanism of infectivity between UV-C irradiation and PDI. Hence, we extended our studies focusing on the viral RNA and proviral DNA loads, as described in Section 3.4.

99.99% of SC2 Spike-pseudovirus particles during 1, 6, and 36 s irradiation, respectively. These results are comparable with the published results on SC2 elsewhere [20], due to the

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**Figure 3.** Study of the effect of photo-inactivation of ssRNA pseudovirus considering the relative luciferase units with a time-dependent manner of UV-C irradiation at 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2, respectively (**A**); and PDZ-based PDI in a serial dilution of 10, 50 and 250 μg/mL in a time-dependent manner of 1, 10 and 20 min which equal the light doses of 1.8, 18 and 36 J/cm2, respectively (**B**). Data are ± means S.E.M. (*n* = 3). **Figure 3.** Study of the effect of photo-inactivation of ssRNA pseudovirus considering the relative luciferase units with a time-dependent manner of UV-C irradiation at 1, 6, and 36 s corresponding to doses of 10, 60, and 360 mJ/cm2 , respectively (**A**); and PDZ-based PDI in a serial dilution of 10, 50 and 250 µg/mL in a time-dependent manner of 1, 10 and 20 min which equal the light doses of 1.8, 18 and 36 J/cm2 , respectively (**B**). Data are ± means S.E.M. (*n* = 3).

#### *3.4. Study the Infectivity Mechanism of UV-C Irradiation and PDI Using qPCR 3.4. Study the Infectivity Mechanism of UV-C Irradiation and PDI Using qPCR*

Viral inactivation could be due to either viral protein or viral genome damage [8,30,31]. We suppose that any damage to the virus spike may lead to loss of the virus binding ability and neutralization of the virus infectivity, while damaging the viral genome may affect the viral and proviral loads of pseudovirus. The results of the viral RNA load showed that both 36 s UV-C irradiation and PDZ-based PDI (10 min irradiation, 50 μg/mL PDZ) can damage ssRNA by 83% and 74%, respectively. The RNA of both control (naked pseudovirus without spike) and spike-positive viral particles were destroyed during irradiation (Figure 4A). By comparing the PDZ-based PDI in two forms of enveloped and non-enveloped (naked) viruses, we found out that PDI may damage the viral genome independently from the virus type. Viral inactivation could be due to either viral protein or viral genome damage [8,30,31]. We suppose that any damage to the virus spike may lead to loss of the virus binding ability and neutralization of the virus infectivity, while damaging the viral genome may affect the viral and proviral loads of pseudovirus. The results of the viral RNA load showed that both 36 s UV-C irradiation and PDZ-based PDI (10 min irradiation, 50 µg/mL PDZ) can damage ssRNA by 83% and 74%, respectively. The RNA of both control (naked pseudovirus without spike) and spike-positive viral particles were destroyed during irradiation (Figure 4A). By comparing the PDZ-based PDI in two forms of enveloped and non-enveloped (naked)viruses, we found out that PDI may damage the viral genome independently from the virus type.

The results of the proviral DNA assay may interpret the virus's ability to complete the subsequent steps of cell binding, internalization, and genome integration after reverse transcription. The proviral DNA load of 36 s UV-C irradiation was as much as the RNA viral load, signifying that the UV-C based viral inactivation is independent of damaging the spike protein. In parallel, the proviral DNA load of PDZ-based PDI (10 min irradiation, 50 μg/mL PDZ) was decreased by 13%, which is half of the RNA viral load (26%), signifying the PDI-treated pseudovirus may lose cell infectivity due to damaging the spike The results of the proviral DNA assay may interpret the virus's ability to complete the subsequent steps of cell binding, internalization, and genome integration after reverse transcription. The proviral DNA load of 36 s UV-C irradiation was as much as the RNA viral load, signifying that the UV-C based viral inactivation is independent of damaging the spike protein. In parallel, the proviral DNA load of PDZ-based PDI (10 min irradiation, 50 µg/mL PDZ) was decreased by 13%, which is half of the RNA viral load (26%), signifying the PDI-treated pseudovirus may lose cell infectivity due to damaging the spike (Figure 4A,B). Presumably, PDZ-based PDI destroys more of the spike than the viral genome, which leads to losing the binding ability of the virus. Unsurprisingly, the naked control particle

showed no DNA load, as the control particle lacks a spike for cell binding. Furthermore, we observed that the cells infected by either UV-C or PDI-treated pseudovirus could not express the luciferase reporter gene (Figure 4C), signifying the total viral inactivation despite the presence of RNA and DNA intact genes. control particle showed no DNA load, as the control particle lacks a spike for cell binding. Furthermore, we observed that the cells infected by either UV-C or PDI-treated pseudovirus could not express the luciferase reporter gene (Figure 4C), signifying the total viral inactivation despite the presence of RNA and DNA intact genes.

(Figure 4A,B). Presumably, PDZ-based PDI destroys more of the spike than the viral genome, which leads to losing the binding ability of the virus. Unsurprisingly, the naked

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**Figure 4.** The pseudovirus has been treated with either 36 s UV-C irradiation or 10 min Photodynamic Inactivation in the presence of 50 μg/mL Photodithazine. The mechanism of infectivity of photo-inactivated pseudovirus particles has been compared on three levels; (**A**) viral RNA load referring to the free-cell virus; (**B**) proviral DNA load referring to the ability of the treated virus to complete the subsequent steps of cell internalization, reverse transcription and genome integration; (**C**) luciferase activity referring to the expression of luciferase reporter after DNA integration. nt/n0 represents the fraction of the targeted genome region that remained intact after treatment. **Figure 4.** The pseudovirus has been treated with either 36 s UV-C irradiation or 10 min Photodynamic Inactivation in the presence of 50 µg/mL Photodithazine. The mechanism of infectivity of photoinactivated pseudovirus particles has been compared on three levels; (**A**) viral RNA load referring to the free-cell virus; (**B**) proviral DNA load referring to the ability of the treated virus to complete the subsequent steps of cell internalization, reverse transcription and genome integration; (**C**) luciferase activity referring to the expression of luciferase reporter after DNA integration. nt/n0 represents the fraction of the targeted genome region that remained intact after treatment.

In this study, nt/n0 represents the fraction of the targeted genome region that remained intact after treatment. In the viral RNA load, the targeted genome region is ssRNA of pseudovirus with LTR sequences. In the proviral DNA load, the targeted genome region is the integrated DNA of pseudovirus genome after reverse transcriptase. Unlike the SC2 virus, the mechanism of infectivity of the SC2 pseudovirus includes DNA integration, which is one of the advantages of utilizing the pseudotyped model. Therefore, we could follow a simple protocol for calculation of RNA and DNA load and compare the qPCR data with luciferase assay results, otherwise to estimate the infectivity based on qPCR data, the infectivity of virus should be assessed by estimation from the qPCR results, according to the protocol published by Sabino et al. [20,32]. In this study, nt/n0 represents the fraction of the targeted genome region that remained intact after treatment. In the viral RNA load, the targeted genome region is ssRNA of pseudovirus with LTR sequences. In the proviral DNA load, the targeted genome region is the integrated DNA of pseudovirus genome after reverse transcriptase. Unlike the SC2 virus, the mechanism of infectivity of the SC2 pseudovirus includes DNA integration,which is one of the advantages of utilizing the pseudotyped model. Therefore, we could follow a simple protocol for calculation of RNA and DNA load and compare the qPCR data with luciferase assay results, otherwise to estimate the infectivity based on qPCR data, the infectivity of virus should be assessed by estimation from the qPCR results, according to the protocol published by Sabino et al. [20,32].

#### *3.5. DLS Measurements before and after Irradiation 3.5. DLS Measurements before and after Irradiation*

DLS measurement demonstrated that 36 s UV-C irradiation on pseudovirus with 18 J/cm2 resulted in a slight decrease in the size distribution compared to the non-irradiated pseudovirus (Figure 5A). On the other hand, in the PDI study, the increase of PDZ concentration from 10 to 50 μg/mL had a significant effect on the size and polydispersity of the virus, and yielded significant aggregated particles (Figure 5B). We assumed that this aggregation may interrupt our results on the cell toxicity therefore we found that PDZ with 10 μg/mL was an appropriate concentration for further studies on flow cytometry and microscopy observations. DLS measurement demonstrated that 36 s UV-C irradiation on pseudovirus with 18 J/cm2 resulted in a slight decrease in the size distribution compared to the non-irradiated pseudovirus (Figure 5A). On the other hand, in the PDI study, the increase of PDZ concentration from 10 to 50 µg/mL had a significant effect on the size and polydispersity of the virus, and yielded significant aggregated particles (Figure 5B). We assumed that this aggregation may interrupt our results on the cell toxicity therefore we found that PDZ with 10 µg/mL was an appropriate concentration for further studies on flow cytometry and microscopy observations.

#### *3.6. Green Fluorescent Measurement by Flow Cytometry*

Furthermore, the cells were infected with viruses, which were treated with either 36 s of UV-C or PDZ (10 µg/mL) of PDI, to measure the expression of the ZsGreen protein. The flow cytometry results showed that 46.3% of the virus-infected cells were emitting green fluorescence, while the cells treated with UV-C or the PDI-treated viruses were not able to express the ZsGreen (Figure 5C).

**Figure 5.** (**A**) Dynamic Light Scattering histograms of hydrodynamic radius (Rh) for pseudovirus showed optimal polydispersity with Rh of 100 nm. During 36 s of UV-C irradiation (360 mJ/cm2), a slight decrease in the size of pseudovirus was observed (Blue arrow) with no significant aggregation; (**B**) PDZ-based PDI significantly affected the size and polydispersity of the virus, resulting in major aggregated particles in a higher concentration of PDZ (50 μg/mL); (**C**) Flow cytometric diagram on the left demonstrated the percentage of pseudovirus-infected cells expressing ZsGreen Fluorescent protein. The middle and right diagrams represent the cells infected by UV irradiated-virus and PDZ-based PDI virus, which do not express ZsGreen protein. FITC rate indicates the green fluorescent emission from ZsGreen. Data are ± means S.E.M. (*n* = 2). **Figure 5.** (**A**) Dynamic Light Scattering histograms of hydrodynamic radius (Rh) for pseudovirus showed optimal polydispersity with Rh of 100 nm. During 36 s of UV-C irradiation (360 mJ/cm2 ), a slight decrease in the size of pseudovirus was observed (Blue arrow) with no significant aggregation; (**B**) PDZ-based PDI significantly affected the size and polydispersity of the virus, resulting in major aggregated particles in a higher concentration of PDZ (50 µg/mL); (**C**) Flow cytometric diagram on the left demonstrated the percentage of pseudovirus-infected cells expressing ZsGreen Fluorescent protein. The middle and right diagrams represent the cells infected by UV irradiated-virus and PDZbased PDI virus, which do not express ZsGreen protein. FITC rate indicates the green fluorescent emission from ZsGreen. Data are ± means S.E.M. (*n* = 2).

#### *3.7. Observation of ZsGreen Expression by Confocal Microscopy*

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*3.6. Green Fluorescent Measurement by Flow Cytometry*

able to express the ZsGreen (Figure 5C).

Forty-eight h after cell incubation with pseudovirus (with no treatment), the ZsGreen expression was observed using confocal microscopy. Figure 6A shows images of the field in spectral mode (Figure 6A—panel (a)), and in channel mode merged with a wide field transmission image (Figure 6A—panel (c)). The two spectral contributions for both ZsGreen emission and the cell autofluorescence can be separated by taking two regions of interest (ROI) in panel (a) (green and red circles), as depicted as two graphs in Figure 6A—panel (b). In Figure 6A—panel (c) demonstrates that the emission detected between 492 and 532 nm (assigned the bright-blue false color) mainly signals the expression of the ZsGreen protein while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm. *3.7. Observation of ZsGreen Expression by Confocal Microscopy*  Forty-eight h after cell incubation with pseudovirus (with no treatment), the ZsGreen expression was observed using confocal microscopy. Figure 6A shows images of the field in spectral mode (Figure 6A—panel (a)), and in channel mode merged with a wide field transmission image (Figure 6A—panel (c)). The two spectral contributions for both ZsGreen emission and the cell autofluorescence can be separated by taking two regions of interest (ROI) in panel (a) (green and red circles), as depicted as two graphs in Figure 6A panel (b). In Figure 6A—panel (c) demonstrates that the emission detected between 492 and 532 nm (assigned the bright-blue false color) mainly signals the expression of the ZsGreen protein while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm.

Furthermore, the cells were infected with viruses, which were treated with either 36 s of UV-C or PDZ (10 μg/mL) of PDI, to measure the expression of the ZsGreen protein. The flow cytometry results showed that 46.3% of the virus-infected cells were emitting green fluorescence, while the cells treated with UV-C or the PDI-treated viruses were not

**Figure 6.** (**A**) Confocal microscopy images showing ZsGreen expression in 293T-ACE2 cells at 48 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone. The images are represented in spectral mode (panel **a**) and channel mode merged with a wide field transmission image (panel **c**). In panel **b**, the green and red curves represent the regions of interest (ROI) of ZsGreen emission and the cell autofluorescence, respectively; (**B**) The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission, indicating the expression of ZsGreen in comparison to negative control cells. The ZsGreen emission was **Figure 6.** (**A**) Confocal microscopy images showing ZsGreen expression in 293T-ACE2 cells at 48 h after incubation with Spike-pseudotyped lentiviral particles with the ZsGreen backbone. The images are represented in spectral mode (panel (**a**)) and channel mode merged with a wide field transmission image (panel (**c**)). In panel (**b**), the green and red curves represent the regions of interest (ROI) of ZsGreen emission and the cell autofluorescence, respectively; (**B**) The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission, indicating the expression of ZsGreen in comparison to negative control cells. The ZsGreen emission was detected between 492 and 532 nm (assigned the bright-blue false color), while the cellular autofluorescence can be differentiated by taking the emission (orange false color) in the spectral range from 585 to 695 nm; (**C**) The results of viruses with 36 s UV-C irradiation (360 mJ/cm2 ) did not show green fluorescent emission, while the cells with 1 s UV-C irradiation (10 mJ/cm2 ) were still showing somewhat ZsGreen expression; (**D**) The viruses were treated with Photoditazine (PDZ) (10 µg/mL) and irradiated in a time-dependent manner of 0 (so-called dark), 1 and 20 min. The cells infected by PDI virus after 20 min do not express ZsGreen protein. (**A**,**B**) scale bar: 20 µm.

To study the expression of ZsGreen protein by confocal microscopy, the cells were infected with pseudovirus treated with UV-C or PDI. The positive control cells incubated with pseudovirus without treatment showed strong green fluorescent emission indicating the expression of ZsGreen in comparison to negative control cells (Figure 6B). The results of viruses with 36 s UV-C irradiation (360 mJ/cm2 ) did not show green fluorescent emission (Figure 6C), while the cells with 1 s UV-C irradiation (10 mJ/cm2 ) were still showing slight ZsGreen expression. The results were in agreement with our luciferase assay (Figure 3A), confirming that the 1 s UV-C irradiation is not sufficient to completely inactivate the viruses.

For the PDI study, the viruses were incubated with 10 µg/mL PDZ, and irradiated for 1 or 20 min, which equals the light doses of 1.8 and 36 J/cm2 , respectively (Figure 6D). The dark control groups were submitted to the same procedure, except for light exposure. No green fluorescent emission was observed in the cells after PDI with 20 min irradiation. In contrast, the dark controls showed fluorescent emission of ZsGreen. Neither PDZ irradiated samples nor dark samples showed toxicity on the cell confluency, while an increase of autofluorescence was observable, compared to the negative control cells. These observations confirm our results of luciferase assay (Figure 3B), and are in agreement with our previous studies on PDZ-based PDI, as described elsewhere [15].

In sum, two distinct strategies (UV-C irradiation and PDZ-based PDI) were applied for the inactivation of SC2 pseudovirus produced using HIV-based lentiviral system which specifically infect ACE2-expressing cells. This specificity was demonstrated using luciferase assay compared to the control negative cells and the control naked viruses, which agreed with previous reports [21,23,33,34]. The viral inactivation could be the consequence of either viral protein damage, which affects the cell internalization, or viral genome damage affecting the viral load. Unlike the SC2 RNA virus with viral reproduction independent of the host genome [7,8,30,35,36], this pseudotyped model enabled us to study not only the RNA viral load, but also the DNA integration, as well as the presence or absence of a spike on the viral particle. Several reports demonstrating the results of viral inactivation assays have a high degree of concordance with a clinical isolate of SC2 [33,34]; however, the results cannot be used for the inactivation of the actual SC2 virus unless tested.

#### **4. Conclusions**

Considering the advantages of pseudovirus over the actual SC2 virus, which was discussed above, we followed a simple protocol for calculating the RNA and DNA load and compared the qPCR data with luciferase assay results. Hence, we studied the viral inactivation by UV-C and PDI in dose and time-dependent manners via biochemical characterizations and quantitative PCR on four levels; virion damage; viral cell entry; DNA integration; and expression of reporter genes. Both UV-C and PDI treatments could destroy ssRNA and the spike protein of the virus in different ratios; however, the virus was still capable of binding and entering into the ACE-2 expressing 293T cells. UV-C irradiation disinfected the virus mainly through viral genome damage, with no apparent effects on the viral size and virus–cell binding ability. On the other side, PDZ-based PDI mostly destroyed the spike and viral membrane. Ignoring the type of viral destruction (ssRNA or spike), the cells infected by the photo-inactivated virus could not express the luciferase reporter gene. Our findings emphasize the advantages of PDI over UV-C viral inactivation. ROSmediated damages on the viral envelope may generate debris or the fragments which could stimulate host immune defense. Moreover, viral PDI has affordability compared to other therapeutics like monoclonal antibodies (e.g., Ronapreve), which can be important factors for preventative use at home [37]. Other advantages of PDI include high repeatability without viral resistance or UV-signature mutations, with fast removal of the virus in a very short time.

The other advantage of this model is comparing the viral particles in two forms of enveloped and non-enveloped (naked) viruses, as a matter of importance for side-by-side comparison. Therefore, comparing two viruses with similar genomes but different in their protein envelope enables us to study the effect of each inactivation strategy on damaging the RNA genome in the presence and absence of a spike. Besides, this pseudotyped model can be used for other radiation-based strategies for understanding their mechanism of viral inactivation, with no need to work in BSL-3.

**Author Contributions:** F.E.G.G., M.M. and M.S. conceptualized and wrote the main manuscript text and prepared figures; F.E.G.G., M.M., R.S.D. and M.S. designed and performed the biochemical and microscopy experiments; F.F.P.J. designed and performed the DLS assay; R.S.D., R.S.F., E.F.d.C., M.S. and J.G. designed and performed the viral assays; L.F., L.Z. and G.C.-M. reviewed and edited the manuscript; M.S., R.S.D. and F.E.G.G. supervised the study. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors acknowledge the support provided by: FAPESP (Sao Paulo Research Foundation)–grant numbers: 2017/10910-5 (M. Sadraeian-Pós-Doutorado-Fluxo Contínuo), 2013/07276-1 (CEPOF–CEPID Program), 2019/14526-0 and 2020/05146-7 (G. Cabral-Miranda, JP FAPESP), and CAPES Project (COMBA TE-COVID1673158P) Program (PHYSICS<PHONE_NUMBER>2P9). The COVID-19 reagents were supported by BEI Resources Repository of ATCC and the NIH.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data available are reported in the article.

**Acknowledgments:** We thank BEI Resources Repository of ATCC and the NIH for supporting the reagent related to COVID-19. We thank Seth Pincus, M.D. for his support in the Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT.

**Conflicts of Interest:** Authors declare no competing interests. Graphical figures were created with BioRender software ((https://biorender.com/ (accessed on 15 July 2021)).

#### **Abbreviations**

#### **References**

## *Article* **Multiple Light-Activated Photodynamic Therapy of Tetraphenylethylene Derivative with AIE Characteristics for Hepatocellular Carcinoma via Dual-Organelles Targeting**

**Chuxing Chai 1,†, Tao Zhou 2,†, Jianfang Zhu 3,†, Yong Tang 1 , Jun Xiong 1 , Xiaobo Min 1 , Qi Qin 1 , Min Li 1,\* , Na Zhao 4,5,6 and Chidan Wan 1,\***

**Abstract:** Photodynamic therapy (PDT) has emerged as a promising locoregional therapy of hepatocellular carcinoma (HCC). The utilization of luminogens with aggregation-induced emission (AIE) characteristics provides a new opportunity to design functional photosensitizers (PS). PSs targeting the critical organelles that are susceptible to reactive oxygen species damage is a promising strategy to enhance the effectiveness of PDT. In this paper, a new PS, 1-[2-hydroxyethyl]-4-[4-(1,2,2 triphenylvinyl)styryl]pyridinium bromide (TPE-Py-OH) of tetraphenylethylene derivative with AIE feature was designed and synthesized for PDT. The TPE-Py-OH can not only simultaneously target lipid droplets and mitochondria, but also stay in cells for a long period (more than 7 days). Taking advantage of the long retention ability of TPE-Py-OH in tumor, the PDT effect of TPE-Py-OH can be activated through multiple irradiations after one injection, which provides a specific multiple light-activated PDT effect. We believe that this AIE-active PS will be promising for the tracking and photodynamic ablation of HCC with sustained effectiveness.

**Keywords:** photodynamic therapy; aggregation-induced emission; organelles targeting; hepatocellular carcinoma

## **1. Introduction**

Locoregional ablation therapy was recommended as an effective treatment for patients with primary hepatocellular carcinoma (HCC) and liver metastases [1,2]. The mechanism of ablation is to cause the destruction of local tumor by physical or chemical damage. Thermal ablation, including radiofrequency ablation and microwave ablation, has been applied as a safe, low-cost, effective alternative of surgery in small and multifocal liver tumor [3]. The ablation procedure often relies on the placement of the needle electrode into target tumor under the guidance of imaging technology which requires additional competence and fails to provide real-time self-monitoring. Multiple treatment sessions and repetitive puncture operation may be required due to difficult tumor conditions and

**Citation:** Chai, C.; Zhou, T.; Zhu, J.; Tang, Y.; Xiong, J.; Min, X.; Qin, Q.; Li, M.; Zhao, N.; Wan, C. Multiple Light-Activated Photodynamic Therapy of Tetraphenylethylene Derivative with AIE Characteristics for Hepatocellular Carcinoma via Dual-Organelles Targeting. *Pharmaceutics* **2022**, *14*, 459. https://doi.org/10.3390/ pharmaceutics14020459

Academic Editors: Udo Bakowsky, Matthias Wojcik, Eduard Preis, Gerhard Litscher and Patrick J. Sinko

Received: 8 December 2021 Accepted: 17 January 2022 Published: 21 February 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/).

limitation of imaging technology, which may cause additional suffering of patients [4]. Alternatively, photodynamic therapy (PDT) is a potential tumor-ablative locoregional therapy [5–7]. Fluorescent molecules with photodynamic effect can provide precise and safe ablation of tumor at the cellular level while enabling dynamic monitoring of tumor lesion, which has great promise in building cancer-targeted theragnostic platforms [8,9]. PDT can induce both cancer cell death and immune response against the tumor [10,11]. Additionally, intraoperative fluorescence image-guided surgery combined with PDT can eliminate residual tumors and reduce the recurrence of cancer [12–15]. The use of PDT has gained much attention in the treatment of HCC, and has been approved as a feasible approach for HCC treatment in preclinical studies [6,16].

The three crucial elements of PDT are photosensitizer (PS), light irradiation, and oxygen. PS is the core element of PDT. The procedure of PDT involves administration of PSs which accumulate in the tumor tissue, followed by local illumination with specific wavelength to active PSs [17]. After exposure to the light, the PSs can transfer energy from light to molecular oxygen, contribute to their change of energy states, to generate reactive oxygen species (ROS), which then induce apoptosis, necrosis, and autophagy in treated cells and cause cell death [18,19]. However, traditional PSs—such as porphyrin, phthalocyanine, and analogs—are prone to aggregate in the treated cells or aqueous solution, which will lead to a dramatic reduction in ROS production and suppression in fluorescence emission [20,21].

In 2001, Tang's group discovered a novel fluorogen with the opposite characteristics, namely aggregation-induced emission (AIE) [22], which is nonemissive in a molecularly dissolved state, but is highly emissive in their aggregate/solid state due to the mechanism of restriction of intramolecular motions/rotations [23]. Meanwhile, as organic molecules, AIE luminogens (AIEgens) were highly editable. The different additional groups and molecular structures can provide different optical, physical, and chemical properties to AIEgens, which enables broad biological medicine application [24–27]. A series of AIEgens have been developed for translational applications in sensing, imaging, and theranostics with excellent performance than conventional fluorescent probes [28,29]. Recently, some AIEgens were designed to exhibit excellent photosensitization and ROS generation ability, which broadens their potential applications in PDT of cancer. The unique features of AIEgens provide new opportunities for the facile design of PS with special function, high accuracy, and efficacy for image-guided PDT [30–33].

A critical limitation of PDT is that the half-life of ROS (<40 ns) is usually very short so that it can only act on the production site (<20 nm) [34], which means simple transportation PSs into tumor tissues or cancer cells are not efficient enough to achieve expected outcomes [34,35]. Designing PSs to target the critical organelles that are susceptible to ROS damage is a promising strategy to enhance the effectiveness of PDT. The mitochondrion is a promising target for PSs owing to its crucial role in ROS generation, oxidation-reduction status modulation, and cell apoptosis mediation. Mitochondria in tumor cells are sensitive to ROS generation induced by PDT, which ultimately induces tumor cell apoptosis by disrupting the balance of mitochondrial ROS [36]. Recently, several AIEgens have been designed for targeting mitochondria and have exhibited remarkable photostability, high brightness, and excellent PDT effect [37–40]. However, multiple doses and repeated injections of PSs are always required for better outcomes in PDT procedures, which may lead to superfluous side effects and additional toxicity to patients [36]. The destruction of tumor vessels of PDT may also reduce the accumulation of PSs in tumor tissue and influence the efficiency of second PDT. Lipid droplets (LDs), also known as adiposomes or lipid bodies, are intracellular lipid-rich organelles for the long-term storage of lipids [41]. The synthesis of lipid droplets is at a lower level in normal cells, whereas tumor cells actively synthesize lipid droplets owing to their high-metabolism state [42,43]. Due to the suitable metabolic activity and stability, as well as their interactions with other organelles, especially mitochondrial, LDs can be valuable targets for PDT [44,45]. Therefore, AIEgens that could dual-target mitochondria and LDs may provide long-term photodynamic effect and better outcomes in PDT treatment procedure after administration of single-dose PSs.

In this work, for developing a new PS with multiple light-activated photodynamic effect and organelle-targeting abilities, the 1-[2-hydroxyethyl]-4-[4-(1,2,2-triphenylvinyl) styryl]pyridinium bromide (TPE-Py-OH) was designed and synthesized. TPE-Py-OH possesses a typical AIE character and exhibits strong fluorescence emission in the aggregated state. It could simultaneously target LDs and mitochondria in living cells. Due to its lipophilic feature, it can aggregate and store in LDs in a relatively stable state for long-term intracellular retention. Meanwhile, TPE-Py-OH also has efficient ROS generation ability and PDT effect both in vitro and in vivo. All these features suggest its superior performance in long-term tracking of living cancer cells and organelles-targeted PDT effect of cancer cells. Benefiting from the strong fluorescence, good photostability, and efficient ROS generation ability of TPE-Py-OH, the long-term cancer cell tracking and multiple light-activated PDT effect of HCC for more than 7 days were achieved after one PS administration. This AIE-active PS exhibit sustained effectiveness in vitro and in vivo, which suggests that the PS can hopefully be applied in long-term intracellular organelle imaging and multiple light-activated photodynamic ablation of HCC.

## **2. Materials and Methods**

### *2.1. Materials and Instruments*

4-Methylpyridine, 2-bromoethanol, piperidine, anhydrous acetonitrile, and ethanol were purchased from Sigma-Aldrich and used without further purification unless specified otherwise. 4-(1,2,2-Triphenylvinyl)benzaldehyde was synthesized according to the literature method [46,47]. Other chemicals and solvents were all purchased from Aldrich and used as received without further purification. Distilled water was used throughout the experiments. HepG2 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle medium, fetal bovine serum (FBS), BODIPYTM 493/503 Dye, and JC-1 Dye were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Mashikimachi, Japan). Reactive oxygen species (ROS) assay kit, calcein AM/PI double stain kit, Annexin V-FITC/PI double stain kit and MitoTracker Red FM was purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/PI apoptosis detection kit was purchased from Qcbio Science & Technologies (Shanghai, China). Tissue culture flask, 96-well plates, and 24-well plates were purchased from Corning Incorporated (Corning, NY, USA). Confocal dishes were purchased from Biosharp Life Science (Hefei, China).

1H and 13C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer (Bruker, Tucson, AZ, USA) using DMSO-*d6* as solvents. High-resolution mass spectra (HRMS) were recorded on a Finnigan MAT TSQ 7000 Mass Spectrometer System (Finnigan MAT, San Jose, CA, USA) operating in a MALDI-TOF mode. UV–vis absorption spectra were recorded on a Biochrom UV visible spectrometer (Biochrom, Berlin, Germany). Photoluminescence (PL) spectra were recorded on a PerkinElmer spectrofluorometer LS 55 (PerkinElmer, Singapore, Singapore). The cells were imaged under LSM7 DUO confocal laser scanning microscope (CLSM; Carl Zeiss Microscopy, Jena, Germany) and fluorescence microscope (IX71, Olympus, Tokyo, Japan). The absorbance was measured using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

#### *2.2. Synthesis of 1-(2-Hydroxyethyl)-4-methylpyridinium Bromide (Compound 1)*

An acetonitrile (20 mL) solution of 4-methylpyridine (0.93 g, 10.00 mmol) had 2 bromoethanol (2.5 g, 20.00 mmol) added to it. The mixture was stirred at reflux temperature (82 ◦C) under nitrogen for 24 h and then cooled to room temperature. Then, the reaction mixture was precipitated in ethyl acetate (EA) (200 mL). The solid residue was filtrated and washed with excess EA. The filtrate was collected and dried in a vacuum oven. Compound 1 was a light brown powder in 86% yield. 1H NMR (400 MHz, DMSO-*d6*, δ): 8.91 (d, 2H, *J* = 6.8 Hz), 7.99 (d, 2H, *J* = 6.4 Hz), 4.64 (t, 2H, *J* = 15.6 Hz), 3.81 (t, 2H, *J* = 15.6 Hz), 2.60 (s, 3H). 13C NMR (100 MHz, DMSO-*d6*) δ: 158.5, 143.9, 127.7, 61.9, 59.7, 21.2. HRMS (LDI-TOF) m/z calcd for [C8H12NO]+ , 138.0919 [M-Br]+; found, 138.0920 [M-Br]+.

#### *2.3. 1-[2-Hydroxyethyl]-4-[4-(1,2,2-triphenylvinyl)styryl]pyridinium Bromide (TPE-Py-OH)*

A solution of 1 (0.5 g, 2 mmol) and 4-(1,2,2-triphenylvinyl)benzaldehyde (1.01 g, 2.8 mmol) was refluxed at temperature (78 ◦C) under nitrogen in dry ethanol (20 mL) catalyzed by three drops of piperidine. After cooling to ambient temperature, the solvent was evaporated under reduced pressure. The residue was purified by a silica gel column chromatography (50 mesh) using dichloromethane/methanol mixture (5:1 *v*/*v*) as eluent to obtain TPE-Py-OH as a yellow solid in 64% yield (0.72 g, 1.29 mmol). 1H NMR (400 MHz, DMSO-*d6*) δ: 8.82 (d, 2H, *J* = 6.8 Hz), 8.16 (d, 2H, *J* = 6.8 Hz), 7.88 (d, 1H, *J* = 16.0 Hz), 7.50 (d, 2H, *J* = 8.4 Hz), 7.42 (d, 1H, *J* = 16.4 Hz), 7.16–6.94 (m, 17H), 4.51 (t, 2H, *J* = 10.0 Hz), 3.82 (m, 2H), 3.5(brs, 1H). 13C NMR (100 MHz, DMSO-*d6*) δ: 152.70, 145.35, 144.50, 142.79, 142.71, 142.56, 141.40, 140.07, 139.72, 133.10, 131.25, 130.53, 130.51, 130.43, 127.79, 127.68, 127.51, 126.69, 126.62, 123.22, 123.02, 61.92, 59.83. HRMS (LDI-TOF) *m*/*z* calcd for [C15H30NO]+ , 480.2327 [M-Br]+; found, 480.2317 [M-Br]+.

#### *2.4. Cellular Uptake and Subcellular Distribution*

HepG2 human HCC cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ◦C in a 5% CO2, 90% relative humidity incubator. For cell imaging, the HepG2 cells were seeded in slide chambers at a density of 105 and cultured overnight for adhesion. Then, cells were incubated with TPE-Py-OH at various concentrations (2, 5, and 10 µM) for 30 min and continued to incubate with fresh medium for 12 h. Nuclei were labeled with DAPI (1 µg/mL) overnight. After rinsing with PBS, the cells were imaged under a confocal laser scanning microscope (CLSM, Nikon Corporation, Tokyo, Japan). To further explore the subcellular distribution of TPE-Py-OH, 2 × 105 HepG2 cells were plated in the 35-mm confocal dish and cultured overnight, then incubated with 5 µM TPE-Py-OH for 30 min. After rising with PBS, the cells were then stained with 50 nM MitoTracker Red FM, or continuously incubated with fresh culture medium and then stained with 1 µg/mL BODIPY for 30 min, respectively. After three washes, the cell images were captured under CLSM, and the data was analyzed by NIS-Elements Imaging Software (Nikon Corporation, Tokyo, Japan) and Image-J (National Institutes of Health freeware, Bethesda, MD, USA). For TPE-Py-OH: λex = 405 nm and band-pass filter λ = 550–600 nm. For DAPI: λex = 405 nm and band-pass filter λ = 425–475 nm. For MitoTracker Red: λex = 581 nm and band-pass filter λ = 600–650 nm. For BODIPY 493/503: λex = 488 nm and band-pass filter λ = 500–550 nm.

#### *2.5. Cytotoxicity Studies In Vitro*

Cell viability after incubation with various concentrations of TPE-Py-OH was evaluated by CCK8 assays. Briefly, the cells at a density of 1 × 104 were seeded into a 96-well plate overnight and then incubated with a series of concentrations of TPE-Py-OH (0, 1, 3, 5, 7, 10, and 15 µM). After pre-treatment, all the cells were incubated with 100 µL fresh medium including with 10 µL CCK8 solution per well and then continued to incubate for 4 h. The absorbance of the solution at 450 nm was measured using a multimode plate reader (Perkin Elmer Pte. Ltd., Singapore, Singapore).

For cytotoxicity effect induced by PDT, the cells were incubated with various concentrations of TPE-Py-OH for 30 min and continued to incubate with fresh medium for 12 h and then exposed to blue laser irradiation with different duration (450 nm, 30 mW/cm2 , 1.8 J/cm2–18 J/cm2 ). The cells were as negative controls without both laser irradiation and TPE-Py-OH treatment. Sequentially, the cells continued to incubate for 24 h and the cell viability was measured according to the above description.

#### *2.6. Intracellular ROS Detection*

Intracellular ROS production was measured by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe according to the manufacturer's instructions. After incubation in the presence or absence of TPE-Py-OH for 30 min, the cells were loaded with 10 µM DCFH-DA at 37 ◦C for 30 min in dark. The cells were replaced with fresh medium and then exposed to 450 nm blue laser irradiation at the power density of 30 mW/cm2 , then immediately imaged under a fluorescence microscope (IX71, Olympus, Tokyo, Japan). For DCFH-DA: λex = 488 nm and band-pass filter λ = 500–550 nm.

#### *2.7. Calcein-AM and Propidium Iodine (PI) Staining Assay*

The live and dead cells were identified using a calcein/PI double stain kit according to the protocol. After treatment, the cells were incubated with 2 µM calcein-AM and 5 µM PI for 30 min at 37 ◦C. The fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

#### *2.8. Flow Cytometry Analysis*

The apoptotic and necrotic cells were detected by Annexin V-FITC/PI dual staining using flow cytometry. The cells were seeded into a 24-well plate overnight and then incubated with TPE-Py-OH for 30 min. After exposure to laser irradiation (450 nm, 30 mW/cm2 , 4 min, or 8 min), the cells were further incubated for 24 h, then collected and resuspended in 100 µL of binding buffer. According to the manufacturer's instructions, the cells were incubated with 5 µL Annexin V-FITC and 10 µL PI for 15 min in the dark at room temperature and then immediately measured by flow cytometry (BD Falcon, San Jose, CA, USA).

#### *2.9. Mitochondrial Membrane Potential (*∆*ψm) Measurement*

The change of mitochondrial membrane potential (∆ψm) was measured by JC-1 Dye according to the manufacturer's instructions. Briefly, the cells were incubated with 2 µg/mL of JC-1 for 20 min at 37 ◦C after different concentrations of TPE-Py-OH administration and different laser irradiation times, washed twice with PBS, and then imaged under CLSM. For JC-1 (monomer): λex = 488 nm and band-pass filter λ = 500–530 nm; (J-aggregate): λex = 585 nm and band-pass filter λ = 590 nm.

#### *2.10. Long-Term Cell Tracking and In Vitro PDT*

The cells were first stained with 10 µM TPE-Py-OH for 12 h and then rinsed with PBS to remove the extracellular molecules. The image was captured as a reference. The cells were incubated continuously and one-third of them were sub-cultured into another dish for continual tracking every 2 days. Other cells were seeded into the 35-mm confocal dish. After attachment for 24 h, the fluorescence of TPE-Py-OH in cells was imaged by CLSM, the ROS level in cells was performed according to the above description. The step was repeated every 2 days until non-fluorescence in cells.

To confirm the long-term localization of intracellular TPE-Py-OH, the HepG2 cells were incubated with 10 µM TPE-Py-OH for 12 h and then cultured with fresh DMEM medium, the daughter cells were sequentially co-stained with 1 µg/mL BODIPY for 30 min on days 1, 3, 5, and 7 and their fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

For long-term PDT in vitro, the HepG2 cells were incubated with 10 µM TPE-Py-OH for 12 h and replaced with fresh medium for following experiments. On days 1, 3, 5, and 7 after TPE-Py-OH treatment, the cells were seeded into 35-mm confocal dishes. After attachment, the cells were exposed to blue laser irradiation with different duration (450 nm, 30 mW/cm2 , 18–45 J/cm2 ) with half of the confocal dish covered by foil paper to avoid light irradiation. Then the calcein/PI double stain was used to detect the effect of PDT, and the JC-1 mitochondrial membrane potential probe was used for mitochondrial membrane

potential measurement. The fluorescence images were captured under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

#### *2.11. Animal Model*

Male BALB/c mice (4 weeks old, 20 g) were purchased from HFK Bioscience Co, Ltd. (Beijing, China). All animal experiments were approved by the Ethics Committee of the Union Hospital of Huazhong University of Science and Technology and conducted in accordance with the guidelines of the Department of Laboratory Animals of Tongji Medical College. H22 cells (5 × 105 ) were subcutaneous injection into the right axilla of each mouse to establish xenograft liver tumor model, and the tumor volume reached 60 mm3 after 5 days.

### *2.12. In Vivo Multiple Light-Activated PDT*