Second Near-Infrared Photothermal Therapy with Superior Penetrability through Skin Tissues

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE5 Sep 2022Second Near-Infrared Photothermal Therapy with Superior Penetrability through Skin Tissues Yanji Chu†, Shenglong Liao†, Hongguang Liao, Yang Lu, Xiaodong Geng, Di Wu, Jian Pei and Yapei Wang Yanji Chu† Key Laboratory of Advanced Light Conversion Materials and Biophotonics, Department of Chemistry, Renmin University of China, Beijing 100872 , Shenglong Liao† Key Laboratory of Advanced Light Conversion Materials and Biophotonics, Department of Chemistry, Renmin University of China, Beijing 100872 , Hongguang Liao Key Laboratory of Advanced Light Conversion Materials and Biophotonics, Department of Chemistry, Renmin University of China, Beijing 100872 , Yang Lu College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 , Xiaodong Geng Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease, Beijing 100853 , Di Wu Department of Nephrology, Chinese PLA General Hospital, Chinese PLA Institute of Nephrology, State Key Laboratory of Kidney Diseases, National Clinical Research Center for Kidney Diseases, Beijing Key Laboratory of Kidney Disease, Beijing 100853 , Jian Pei College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 and Yapei Wang *Corresponding author: E-mail Address: [email protected] Key Laboratory of Advanced Light Conversion Materials and Biophotonics, Department of Chemistry, Renmin University of China, Beijing 100872 https://doi.org/10.31635/ccschem.021.202101539 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Photothermal therapy (PTT) triggered by second near-infrared (NIR-II) light (1000–1400 nm) has shown great potential in tumor ablation because of its good tissue penetrability. However, NIR-II PTT still cannot treat tumors underneath skin because of the light scattering effect of skin components. This research aims to promote the NIR-II penetrability of skin tissue by weakening the light scattering effect from the refractive index inhomogeneity among skin constituents. This strategy allows for a notable improvement of NIR-II transmittance in vivo from 30% to 70% through mouse skin. In animal experiments, the local temperature of tumor tissue in the experiment group is 14.1 °C higher than that in the control group due to superior tissue penetration, which is thus responsible for the excellent therapeutic effects of complete ablation without any reoccurrence. Such a strategy not only achieves a perfect PTT effect, but also benefits the development of other light-related biological applications. Download figure Download PowerPoint Introduction Photothermal therapy (PTT), which refers to the hyperthermia phenomenon induced by photothermal conversion under light irradiation, is arousing increasing attention regarding its great potential for tumor ablation.1–5 Because of few side effects, PTT is regarded as a powerful substitute to traditional tumor treatment strategies, such as chemotherapy and radiotherapy.6,7 Additionally, light irradiation affords great convenience to control temporal and spatial precision in the process of tumor treatment.8 Combined therapy based on PTT is also worth exploring due to the synergistic effect between photodynamic therapy (PDT), immunotherapy, chemotherapy, ferrotherapy, and PTT.9 However, the inevitable light absorption by hemoglobin, water, and melanin10–12 significantly weakens the light penetration depth through the skin tissue, thereby restricting the adaptability and breadth of PTT in treating deep tumors underneath the skin tissue (Figure 1a). Compared with other wavelengths, near-infrared (NIR) light with wavelength between 780 and 1400 nm is less absorbed by skin compositions and considered the tissue transparent window.13–19 In particular, NIR light with wavelength within 1000–1400 nm, well known as the second NIR (NIR-II) window, can penetrate into deeper tissue than that of the first NIR (NIR-I) window (780–1000 nm) because of less light scattering by biointerfaces.20–27 More importantly, skin cells exhibit higher NIR-II light tolerance in comparison with shorter-wavelength light according to the American National Standard for Safe Use of Lasers, ANSI Z136.1-2007, suggesting that the higher exposure power density of NIR-II light is allowed for deep tissue phototherapy.28,29 Hence, these attractive advantages have stimulated worldwide interests in the design of NIR-II photothermal materials and techniques.8,29–36 Figure 1 | Screening of efficient HRI agents. (a) The interaction of light with skin tissues when incident light is subjected to the skin. (b) Light scattering due to refractive index inhomogeneity. (c) The scattering reduction effect induced by HRI agents in the skin tissue. (d) In vitro experiments of reducing light scattering through the porcine skins treated with different HRI agents. The thickness of porcine skins for each group is about 1.7 mm with minor deviation. Scale bar in the enlarged images is 1 cm. (e) Light (1064 nm) transmittance changes of the porcine skin treated with various HRI agents and saline. (f) Real-time light (1064 nm) transmittance of the porcine skin consecutively treated with glycerol and saline. Download figure Download PowerPoint However, due to light scattering within skin tissue, the effective depth of PTT via NIR-II is still only several millimeters beneath the surface of skin tissue.11,37 Skin tissue is densely packed with a great number of substances with different refractive indexes. For example, collagen and cells have a higher refractive index of 1.39–1.47, while the surrounding media of interstitial fluid and cytoplasm exhibits a lower refractive index of 1.35–1.37.38 The incident light is deflected once it encounters each substance interface, accounting for the light scattering effect that restrains the penetration depth of light (Figure 1b). In principle, only when the refractive indexes of those tissue components match, can the light scattering effect be reduced to allow for a deeper NIR-II penetration.39,40 A pioneering work led by Tuchin et al.41 illustrated that visible light scattering could be notably reduced by replacing the tissue surrounding media with high refractive index (HRI) agents, such as dimethyl sulfoxide,42 glycerol,43 glucose,44 polyethylene glycol (PEG),45 and so forth, which demonstrated a new paradigm to extend the microscopic imaging depth of in vitro biological tissues.46–49 Zhu and co-workers43,45,48,50 successfully extended such a tissue optical clearing technology to in vivo studies on mouse skin. Because HRI agents have similar refractive indexes with the particulate collagen and cells, the number of optical interfaces can be reduced once the surrounding media is replaced by HRI agents (Figure 1c). This work demonstrates the great potential of the tissue optical clearing technology for deepening the NIR-II penetration. On the one hand, NIR-II is a peculiar light window where skin tissues show the weakest absorption, but the introduction of HRI agents can reduce the light scattering effect caused by the refractive index inhomogeneity of skin constituents. According to our studies, the combination of reduced light absorption and scattering effect is a significant achievement for improving the efficacy of NIR-II PTT under a relatively low light power density. To be specific, nanoparticles (NPs) composed of a NIR-II absorbing conjugated polymer (TBDOPV-DT) were used as an NIR-II photothermal material to verify the promotion efficacy of HRI agents.31,33 In the presence of HRI agents within the skin, the NIR-II NPs generated a superior hyperthermia effect in comparison with the control group without HRI treatment. Both in vitro and in vivo studies prove that this strategy exhibits higher efficiency in killing tumor cells and ablating tumor tissues under NIR-II irradiation with a relatively low power density than traditional PTT. Experimental Methods Materials Glycerol (analytical reagent grade [AR]), PEG, and d-(+)-glucose (99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); formaldehyde solution (AR, 37%) and sodium dodecyl sulfate (SDS, chemically pure grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); sodium chloride was purchased from Shanghai Energy Chemicals Co., Ltd. (Shanghai, China). Chloroform was purchased from Beijing Tong Guang Fine Chemicals Company (Beijing, China). Deionized water was obtained by a Milli-Q water-purification system. Roswell Park Memorial Institute (RPMI) 1640 medium and Dulbecco's modified eagle medium (DMEM) were purchased from Cytiva (Marlborough, Middlesex, MA, United States). 0.25% Trypsin-EDTA was obtained from Life Technologies Corporation (Carlsbad, San Diego, CA, United States). Penicillin/streptomycin (P/S) solution (100X) and fetal bovine serum (FBS) were purchased from Mediatech (VA, United States). Phosphate-buffered solution (PBS) was purchased from Biotopped (Beijing, China). The conjugated polymer TBDOPV-DT was synthesized according to the previous method.33 Characterization All UV–vis–NIR spectra were obtained on a Shimadzu UV-3600 spectrometer (Shimadzu Corp., Kyoto, Japan). The power density of NIR laser was measured by a digital handheld optical power and energy meter (Thorlabs, PM100D; Thorlabs Inc., Newtown, NJ, United States). The TiX660 infrared camera (Fluke Corp., Everett, WA, United States) was utilized to monitor the surface temperatures in both in vitro and in vivo experiments. The average diameter of NPs fabricated via emulsification was determined by the Zetasizer Nano ZS90 (Malvern Panalytical, Malvern, United Kingdom). The thermocouple (UNI-T, UT325) was introduced to record the temperature of the NP aqueous suspension. The optical absorption of the formazan dye at 450 nm in the cellular cytotoxicity experiment was detected by an EPOCH2 microplate spectrophotometer (BioTek Instruments Inc., Winooski, VT, United States) after adding the Cell Counting Kit-8 (CCK-8) reagent. Fluorescent images of HeLa cells stained by calcein-AM and propidium iodide (PI) were captured by the DMi8 inverted fluorescence microscope (Leica, Wetzlar, Germany). Preparation of TBDOPV-DT NPs The TBDOPV-DT NPs were fabricated through a one-step emulsion method. Typically, TBDOPV-DT dissolved in chloroform (1 mg/mL) was mixed with SDS aqueous solution (0.2 wt %) at a fixed volume ratio of 1:3. An ultrasonic cleaner was applied for emulsifying the mixed liquid for 120 min. After that, the chloroform was removed by evaporation at 55 °C for 60 min. The TBDOPV-DT NPs were centrifuged and washed three times with deionized water to remove free SDS from the solution. Evaluation of photothermal conversion performance The photothermal performance of TBDOPV-DT was assessed by exposing the TBDOPV-DT film to 1064 nm laser illumination at varied power densities. The real-time temperature and in situ thermal image of the TBDOPV-DT film were monitored using the infrared camera. The photo- and thermal stability of TBDOPV-DT were evaluated by monitoring the real-time temperature of TBDOPV-DT film upon 1064 nm laser exposure (0.64 W/cm2) over several cycles of heating and cooling. In addition, the photothermal conversion performance of TBDOPV-DT NPs aqueous suspension at different concentrations was also investigated by recording the temperature with a thermocouple. Cell culture Mouse breast carcinoma cells (4T1) were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% P/S. HeLa cells were cultured in DMEM high-glucose medium supplemented with 10% FBS and 1% P/S. All cells were cultured at 37 °C in a 5% CO2 humidified chamber. Cellular cytotoxicity evaluation The cytotoxicity assay was carried out using a CCK-8 assay, and HeLa cells were selected as the tumor cells. Typically, cells were cultured in DMEM supplemented with 10% FBS and 100 μg/mL P/S, then cells were seeded in a 96-well plate and incubated for 24 h at 37 °C with 5% CO2. Subsequently, TBDOPV-DT NPs at different concentrations were added into the wells, and the cells were incubated for 24 h. To perform the CCK-8 assay, the culture medium was removed, and the wells were washed with PBS three times. Then the culture medium containing the CCK-8 reagent (10 μL) was added, followed by incubation for 30 min at 37 °C to allow the formation of formazan dye. Finally, the optical absorption of the formazan dye at 450 nm was measured on a plate reader. Live and dead cell staining HeLa cells were seeded onto petri dishes and incubated overnight. Subsequently, TBDOPV-DT NPs at 50 and 100 μg/mL were added into the wells. After 4 h incubation, the cells were divided into five groups and subjected to different treatments. The laser power density is 1.00 W/cm2, and the irradiation time is 10 min. Then the calcein-AM/PI staining kit (C2015S; Beyotime, Shanghai, China) was used to evaluate cell viability. The cells were treated by calcein-AM/PI solutions (1 μM, PBS) for 30 min. After being washed by PBS three times, cells were observed by inverted fluorescence microscopy. In vivo photothermal cancer treatment Four-week old mice (female, BALB/c) were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in the Peking University Health Science Center, Department of Laboratory Animal Science. Tumor-bearing mice were prepared by implanting 100 μL of 4T1 cell suspension (5 × 106 cells/mL) at the right hind hip and raised for about 1 week until the tumor volume reached approximately 200 mm3. All the mice bearing 4T1 tumors were divided into four groups: NPs, Laser, NPs + Laser, and NPs + Laser + Gly. On day 0, the mice in NPs, NPs + Laser, and NPs + Laser + Gly groups were given an intratumor injection with 100 μL of NPs (0.8 mg/mL). The mouse skin tissues above the tumor in NPs + Laser + Gly group were treated with glycerol. After that, the mice in Laser, NPs + Laser, and NPs + Laser + Gly groups were irradiated with an NIR-II laser (1064 nm, 0.50 W/cm2). During the treatment, an infrared thermal camera was used to record the tumors temperature changes. The tumor condition and the mouse weight were checked every other day. The tumor volumes in mice bodies were estimated according to the formula: Tumor volume = Length × (Width)2/2. The lengths and widths of tumors were measured by a Vernier caliper. It should be noted that to alleviate the pain of the experimental mice caused by tumor development, the mice in control groups were sacrificed on the 8th day while the mice in experiment groups were sacrificed on the 20th day. All the tumors and major organs were collected, weighed, photographed, and stored in formalin. Finally, the histological examination of these tumors and major organs were performed by hematoxylin and eosin (H&E) staining according to a standard procedure. All animal experiments were handled in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) in compliance with Chinese law for experimental animals. The experiments were performed in compliance with institutional guidelines established by the Laboratory Animal Research Center of Peking University, which approved our experimental protocols and procedures. Results and Discussion Screening of efficient HRI agents Ideal HRI agents are expected to have HRI, rapid diffusion, and good biocompatibility characteristics. Herein, several liquid formulas, including glycerol, PEG (MW = 400 g/mol), and saturated glucose solution (glucose), were chosen as experimental groups, and the saline was used as the control group to investigate their light scattering reduction effect on the porcine skins. As shown in Figure 1d, the porcine skin region that was treated with glycerol or PEG tended to become optically transparent over time. In particular, the skin treated with glycerol for 3 h was visually transparent and the pattern under the porcine skin could be observed clearly. In contrast, there was limited change in the glucose group. Moreover, the commercially available porcine skin treated with saline became turbid rather than transparent, which was attributed to tissue dehydration during long-term cold storage, and it could be rehydrated via saline treatment. To better demonstrate the light scattering reduction and successful optical clearing of the tissue caused by glycerol, a plant stem specimen covered with the glycerol-treated porcine skin was observed by a microscope ( Supporting Information Figure S1). At a given exposure time, higher imaging brightness was obtained in the case of porcine skin with longer glycerol treatment time ( Supporting Information Figure S2), which is a forceful indication of the enhanced transparency in the light pathway. Besides optical clearing within visible light, such as optical visualization and microscope observation, the tissue optical clearing technology is also applicable for improving the tissue transmittance in the NIR window. According to the light (1064 nm) transmittance (T1064) of porcine skins (Figure 1e and Supporting Information Figure S3), glycerol is superior to other HRI formulas in reducing light scattering within the skin tissues. Concretely, T1064 of the porcine skin was significantly increased from 37% to 67% after the treatment of glycerol for 120 min. Among the three experimental groups, their capability of reducing the light scattering effect follows the order of glycerol, PEG, and glucose. Thus, glycerol was mainly used in the following studies unless otherwise specified. Noting that light with a longer wavelength is generally less scattered through media, the NIR-II exhibits much better tissue penetration than the intensively investigated NIR-I (808 nm), though the latter could also penetrate the skin tissue to a greater depth with the assistance of HRI agents ( Supporting Information Figure S4). It is also worth mentioning that the HRI-treated skin could be fully recovered by replacing HRI agents with saline. As shown in Figure 1f, T1064 of porcine skins increase from 38.6% to 60% under 120 min of glycerol treatment and then decreases back to 39.8% after treatment with saline for 200 min. In vitro enhanced NIR-II photothermal conversion In light of improved tissue transparency in the NIR-II window, skin tissue optical clearing by HRI agents was exploited for enhancing the NIR-II photothermal conversion beneath the skin tissue. As shown in Figure 2a, TBDOPV-DT, a donor–acceptor type of conjugated polymer, was chosen as the material to absorb NIR-II light because its main absorption peak at 1040 nm exactly locates in the NIR-II region with a molar extinction coefficient of 2.68 × 106 L·mol−1·cm−1 (Figure 2b and Supporting Information Figure S5). As a result of the highly efficient internal conversion and vibrational relaxation,33 TBDOPV-DT exhibited a high photothermal conversion efficiency (47.8%, Supporting Information Note S1, Figure S6, and Table S1). As recorded by an infrared camera, the surface temperature of a TBDOPV-DT film rapidly increased under the irradiation of a 1064 nm laser, which is positively affected by the light power density (Figure 2c). As specifically quantified in Figure 2d, the surface temperature reaches 110 °C within 1 min at the power density of 1.52 W/cm2. The temperature increment is still remarkable when the light power density drops to 1.00 W/cm2, which is generally regarded as the tolerance threshold of NIR-II for The changes of surface temperature the during 10 cycles of light irradiation, the of high photo- and thermal stability ( Supporting Information Figure Figure | In vitro enhanced NIR-II photothermal (a) Chemical of The and the and the (b) UV–vis–NIR spectra of TBDOPV-DT dissolved in (c) images of TBDOPV-DT film under 1064 nm laser irradiation at different light power densities. Scale 1 cm. (d) temperature of TBDOPV-DT film during NIR-II illumination for 1 min under different light power followed by for 1 min. and change of a TBDOPV-DT film covered by a or glycerol-treated porcine skin when NIR-II irradiation (e) or NIR-II irradiation W/cm2) with 10 min Download figure Download PowerPoint The in vitro NIR-II photothermal conversion of TBDOPV-DT in combination with the skin tissue optical clearing technology was investigated based on the as shown in Supporting Information Figure Typically, a TBDOPV-DT film was subjected to 1064 nm laser irradiation through a of porcine skin with a thickness of 1.7 The surface temperature of the TBDOPV-DT film was monitored by an infrared camera in time. As in Figure the temperature change increased when the porcine skin was treated with glycerol, while it was when the skin was treated with saline. This in photothermal effect is with the in transmittance when the porcine skin is treated with glycerol or saline. to the studies in Figure increased under NIR-II irradiation with the treatment of glycerol, that the successful reduction of light scattering is of the laser irradiation (Figure the of glycerol on porcine skin significantly enhanced the efficiency of in vitro NIR-II photothermal conversion underneath skin tissue. In vitro NIR-II photothermal conversion for of cancer cells To cellular TBDOPV-DT was into NPs with an average diameter of nm via an emulsion (Figure As assessed in Figure these NIR-II NPs exhibited low cytotoxicity to HeLa cells as assessed by the CCK-8 significant cytotoxicity was observed at a high of 100 μg/mL after for 24 h. to TBDOPV-DT NPs also an photothermal conversion under NIR-II At the light power density of 1.00 W/cm2, the temperature of an NP aqueous suspension monitored by a thermocouple is positively with the NPs (Figure The temperature increase induced by photothermal conversion of NIR-II NPs could also be through the of light power density ( Supporting Information Figure Figure 3 | In vitro NIR-II photothermal conversion for of cancer cells. (a) light scattering result of TBDOPV-DT NPs prepared via an emulsion method. (b) Cell of HeLa cells after incubation with TBDOPV-DT (c) NIR-II photothermal effect of TBDOPV-DT NPs aqueous suspension at various (d) and of different on HeLa cells. (e) images of HeLa cells mixed with NPs by calcein-AM and propidium iodide after various treatments. Scale 100 Download figure Download PowerPoint Because of its NIR-II photothermal effect, TBDOPV-DT NPs have great as photothermal conversion agents for killing cancer cells by NIR-II As in Figure five groups of HeLa cells different including only NPs, only NIR-II light NPs with NIR-II and NPs with NIR-II illumination in the presence of saline or glycerol-treated porcine skin on the cell According to the and staining both NPs and NIR-II irradiation are to cells when are used (Figure NPs and NIR-II irradiation were applied at the time, HeLa cells were fully due to the photothermal However, when the cell medium was covered by a of porcine skin treated with the photothermal effect was and cells This that the NIR-II photothermal effect beneath the mm porcine skin was to hyperthermia to cell once the saline was replaced with glycerol, which the porcine skin mm transparent, the photothermal effect was obtained and cells were under the NIR-II it was that the hyperthermia was induced by NIR-II photothermal conversion of NPs rather than thermal irradiation from the laser because a number of cells when the NPs from 100 to 50 μg/mL ( Supporting Information Figure In vivo of skin tissue optical clearing technology As a of the of and substance caused by the between in vivo and in vitro skin tissues to a in the tissue optical clearing The effective in vitro scattering reduction induced by HRI agents be on in vivo skin tissues. the NIR-II photothermal conversion was underneath skin treated with HRI agents of As illustrated in Supporting Information Figure a thermal composed of a TBDOPV-DT polymer film and a thermocouple was under the skin of so the of tissue transparency could be by the temperature change of polymer film under NIR-II the are different from the phenomenon of in vitro skin treated with HRI agents. In when the HRI agents were on skin tissue and allowed to from the surface to NIR-II photothermal conversion was not as improved as that of the in vitro It was shown that the of skin the of HRI agents into skin However, the limited reduction of light scattering and the potential of skin after a of the this was not applied a strategy of HRI agents into deep skin tissues was to the of HRI agents. an improvement on the photothermal effect was still not significant ( Supporting Information Figure which with the T1064 of those skins after ( Supporting Information Figure At this time, an phenomenon that the tissue the to be so that the the of HRI agents into the To prove this the was removed the mice skins were treated with HRI agents. As T1064 increased over time, a significant in comparison with previous on the skin tissue (Figure As quantified in Figure the T1064 of those in vivo mice skins with different HRI agents in their is fully with the in vitro (Figure For example, T1064 from to after treatment with glycerol for 30 min. on the that the is the the of HRI agents, of HRI agents into the should and the skin As shown in Figure a mouse skin became optically transparent with the of visible after of glycerol in the