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Research Articles

Lanthanide-semiconductor probes for precise imaging-guided phototherapy and immunotherapy

Wang, Yanxinga; Lü, Weidongb; Dai, Ruiyia; Lin, Bia; Lv, Ruichana,∗

Author Information
doi: 10.1097/JBR.0000000000000083

Abstract

Introduction

Upconversion nanoparticles (UCNPs) are commonly used in cancer diagnostics, bioimaging, bioassays, and phototherapy owing to their large Stokes shift, good stability, and high emission intensity.[1–6] For rare-earth-doped upconversion nanoparticles, the luminous intensity and emission peak position can be adjusted by changing the amount and ratio of the doping elements.[7–10] There are many preparation methods for these UCNPs, such as high-temperature pyrolysis, and hydrothermal and co-precipitation methods.[11–15] Hydrothermal processing has the advantage of a high product yield, and leads to nanoparticles with pure crystallinity and a suitable morphology for both biological and energy applications. More importantly, hydrophilic UCNPs can be modified with biological species, such as, proteins, peptides, and antibodies, making them attractive theranostic materials.

Anticancer phototherapy is a precise cancer treatment with fewer side effects in normal cells than traditional tumor treatment methods, such as surgery, radiotherapy, and chemotherapy. In particular, photodynamic therapy (PDT) has emerged as an important research focus in recent years.[16–21] This method imparts only a small trauma, and the PDT agents have low toxicity. When the drug is enriched to a certain concentration, a phototoxic reaction will occur under the irradiation of laser light, thereby generating reactive oxygen species (ROS) to kill cancer cells.[22,23] However, PDT can react and kill only the drug-rich parts of the tumor, and the surrounding tissue is only slightly damaged. Another type of phototherapy is photothermal treatment (PTT). This can be achieved by combining UCNPs with semiconductor elements, which produce fluorescence resonance energy transfer.[24–29] The photothermal effect of fluorescence resonance energy transfer produces a photothermal effect, which can be used to kill cancer cells.[26,30]

Immunotherapy is another emerging tumor treatment strategy.[31–33] The human immune system can intercept bacteria and viruses that invade the human body, but does not effectively kill cancer cells.[34–39] Immunotherapy activates the immune system, enhances the body's immune regulation ability, cuts off the escape mechanism of cancer cells from the immune system, and facilitates the destruction of cancer cells by the immune system. Specifically, cancer cells express PD-L1, which binds to PD-1 molecules on the surface of T-lymphocytes (effectively ‘turning them off’) and causes the T-lymphocytes to protect themselves.[40–43] The use of monoclonal antibodies to bind to PD-1 on the surface of T-lymphocytes or to bind to PD-L1 on the surface of cancer cells can allow T-lymphocytes to retain their function and kill cancer cells.[44–46] The therapeutic effect of immunotherapy for treating metastasis and tumor growth is superior to other therapy strategies and has less harmful side effects. Moreover, immunotherapy can be synergistically combined with another anticancer therapy strategy, such as chemotherapy, PDT, or PTT and is ideal for long treatment periods together with other treatments.[47–55]

In this study, we combined spherical, mesoporous UCNPs with semiconducting manganese nanoparticles as a nanoplatform with good photodynamic and photothermal effects. Fluorescence or absorption methods can be used for detecting glutathione (GSH) and H2O2. When anti-PD-L1 was modified on the surface of the manganese composite particles (USMs) to target cancer cells, the combination therapy was demonstrated to be more effective than that of the single phototherapy or immunotherapy.

Materials and methods

Reagents and chemicals

Y(NO3)3 (99.99%), Yb(NO3)3 (99.99%), Tm(NO3)3 (99.99%), urea, and potassium fluoride, 1,3-diphenylisobenzofuran (DPBF), ethylene diamine tetraacetic acid, sodium fluoride, tetraethoxysilane, manganese chloride tetrahydrate (MnCl2·4H2O), poly allyamine hydrochloride (PAH), polyetherimide, GSH, potassium permanganate (KMnO4), and indocyanine green (ICG) were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China. Hydrogen peroxide 30% (H2O2) was purchased from Tianjin Hongyan Chemical Reagent Factory, Tianjin, China. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Mito-tracker, 4T1 cancer cells and A549 cancer cells were purchased from Beyotime (China), and Cal27 human tongue cancer cell line was purchased from ATCC (USA). All chemical reagents were used as received without any further purification.

Synthesis

Synthesis of UCNPs

The 0.5 mmol of ethylene diamine tetraacetic acid was weighed and dissolved in 15 mL of ultrapure water, and 0.5 mmol of Y(NO3)3, Yb(NO3)3, or Tm(NO3)3 were added to the beaker. After being stirred at room temperature for 1hour, 10 mL of 0.5 M sodium fluoride was added. The mixture was then stirred at room temperature for 1hour, before the solution was added to a reaction kettle in an oven at 180 °C for 3 hours. The mixed solution was centrifuged and the precipitate was dried to obtain the final UCNPs.

Synthesis of [email protected]

The 40 mg of UCNPs and 80 mg of PAA were weighed and dissolved in 25 mL of ultrapure water. After reaction at 70°C for 4 hours, 60 mL of ultrapure water, 300 μL of NaOH (2 M), and 6 mL of absolute ethanol were added. After the temperature stabilized, 200 μL of tetraethoxysilane (mixed with the same amount of absolute ethanol) was added and the mixture was stirred for 10 minutes. The [email protected]2 (denoted as US) precipitate was prepared after centrifuging three times.

Synthesis of [email protected]2@MnO2 (P-USM)

The 50 mg of US and 100 mg of PAH were weighed and dissolved in 10 mL of ultrapure water. After the mixed solution was stirred for 2 hours, it was centrifuged (4000 revolutions/minute at room temperature). The precipitate was then dispersed in 10 mL of ultrapure water, and 10 mL of KMnO4 solution (10 mg/mL) was added. After being stirred at room temperature for 2 hours, P-USM was obtained after centrifuging three times.

Synthesis of P-USM/anti-PD-L1 (P-USM-A)

The 30 mg of P-USM and 30 mg polyetherimide were weighed and dispersed into 10 mL of ultrapure water. After the mixed solution was stirred for 8 hours, the solution was centrifuged (4000 revolutions/minute at room temperature), and the precipitate was re-dispersed in 2 mL of phosphate buffer saline (PBS) solution. 20 μL of PD-L1 (9.637 mg/mL) was then added and the mixture was stirred in an ice bath overnight, before being centrifuged and lyophilized to obtain P-USM-A.

Characterization

The nanoparticles were imaged using scanning electron microscopy and transmission electron microscopy with energy dispersive spectrometer (FEI Tecnai G2 STwin, FEI Company, USA). Their absorbance spectra were recorded with an Ultraviolet (UV)–visible spectrophotometer (UNICO 2355, UNICO Shanghai, China). Their upconversion luminescence (UCL) emission spectra were obtained using an Edinburgh FLS1000 apparatus (Edinburgh, UK) with a 980-nm laser diode module (MDL-III-980-2W, Changchun, China) as the irradiation source. The MTT assay was recorded by a microplate reader Biotek Synergy H1/Synergy2 (BioTek, USA).

Cell co-localization analysis

Different cell lines including the 4T1 mouse breast cell line (Beyotime, China), C27 tongue cancer cells (ATCC, USA), and the A549 lung cancer cell line (Beyotime, China) were separately and evenly dispersed in a Petri dish and cultured for 12 hours, during which the materials were added at different time points (1, 3, and 5 hours). After washing twice, the cells were stained with Mito-tracker for 20 minutes and observed under a confocal microscope.

ROS detection of P-USM under 980-nm laser irradiation

Ultrapure water (2 mL) was added to a quartz dish and tested with a UV–visible spectrophotometer (UNICO 2355, UNICO Shanghai, China). The resulting spectrum was used as the background curve. 2 mL of P-USM (1 mg/mL) and 20 μL of DPBF solution (1 mg/mL) were added to the dish. The mixture was then irradiated by a 980-nm laser (MDL-III-980-2W, Changchun New Industry Optoelectronic Technology Co. Ltd., Changchun, China) with a pump intensity of 1W/cm2. The UV–visible curve of the solution was recorded at 0, 2, 4, 6, 8, and 10 minutes of near-infrared (NIR) laser irradiation. Finally, the relative amount of ROS generated was determined from the normalized curve. The ROS detection of US (laser irradiation for 0, 2, 4, 6, 8, and 10 minutes) was carried out using processes similar to that previously reported.[56]

In vitro viability of cells incubated with P-USM

4T1 cells were incubated in 96-well plates to obtain monolayer cells in experimental wells. Then, the cells were separately treated as the control group and phototherapy group. (i) The control group without laser treatment was also used to evaluate the biocompatibility evaluation. The cells were incubated with 100 μL of P-USM with different concentrations (500, 250, 125, 62.5, and 32.25 μg/mL) for 12 hours. (ii) For evaluating the phototherapy effect by MTT experiments, the cells were incubated for 12 hours with 100 μL of P-USM nanoparticles (62.5 μg/mL). This group was then irradiated with the 980-nm laser for 0, 1, 2, 3, 4, and 5 minutes.

In the presence and absence of treatment, 20 μL of MTT was added to each well. After 4 hours, the solution in the wells was aspirated and 150 μL of DMSO was added. After the 96-well plate was shaken on a shaker for 10 minutes, the absorbance at 490 nm was measured using a microplate reader (Biotek Synergy H1/Synergy2, BioTek, USA).

In vivo anticancer therapy

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Xi’an Jiaotong University and approved by the School of Pharmacy's Ethics Committee of Tumor Hospital of Shaanxi Province, Xi’an Jiaotong University (No. XJTULAC2020-585, approved from April 2, 2020). A549 cells were dissolved in PBS and injected into the right hind limbs of mice (female BALB/c mice with a weight of about 20 g). After 4 to 5 days of growth, the tumor size was approximately 4 to 7 mm. The tumor-bearing mice were randomly divided into four groups: a control group without any treatment; a group injected with P-USM-A material under laser irradiation (‘material + NIR’); a group injected with anti-PD-L1 under laser irradiation (‘anti-PD-L1 + NIR’); and a group injected with P-USM-A under laser irradiation and separately injected with anti-PD-L1 after phototherapy (‘anti-PD-L1 + NIR + material’). The laser irradiation power was 1W/cm2, and the irradiation time was 10 minutes. Tumors were treated every 3 days for a total of 21 days. On day 21, the mice were euthanized after inhalation with isoflurane, and the organs of heart, lung, liver, spleen, kidney, and tumors were collected for histological analysis. Tissues were sliced and stained with hematoxylin and eosin. The stained slices were observed under an optical microscope (FV3000, Olympus, Tokyo, Japan).

Statistical analysis

The tumor sizes of the mice after treatment with the different methods were statistically analyzed using a Student's t test. The data for the tumor size and the standard error in two different groups were compared separately with a two-tailed distribution and adopted with the two-sample equal variance assumption. The P value between two groups was calculated using Microsoft Excel. P < 0.05 was considered statistically significant.

Results

Morphology, optical absorption, and luminescence of P-USM

A schematic diagram of the design and combined immuno-, photodynamic, and photothermal therapy of USM is shown in Figure 1. The [email protected]@Mn was used as a photodynamic therapy agent under NIR laser irradiation and emitted bright blue light for imaging. Anti-PD-L1 modification enabled combined immuno-, photodynamic, and photothermal therapy for the treatment of lung cancer.

Figure 1
Figure 1:
Schematic diagram of the design of [email protected]@Mn and its use in immuno-, photodynamic, and photothermal therapy. NIR=near infrared, PDT=photodynamic therapy, PTT=photothermal therapy, UCNP=upconversion nanoparticles.

The morphology of the nanoparticles is shown in Figure 2 and Additional Figure 1, http://links.lww.com/JR9/A23. The spherical and porous UCNPs can be seen in Figure 2A and Additional Figure 1A, http://links.lww.com/JR9/A23, and the coating with SiO2 and MnO2 can be seen in Figure 2B, and Additional Figure 1B–D, http://links.lww.com/JR9/A23. After the surface was coated with SiO2, it became porous. As seen in Figure 2B, a shell formed on the surface of the material, increasing the average particle size to 170 nm. The energy dispersive spectrometer spectrum (Fig. 2C) and elemental mapping (Fig. 2D) show that all elements were evenly distributed in the P-USM nanoparticles, further indicating their uniformity. The FTIR spectra indicate that the ICG and anti-PD-L1 were successfully loaded on the surface of P-USM (Fig. 2E).

Figure 2
Figure 2:
Morphology, elemental distribution, and composition of the as-synthesized biomaterials. (A, B) Transmission electron microscopy images of [email protected] (denoted as US) and polymer combined [email protected]@Mn (denoted as P-USM), respectively. (C) Energy dispersive spectrometer spectrum and (D) elemental mapping images of the P-USM-A nanocomposite. (E) FT-IR spectra of the UCNP, US, P-USM, anti-PD-L1, and P-USM with antibody modification (denoted as P-USM-A). P-USM=polymer-modified upconversion [email protected]@manganese dioxide, P-USM-A=antibody modified P-USM, UCNP=upconversion nanoparticle, US=upconversion [email protected]

We then compared the UV–visible absorption and upconversion luminescence of P-USM. The material had a strong absorption in the 400–500-nm range when no GSH was added (Fig. 3A). The upconversion luminescence properties of US and P-USM are shown in Figure 3B. The UCL intensity decreased owing to the MnO2 coating on the surface of UCNPs. Because of the strong absorption of MnO2 in the visible region (400–500 nm), the UCL intensity of P-USM at 470 nm was lower than that of the US.

Figure 3
Figure 3:
Absorbance spectra, photoluminescence spectra, reactive oxygen species generation, and glutathione-response effects of the as-synthesized P-USM. (A) UV–visible absorbance spectra of [email protected]@Mn (denoted as P-USM) (10 mg/mL) with different added amounts of glutathione (GSH). (B) Upconversion luminescence (UCL) spectra of [email protected] (denoted as US) and P-USM in water under 980-nm laser irradiation with the same conditions. Absorbance spectra of the 1,3-diphenylisobenzofuran (DPBF) solution with (C) P-USM and (D) US (both 1 mg/mL) under 980-nm NIR laser irradiation (1.0W/cm2). (E) UCL spectra recovery and (F) the corresponding color change of the P-USM solution (10 mg/mL) after adding different amounts of GSH. P-USM=polymer-modified upconversion [email protected]@manganese dioxide, UCNP=upconversion nanoparticle.

Stimuli-responsive UCL/NIR-II imaging and ROS generation using P-USM

We studied the response of P-USM to GSH. The photodynamic effects of US and P-USM were first detected using DPBF as the fluorescent probe for detecting ROS. With increasing irradiation time, the characteristic absorption of P-USM decreased markedly (Fig. 3C) (in contrast with US, which underwent little change in absorption (Fig. 3D)), indicating its good photodynamic effect under NIR irradiation. Then, we measured the fluorescence recovery of P-USM in the presence of GSH. With increasing amount of GSH, the fluorescence intensity at 477 nm gradually increased, and the color of the material gradually faded (Fig. 3E and F). P-USM had a linear response at 477 nm; the lower detection limit of GSH calculated after fitting was 55 μg/mL (Fig. 4A). The absorbance of P-USM (500 μg/mL) decreased with increasing amount of GSH. After fitting, the upper GSH detection limit of P-USM was determined to be 1.2 mg/mL (Fig. 4B). Thus, the sensitivity of the measurement of luminescence intensity was higher than that based on the absorbance.

Figure 4
Figure 4:
GSH-response results using photoluminescence analysis and absorbance analysis. (A) Linear fitting of the fluorescence intensity recovery of P-USM at 477 nm and (B) linear fitting of the absorbance of P-USM at 410 nm as a function of the amount of GSH. GSH=glutathione, P-USM=polymer-modified upconversion [email protected]@manganese dioxide.

We then studied the NIR-II imaging and upconversion luminescence of MnO2-ICG using the same PAH-supported method (Fig. 5A). Both US and P-USM showed strong visible light emission under 980-nm laser excitation (Fig. 5B). The NIR-II and visible light recovered after the addition of GSH, which provides a basis for the application of P-USM in NIR-II imaging in cancer diagnosis. Moreover, the morphology of MnO2 and the corresponding MnO2-ICG were uniform in shape and monodisperse, which is important for their use in medicine (Fig. 5C).

Figure 5
Figure 5:
Upconversion luminescence and NIR-II luminescence recovery after exposure of the material to GSH. (A) NIR-II image recovery of MnO2-ICG under 808-nm laser irradiation. (B) Upconversion luminescence of P-USM under 980-nm laser irradiation before and after the addition of GSH. (C) Transmission electron microscopy images of MnO2 and MnO2+ICG. GSH=glutathione, ICG=indocyanine green, NIR=near infrared, P-USM=polymer-modified upconversion [email protected]@manganese dioxide.

In vitro specificity of P-USM-A and its use in treating lung cancer

Before biological use, we tested the stability of P-USM in different solutions (Additional Fig. 2, http://links.lww.com/JR9/A23); the USM had good stability in serum. The phagocytic effect of P-USM-A and P-USM incubated with different cell lines was monitored using confocal microscopy. Pearson's co-localization coefficient (R) was calculated from the microscope images to quantify the intracellular effect of the materials. The Mito-tracker (marked as green) entered the mitochondria as the intracellular control, and the luminescence intensity of P-USM or P-USM-A (marked as blue) correlated with the amount of the intracellular material. The Pearson's R values were high for P-USM-A (0.91–0.96; Fig. 6) and P-USM (0.87–0.92; Additional Fig. 3, http://links.lww.com/JR9/A23) in human lung cancer A549 cells, indicating high intracellular uptake into this cell line. By contrast, the Pearson's R values for the phagocytosis of the nanomaterials into human tongue cancer C27 cells (0.7–0.84; Additional Figs. 4, 5, http://links.lww.com/JR9/A23) and mice breast cancer 4T1 cells (0.57–0.74; Additional Figs. 6, 7, http://links.lww.com/JR9/A23) were low. In all cases, the content of intracellular materials increased over time. We attribute the high specificity of P-USM-A to A549 cells to the higher expression of PD-L1 on these lung cancer cells than the C27 and 4T1 cell lines. In addition, the human cancer cells had higher expression than mice cancer cells. These results provide a basis for further research on the imaging and precise therapy using antibody targeting.

Figure 6
Figure 6:
The intracellular effect of antibody-modified material (P-USM-A). (A) Confocal microscopy images of A549 cells co-incubated with P-USM-A (500 μg/mL) for different durations (1, 3, and 5 hours), (B) and the corresponding Pearson's R value of the A549 cells cultured with P-USM-A for different times (1, 3, and 5 hours). Note that the Pearson's R value relates to the intracellular effect. All of the images have the same magnification. P-USM=polymer-modified upconversion [email protected]@manganese dioxide, P-USM-A=antibody modified P-USM.

We demonstrated the in vitro biocompatibility, ROS generation, and phototherapy effect of P-USM. The fluorescent probe DCFH-DA was used to detect intracellular ROS. The ROS production of the nanoplatform under laser irradiation was first detected at the cell level (Fig. 7A). The intensity of green fluorescence and the overall average fluorescence intensity (which indicates the amount of ROS produced) increased with laser irradiation time. P-USM was incubated with A549 cells for 12 hours with and without NIR laser irradiation, and an MTT assay was performed. As shown in Fig. 7B, the cell survival rate was high from P-USM concentrations from 31.25 to 500 μg/mL, indicating that the material without laser irradiation was biocompatible. After co-incubating P-USM (250 μg/mL) and cells with laser irradiation treatment (Fig. 7C), the survival rate gradually decreased with laser irradiation time, demonstrating that the material has a good phototherapy effect. We then used Trypan blue to stain dead cells to determine the photodynamic effect of the material (Additional Fig. 8, http://links.lww.com/JR9/A23). After 10 minutes of laser irradiation, a large number of cells died, which indicates that the material has a good photodynamic effect under NIR irradiation and can kill cancer cells. The effect of the material on the invasion and metastasis of cancer cells was verified by scratch experiments (Fig. 8). Relative to the control group, the application of the material with irradiation had an inhibitory effect on the invasion and metastasis of cancer cells, whereas the control group without treatment and the group with P-USM materials intracellular had almost no effect on cell growth.

Figure 7
Figure 7:
In vitro ROS generation and phototherapy effect of P-USM. (A) Quantification of the amount of ROS produced in cells under laser irradiation at different time points. (B, C) Viability of cells incubated with P-USM at different concentrations without (B) and with (C) near-infrared laser irradiation measured at different time points using an MTT assay. The scale bar is 50 μm. MTT=(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, P-USM=polymer-modified upconversion [email protected]@manganese dioxide, ROS=reactive oxygen species.
Figure 8
Figure 8:
In vitro phototherapy of P-USM using the cell scratch method. The microscopy images show A549 cells at different incubation time points. NIR=near infrared, P-USM=polymer-modified upconversion [email protected]@manganese dioxide.

These results indicate that the nanoplatform had good biocompatibility, the ability to inhibit cell invasion, and can generate ROS under laser irradiation, leading to a good phototherapy effect.

In vivo immuno-, photothermal, and photodynamic therapy

The photothermal effect of P-USM was demonstrated, first in vitro and then in vivo. As shown in Additional Fig. 9, http://links.lww.com/JR9/A23, we irradiated P-USM for 0, 1, 2, and 5 minutes. The temperature (measured using a thermal trigger) increased with time. Relative to PBS, the absolute temperature of the P-USM increased by 6°C in 5 minutes, which proved that P-USM has a photothermal effect. The same temperature rise of 6°C for irradiation for 5 minutes was also recorded in vivo (Additional Fig. 10, http://links.lww.com/JR9/A23).

Figure 9 shows the effect of in vivo therapy for treating tumors in mice. We set up four groups: a control; administration of the P-USM-A material under laser irradiation (‘material + NIR’); administration of anti-PD-L1 combined with laser irradiation (‘anti-PD-L1+NIR’); and the material (P-USM-A) with NIR laser and anti-PD-L1 separately injected after the phototherapy treatment (‘anti-PD-L1+NIR+material’). In Figure 9A, it can be seen that the weight of the mice in each group gradually increased with time, indicating that the material had no serious side effects on growth.

Figure 9
Figure 9:
In vivo anticancer therapy effect. (A) Body weight curves and (B) tumor size curves of mice in the four groups. (C) Hematoxylin-eosin stained images of the heart, liver, spleen, lung, and kidney in the four groups. Scale bars: 50 μm. NIR=near infrared, PDT=photodynamic therapy.

After the tumor-bearing mice were treated for 21 days (Fig. 9B), the tumor size of the material + NIR group increased more slowly than the control group over the first 7 days, and more quickly than the control group after day 10. This indicates that the material had a certain inhibitory effect on tumor growth under laser irradiation but single phototherapy was not particularly effective. The tumors in the anti-PD-L1 + NIR group grew more quickly than those of the control group, which indicates that the single anti-PD-L1 immunotherapy did not impair the treatment efficiency on tumor growth and that NIR irradiation may have stimulated tumor growth and partly destroyed the antibodies. In contrast with the other three groups, the tumor growth was significantly inhibited by the combined treatment of the anti-PD-L1 + NIR + material group. We note that we injected the anti-PD-L1 separately because we found that the immunotherapy was more effective without irradiation of the antibody. We also monitored the growth of the mice in each treatment group (Additional Fig. 11, http://links.lww.com/JR9/A23). One mouse in the combination treatment group and one mouse in the immunotherapy group died early owing to intravenous injection of anti-PD-L1, indicating that there may be some risk of stimulating the immune system with this antibody. After treatment, the organs (heart, liver, spleen, lung, and kidney) of the mice were removed for hematoxylin and eosin staining analysis. As shown in Figure 9C, no abnormalities were observed. These results indicate that the combination of P-USM and anti-PD-L1 had a good therapeutic and inhibitory effect on mouse tumors, and that the single immunotherapy and single phototherapy were less effective for larger tumors. In summary, the strong inhibition effect of the synergistic immuno-phototherapy treatment is due to the early phototherapy effect and longer-term effects of immunotherapy.

Discussion

We optimized UCNPs and manganese composite particles with a porous structure as a theranostic nanoplatform for synergistic immunotherapy and phototherapy. This material displayed a good response to GSH and H2O2, with photoluminescence recovery analysis being more sensitive than absorbance analysis. We used the immune checkpoint inhibitor anti-PD-L1 to specifically bind to the PD-L1 receptor on the surface of cancer cells. For in vivo tumor treatment, the combined immuno-phototherapy group achieved excellent results. In particular, the immunotherapy is important when the laser intensity in phototherapy treatment is low, whereby there is insufficient energy transfer between the energy donor and accepter. In this case, the immunotherapy overcomes the limitations of the single treatment.

Our immuno-phototherapy platform three notable advantages: (i) The phototherapy has a rapid and targeted (by the laser) anticancer effect. When combined with long-term immunotherapy, the overall anticancer effect can be enhanced. (ii) The phototherapy has less severe side effects on normal cells and organs than conventional treatments, such as chemotherapy. (iii) Phototherapy can be used for early diagnosis and surgery navigation, which is an advantage over chemotherapy and targeted therapy. Thus, our results provide a good basis for the future use our imaging-guided immuno-phototherapy nanoplatform for the diagnosis and treatment of lung adenocarcinoma.

There are several limitations of our platform:

  • (1) Although the glutathione-sensitivity of photoluminescence analysis was higher than that of absorbance analysis, the real sensitivity of the lanthanide-semiconductor is lower than other molecular dyes owing to the structure of the inorganic nanomaterials.
  • (2) The immuno-phototherapy results were not as reliable as in our previous studies.[56] Moreover, the immuno-phototherapy was not as effective as other combination strategies, such as the immuno-chemotherapy.[57] This is may be due to the different cancer cells used. Moreover, the type of photosensitizer and laser, and pump powers may play an important role in determining the effectiveness of the platform.

Acknowledgments

None.

Author contributions

YW, WL, and RD did the synthesis experiments. YW and BL participated in the material characterization. YW and WL wrote the manuscript together. RL managed the biological characterization and revised the paper. All of the listed authors have given approval to the manuscript.

Financial support

This work was supported by the National Key Research and Development Program of China Grant (Nos. 2017YFC1309100, 2018YFC0910602, and 2017YFA0205202) and the Natural Science Foundation of China (Nos. 81801744 and 91859202).

Institutional review board statement

This study was approved by the School of Pharmacy's Ethics Committee of Tumor Hospital of Shaanxi Province, Xi’an Jiaotong University, China (approval No. XJTULAC2020-585) on April 2, 2020.

Declaration of participant consent

The authors certify that they have obtained the patient consent forms. In the form, patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity.

Conflicts of interest

The authors declare that they have no conflicts of interest.

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Keywords:

Anti-PD-L1 antibody; glutathione; immunotherapy; phototherapy; upconversion nanoparticles

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