| Cite this article as: |
Yi Tian, Zhiguang Fu, Xiaosheng Zhu, Chunjing Zhan, Jinwei Hu, Li Fan, Chaojun Song, Qian Yang, Yu Wang, and Mei Shi, Establishment of NaLuF4:15%Tb-based low dose X-PDT agent and its application on efficient antitumor therapy, Int. J. Miner. Metall. Mater., 31(2024), No. 3, pp.599-610. https://dx.doi.org/10.1007/s12613-023-2717-3
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Colorectal cancer is one of the most widespread and lethal forms of cancer. The worldwide prevalence of colorectal cancer has escalated to rank third in terms of overall cancer incidence, with 1.93 million new cases worldwide in 2022 [1]. In spite of decades of laboratory, epidemiological, and clinical research, colorectal cancer rates continue to rise. In recent years, novel tumor therapy techniques, including photodynamic therapy (PDT), have emerged and introduced innovative approaches for the management of colon cancer [2].
Conventional PDT uses visible light to excite photosensitizer to generate single oxygen or active oxygen species for non-invasive tumor therapy [3–4]. In the past 60 years, PDT has become a major non-invasive therapeutic method in the treatment of superficial or endoscopically accessible tumors [5−6]. However, due to its limitation of penetration depth, PDT cannot be applied in deep-seated or big tumors [7]. For the past few years, X-ray excited photodynamic therapy (X-PDT), which uses X-ray instead of visible light as the irradiation source, has overcome the limitation of light penetration depth of conventional PDT, becoming the bravo solution of photodynamic therapy (PDT) for deep-seated or big tumor [8–10].
In the X-PDT research field, X-ray excited luminescence nanoparticles (XLNPs) were usually employed as X-ray irradiation receptor and internal illuminant to excite the coupled photosensitizer to produce singlet oxygen and eliminate tumor cells [11–12]. However, high X-ray irradiation dose caused organ lesions and side effects, which became the major barrier to X-PDT application in vivo. Numerous researches focused on preparing XLNPs with the high quantum yield to reduce the X-ray irradiation dose [13]. In our previous research [14], β-NaLuF4:Tb3+ nanoparticles (NPs), coupling with a photosensitizer rose bengal (RB), have been well established as the low dose X-PDT system, which showed significant antitumor efficiency up to 80% ± 12.3% with a total X-ray dose of only 0.19 Gy, demonstrating the feasibility of low-dose X-PDT in vivo for the first time. It was also reported in the literatures that researches focused on low dose X-PDT in antitumor therapy has received extensive attention especially in the X-ray irradiation excitation range from 0.1 to 0.5 Gy [15–16]. In such a specific range of low dose X-ray excitation (0.1–0.5 Gy, far below the radiation therapy dose), X-PDT can obtain equivalent or better efficacy than high dose irradiation of X-PDT [9].
In order to optimize the low dose X-PDT system to maximize the therapeutic efficacy, based on our previous study, the new low dose X-PDT system NaLuF4:15%Tb3+ (NLF)-MC540 was constructed which was formed by β-NaLuF4:Tb3+ NPs employed as XLNPs to excited photosensitizer MC540 (Fig. 1). The therapeutic efficacy in vitro revealed that the system could induce well antitumor efficacy in the relative low dose X-ray irradiation range (0.1–0.3 Gy). In vivo experiments demonstrated that NLF-MC540 irradiated by 0.1 Gy X-ray also resulted in significant tumor inhibition percentage. Besides, the impacts of low dose X-PDT on the tumor microenvironment and potential anti-tumor immune mechanisms were discussed.
Cysteamine (Cy), sodium hydroxide (NaOH, 98%), merocyanine (MC540), LuCl3·6H2O (99.9%), and TbCl3·6H2O were from Sigma-Aldrich. Oleic acid (OA, 90%), anhydrous ethanol (EtOH), and ammonium fluoride (NH4F, 98%) were also from Sigma-Aldrich. Enzyme-linked immunosorbent assay (ELISA), singlet oxygen sensor green (SOSG), and enzyme-linked immune spot assay (ELISPOT) were supplied from Thermo Fisher.
Based on our previous study [14], NLF were synthesized via a classical co-precipitation reaction. Briefly, 30 mL ODE and 12 mL OA were added to a flask, to which 1.7 mmol of LuCl3·6H2O and 0.3 mmol of TbCl3·6H2O were then added, and argon gas was passed through to create an inert gas atmosphere. The solution was then heated to 160°C for 1 h. After cooling to room temperature, the solution was stirred at 300 r·min–1. Add 20 mL of methanol solution dissolved in 8 mmol NH4F and 5 mmol NaOH drop by drop to the round bottom flask and continue stirring for 1 h. Slowly heat the solution to 60°C and hold for 30 min to remove the methanol, then heat the solution to 120°C under evacuation for 20 min. When the solution was cooled to room temperature, excess ethanol was added to precipitate the NLF, which was collected by centrifugation and washed three times with a 1:1 mixture of ethanol and cyclohexane, each wash followed by centrifugation at 12000 r·min–1 for 10 min. Dissolved the washed NLF in cyclohexane.
This NLF was characterized by transmission electron microscopy (TEM) at 200 kV acceleration voltage. The luminescence spectrum of NLF was measured using the fluorescent spectrometer.
According to our previous research [17], as conventional hydrophilic ligands, Cy ligands were utilized to decorate the NLF via place exchange reaction. The previously prepared NLF–cyclohexane solution was centrifuged to remove a small amount of residual OA, and the NLF precipitate was dissolved in cyclohexane. After the cyclohexane was vapored, the solid was weighed and add dichloromethane (DCM) to it. Cy was weighed into another glass vial also dissolved by DCM. The final mass ratio of NLF:Cy is 1:4. Argon gas was introduced into the two vials for 1 min, the DCM containing Cy is then quickly poured into the NLF solution, the argon gas is continued into the vials for 1 min, and the gas tip is poured deep below the liquid surface for 5 min. After stirring for 48 h, the solution of 10 mg·mL–1 NLF–Cy was obtained. The NLF–Cy solution was dialyzed in 5 L of water for 24 h using a dialysis membrane with a pore size of 800 D to give a final NLF–Cy solution of 10 mg·mL–1. The luminescence spectrum of NLF–Cy was also measured using the fluorescent spectrometer.
MC540 as the photosensitizer was coupled with NLF via electrostatic adsorption to form the X-PDT system NLF–MC540. Briefly, a solution of 50 mg MC540 in 10 mL water was prepared. Then 0.1, 0.2, and 0.4 mL MC540 solution were separately added in 10 mg NLF. After the solution was filled with water to 10 mL, the solution was stirred for 24 h at 300 r·min–1.
Add 33 mL methanol to 100 mg SOSG to make a work stock. NLF–MC540 was added into a 96-well plate (100 μg·mL–1) with 10 mM SOSG stock. The radiation dose rate of the X-ray source was 0.05 Gy·min–1. Then the plate was immediately tested.
NLF–Cy–MC540 was incubated with CT26 cells for 12, 24, 36, and 48 h, respectively. Excessive NLF–MC540 was then removed by three times phosphate buffer saline (PBS) wash.
Intracellular uptake was observed by live-cell confocal microscopy. Nuclei were stained with Hoechst 33342 (excitation/emission: 350/461 nm). Lysosomes were stained with LysoTracker Green DND (excitation/emission: 488/511 nm). MC540 (excitation/emission: 533/572 nm) can be used to monitor nanoparticles. Following treatment with NLF–MC540 for 12, 24, 36, and 48 h, cells were washed and observed.
TEM also provided evidence for cellular uptake of NLF–MC540. Cells were treated three times with NPs, fixed in 2.5% glutaraldehyde solution at 4°C for 24 h, and then soaked in PBS. Then, cells were stained for 2 h until cell granules were completely darkened with 1vol% Osmium tetraoxide. Cells were washed three times with PBS. Stained cells were dehydrated with increasing concentration gradients of ethanol and acetone for 5 min. The sections of 90 nm thickness were made with an ultra-microtome. Finally, TEM was used to observe the morphology of the cells.
After cells were incubated to apposition, 50, 100 µg·mL–1 NLF–Cy, 5, 10 µg·mL–1 MC540, 50, 100 µg·mL–1 NLF–MC540, and a blank medium control group were added for a total of 10 groups with 5 replicate wells in each group. The 96-well plates were then incubated for 12 h at 37°C in an incubator containing 5vol% CO2. The X-ray source parameters were set to 80 kV, 0.2 mA (0.1 Gy), 40 cm from the sample, and an X-ray dose rate of 0.05 Gy·min–1. Two 96-well plates were irradiated for 2 and 6 min, 0.1 and 0.3 Gy, respectively, and the other plate was not irradiated as a control group. Finally, cell viability was measured by the CCK-8 assay.
According to the results in vitro, the experiment in vivo to evaluate the efficacy of NLFin vivoMC540 with 0.1 Gy X-ray irradiation treatment was conducted. Firstly, a mouse subcutaneous tumor model was constructed using Balb/c mice and CT26 cells. The mice were divided into 8 groups (5 mice/group) after the volume of the tumor reached 50 m3: (a) saline, (b) saline+0.1 Gy X-Ray, (c) NLF, (d) NLF+0.1 Gy X-Ray, (e) MC540, (f) MC540+0.1Gy X-Ray, (g) NLF–MC540, and (h) NLF–MC540+0.1Gy X-Ray. After injected for 12 h, X-ray of the 0.1 Gy group were irradiated 0.1 Gy dose (0.05 Gy·min–1 for 2 min). The tumor volume was observed and measured every 3 d. All of the animal experiments were approved by the Animal Experiment Administration Committee of the Air Force Medical University, Shaanxi, China.
Tumor volume was calculated using the Eq. (1):
|
V=0.5×a×b2 |
(1) |
where a represents the long diameter of the tumor, and b represents the short one. To facilitate the comparison of tumor volume across various groups, the initial volume was standardized to 1. Consequently, the tumor volume (Vr) was calculated using the Eq. (2):
|
Vr=ViV0 |
(2) |
where Vi represents the volume of the tumor on the day i, and V0 represents the initial tumor volume. The mice weight was also measured every 3 d. Meanwhile, we took blood samples from the mice tail vein for blood routine examination every 3 d.
After the animal experiments, three mice were randomly selected from each group to obtain the tumor tissue. The tumors of mice had been surgically removed and then sliced after fixation and embedding. Then the sections were incubated with the target primary antibody overnight. According to the literature, Ly6G, CD11c, and CD8 as specific primary antibody were selected for finding neutrophils, dendritic cells, and cytotoxic T lymphocytes in the tumor tissue. The next day, the slides were rinsed and incubated with the secondary antibody followed by 3,3-diaminobenzidine (DAB) and hematoxylin staining, respectively.
In order to demonstrate the tumor immunity effects on antitumor efficacy in low dose X-PDT treatment, a bilateral murine tumor CT 26 model was utilized to study the inhibition of tumor metastasis with NLF–MC540 mediated low dose X-PDT.
On 7 and 14 d, CT26 cells were subcutaneously injected on the right and left side to establish primary and secondary tumors. The mice were also divided into 8 groups consisting of 5 mice per group, consistent with the groups outlined in section 2.9. The changes in tumor volume on the left and right sides were still measured at 3, 6, 9, and 12 d.
To determine whether immune cells had been activated after treatment, splenocytes were obtained from mice spleens through surgical removal and milling, followed by the preparation of single-cell suspensions. The splenocytes were stimulated based on the method in literature [18].
Afterward, the splenocytes were enumerated and co-cultured with CT26 cancer cells at ratios of 5:1, 10:1, 25:1, and 50:1 (splenocytes/CT26). Then analyzed the cell viability by the CCK-8 assay.
ELISA kits were used to measure the level of cytokine in mice serum including tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) following the kits instructions.
ELISPOT assay was used to evaluate the specific tumor immunity of isolated splenocytes. The ELISPOT plates were washed three times in PBS. Condition the plates with medium (200 μL·well–1) containing 10% serum and incubate for at least 30 min at room temperature. After removing the medium, the stimuli and splenocytes were added into the well. The following groups were studied: (1) experiment group (splenocytes+cell lysates CT26 [40 µg·mL–1]), (2) negative control group (splenocytes), (3) positive control group (splenocytes+non-specific stimulant [2 µg·mL–1 PHA]), and (4) plain control (culture medium). Then the cells were removed by emptying the plate. The detection antibody (0.5 µg·mL–1) was added 100 µL·well–1 for 2 h of incubation at room temperature. Then the plate was washed as above. Then the streptavidin-HRP (1:1000) was added 100 µL·well–1 into the plates, which were incubated for l h at room temperature. After washing as above, the plates were added 100 µL·well–1 of the 3,3,5,5-tetramethylbenzidine (TMB) substrate solution and developed until a distinct spot emerged. The color development was stopped by washing extensively. The plates were inspected and counted spots in an ELISPOT reader.
All of these experiments had three or more parallel control experiments. For independent groups, one-way analysis of variance (ANOVA) was performed using GraphPad Prism 7.0 software. The t-test was used for the comparison of two groups. When the P was less than 0.05, the analyzed groups were considered statistical differences (Statistical significance analysis P: **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05 in figures).
The NaLuF4:15%Tb3+ (NLF) were successfully synthesized via a classical co-precipitation reaction and then were characterized by TEM, high-resolution TEM (HRTEM), and X-ray Diffraction (XRD). Thus, the Fig. 2(a) depicted NLF with the average particle size of (23.48 ± 0.91) nm calculated by TEM images of over 100 nanoparticles.. In Fig. 2(b), HRTEM measured 0.294 nm interplanar spacing in the plane. By element mapping shown in Fig. 2(c), the elemental composition of NLF was characterized, which showed that the NLF consist of Na, Lu, F, and Tb elements without impurities. The frequency of diameter distribution (Fig. 2(d)) was consistent with the result of TEM image. In Fig. 2(e), XRD was used to examine the crystal structure and phase of the NLF. The diffraction peaks matched well with the values for standard reflections of β-NaLuF4 (JCPDS No. 27-0726).
The synthesized NPs could not be dispersed in water because they were wrapped with oleic acid (OA) groups, which were hydrophobic and unsuitable for biomedical applications. The mass ratio 1:4 of NLF to Cy was chosen based on our previous studies. As expected, NLF–Cy also had good hydrophilicity and Tyndall effect, as shown in Fig. 2(f)–(g).
To demonstrate the hydrophilic decoration could not influence the luminescence spectrum, the emission spectra of NLF and NLF–Cy were measured and illustrated in Fig. 3(a)–(b), respectively. The XEOL spectrum of NLF and NLF–Cy was all at the 545 nm peak. Attaining a satisfactory spectral match was also a crucial factor in achieving optimal therapeutic efficacy in X-PDT. As shown in Fig. 3(c), MC540 (characteristic absorption peak around 535 nm) had a perfect overlapping area with the emission spectra of the NLF–Cy (545 nm). So MC540 as the photosensitizer was coupled with NLF via electrostatic adsorption to form the X-PDT system NLF–MC540. As shown in Fig. 3(d), the absorption spectrum NLF–MC540 solution also had an absorption peak around 535 nm which was similar to MC540. The system NLF–MC540 was successfully constructed.
According to the above results, the NLF with desirable appearance was successfully synthesized. The reduced particle size could enhance the uptake by tumor cells. The addition of the lutetium element with high X-ray mass absorption efficiency resulted in high quantum yield of NLF at 545 nm [14]. Besides, the hydrophilic decoration by Cy did not significantly affect the luminescence quantum yield of NLF. Therefore, the system with high X-ray excited optical luminescence efficacy was constructed.
The X-PDT system NLF–MC540 was formed via electrostatic adsorption. When absorbing emission photons around 535 nm, MC540 transferred the energy to the oxygen to yield SO to finish the X-PDT. The detector SOSG of SO was employed to determine the SO generation of NLF–Cy–MC540 after X-ray excitation.
As shown in Fig. 3(e), the production of SO elevated continuously with the increase of MC540 amount in the coupling process under the irradiation of 0.3 Gy. But when the weight ratio of NLF:MC540 reached 5:1, the NLF–MC540 solution got precipitated. Therefore, 10:1 was chosen as the weight ratio of NLF:MC540.
Besides, the other crucial component of the SO production is the dose of X-rays. As shown in Fig. 3(f), the amount of SO produced by NLF–MC540 was significantly enhanced with the increase of X-ray irradiation dose. The yield of SO increased nearly linearly as the irradiation dose gradually increased. The growth rate of SO production slowed down significantly in the spectra of 0.1–0.3 Gy and reached a platform in 0.3–0.5 Gy. In summary, the system NLF–MC540 had high luminescence quantum yield and overlap in the absorption spectrum of the photosensitizer, which led to high SO production.
Due to SO having the potential to induce tumor cell necrosis and apoptosis, the production of SO is the primary factor that influences the efficacy of X-PDT. It was demonstrated that high SO production resulted in a high X-PDT efficacy of NLF–MC540. This was because the absorption wavelength of MC540 had a perfect overlapping area with the emission spectra of the NLF, thereby ensuring high X-PDT efficacy and ultimately leading to a perfect therapeutic outcome with low dose X-PDT. Besides, MC540 and some other cyanine dyes were also used in the PDT treatment of lymphoma and leukemia [19].
Effective X-PDT requires intracellular SO generation. Consequently, the initial stage of this biological process involves the endocytosis of NPs. Previous studies have indicated that the entry of NPs into cancer cells occurred through the endocytic pathway, whereby NPs were initially enclosed by membrane-bound vesicles and subsequently conveyed to endosomes or lysosomes [20]. As such, it is hypothesized that NLF–Cy-MC540 may be situated within the endosomes or lysosomes of CT26 cells.
In Fig. 4(a), a cell structure was shown in TEM images, which was the globular organelles containing a large number of intracellular dense material particles, which proved that NLF was taken up by CT26 cells and located in the lysosome. Furthermore, to test the hypothesis mentioned, immunofluorescence staining was used for the presentation of cellular uptake. The Fig. 4(b) illustrated that NLF–MC540 co-localized well with lysosomes (green spots) at 12 h. The overlapping rates of green and red pixels were around 90% (Fig. 4(c)). Thus, NLF-MC540 could be endocytosed by CT26 cells and located mainly in the lysosome.
Safety issues were addressed in vitro by CCK-8 assay. In the range of 0–100 μg·mL–1, the cell survival rate remained around 80% as NLF concentration rose. And LC50 (NLF) was estimated to be (149.8 ± 1.59) μg·mL–1 (Fig. 5(a)).
In Fig. 5(b), the therapeutic efficacy in vitro was demonstrated by cell viability evaluation under series X-ray irradiation dose excitation. The results revealed that the X-PDT system NLF–MC540 could induce well antitumor efficacy in relative low dose X-ray irradiation range (0.1–0.5 Gy). And the cell viability decreased as a function of X-ray dose, which is consistent with the well-known X-PDT effect mechanism.
At the irradiation dose of 0.1 Gy, the cell viability of the NLF–MC540 group was significantly decreased to about 49% and 28% at concentration of 50 and 100 µg·mL–1 compared with the control group, respectively (Independent-samples t-test, P < 0.001). At the irradiation dose of 0.3 Gy, the cell viability declined to 39% and 24% at the two concentrations, respectively (Independent-samples T Test, P < 0.001). Besides, no difference of cytotoxicity was observed between different irradiation dose.
In order to maximize the bio-safety extent, 0.1 Gy was selected as the irradiation dose in the in vivo X-PDT efficacy evaluation.
Surprisingly, after 12 d, the tumor inhibition percentage reached 89.5% ± 5.7% with only 0.1 Gy X-ray irradiation in NLF–MC540 treatment group. Comparatively, tumor inhibition percentage with 0.1 Gy X-ray irradiation in NLF without MC540, free MC540, and PBS were 54.6% ± 12.7%, 46.7% ± 13.6%, and 20.6% ± 16.2% (Fig. 6(a)). Upon further analysis, we found a significant difference in tumor growth between the 0.1 Gy X-ray irradiation in NLF–MC540 treatment group and the 0.1 Gy irradiation in PBS group (Two-way ANOVA, P = 0.0170), and the negative control group (Two-way ANOVA, P = 0.0007). The in vivo antitumor efficacy results demonstrated the low dose X-PDT could produce well tumor inhibition efficacy. Fig. 6(b) showed a visualization of the tumor bodies that were reduced in each group.
As shown in Fig. 6(c), during the observation period, no significant decrease in mice body weight occurred in the different groups. In Fig. 6(d), comparing hematoxylin and eosin staining (H&E staining) of major organs of 0.1 Gy X-ray irradiation in NLF–MC540 treatment group and negative control group, the system NLF–MC540 with 0.1 Gy irradiation could not be detected obvious toxicity. The systemic toxicity of the X-PDT system has not been significantly observed, indicating that the X-PDT system we constructed is basically safe for mice.
As is known to all, safety issues invalidate the X-PDT application. The safety issues mainly come from high X-ray dosage in previous research [21–22]. Thus, numerous researches focused on preparing XLNPs with high quantum yield to reduce the X-ray irradiation dose and concentration of NPs [23]. In our study, based on the high luminescence quantum yield of NLF and perfect overlap in the absorption spectrum of photosensitizer leading to high X-PDT efficacy, the system we obtained, NLF–Cy–MC540 presented excellent efficacy, even in the 0.1 Gy X-ray irradiation. By establishing NLF–MC540 system, a new platform will be provided for safe and efficient applications of X-PDT.
It was well accepted that the effect of X-PDT came from the combination or synergy of PDT, radiotherapy (RT), and the radiosensitization of nanomaterials. However, the in vivo antitumor efficacy results demonstrated the low dose X-PDT could produce well tumor inhibition efficacy again, compared with our previous studies and literature reports [14–15]. This phenomenon was not followed the X-PDT mechanism mentioned above, indicating that new therapeutic mechanism may exist in low dose X-PDT [24–25]. Tumor immune activation and microenvironment involvement may play an important role in it.
Thus, the test of the level of neutrophil in mice blood after treatment showed the significant increase of neutrophil on the third day after treatment. As shown in Fig. 6(e), the peripheral blood neutrophil level in 0.1 Gy irradiation in NLF–MC540 treatment group was significantly increased in 3 d and far exceeded the standard value of neutrophils in mice (0.1Gy NLF–MC540 group vs. 0.1Gy PBS group: One-way ANOVA, P = 0.0057; 0.1Gy NLF–MC540 group vs. 0 Gy negative control group: One-way ANOVA, P = 0.0175).
The recruitment of neutrophils in the tumor tissues indicated that acute inflammatory reaction occurred in tumor microenvironment [26]. However, it is inevitable that peripheral blood analysis contains biomarkers secreted from tissues other than tumors, which limits its specificity for the tumor microenvironment.
In order to find the changes of tumor microenvironment, the cells infiltrated in the tumor tissues by immunohistochemical staining of tumor sections was studied. In Fig. 6(f)–(h), a large number of Ly6G+, CD8+, and CD11c+ cells were infiltrated in the tumors in the group of NLF–MC540 with 0.1 Gy X-ray irradiation, which were significantly different from those in other groups (Fig. 6(i)–(k)). The number of Ly6G+ in 0.1Gy NLF–MC540 group vs. all of other group: One-way ANOVA, P ＜ 0.0001. The number of CD8+ in 0.1Gy NLF–MC540 group vs. all of other group: One-way ANOVA, P ＜ 0.01. The number of CD11c+ in 0.1Gy NLF–MC540 group vs. all of other group: One-way ANOVA, P ＜ 0.001.
As reported in literature [27], the Ly6G+ antigen was mainly expressed in neutrophils. The neutrophils especially N1 neutrophils were highly cytotoxic toward cancer cells and secreted high levels of inflammatory cytokines [27–28]. Besides, a large number of CD8+ and CD11c+ cells were infiltrated in the tumors. The CD11c+ antigen was mainly expressed in dendritic cell (DC) [29]. The CD8 antigen was mainly expressed in cytotoxic T lymphocyte (CTL) [30]. The infiltration of DCs and CTLs in tumor microenvironment may reveal that the cell fragments generated by X-PDT and the local inflammatory response were taken up by dendritic cells as antigens, which induced the maturation of DCs, and then presented antigen to activate CD8+ T cells, causing specific immunity [31].
In order to demonstrate the tumor immunity effect on antitumor efficacy in low dose X-PDT treatment, a bilateral murine tumor CT 26 model was utilized to study the inhibition of tumor metastasis with NLF–MC540 mediated low dose X-PDT (Fig. 7(a)).
Fig. 7(b)–(c) showed that low dose X-PDT inhibit the growth of both the primary tumor and the distant tumor. In the NLF–MC540 with 0.1 Gy X-ray irradiation group, the inhibition efficiency of the primary tumor reached 90.7% ± 6.3%, and the distant one reached 75.8% ± 10.1%. On the opposite, neither the primary tumors nor the distant ones were inhibited in all other treatment groups. The inhibition of the primary tumor and the distant tumor growth indicated that antitumor immunity had been efficiently triggered by low dose X-PDT.
The cancer cell killing experiments with spleen cells were performed to further verify the antitumor immunity at the cellular level. As shown in Fig. 7(d), the spleen cells from NLF–MC540 with 0.1 Gy X-ray irradiation group exhibited a superior ability to kill cancer cells. The highest cell killing rate of spleen cells was 82.51% ± 1.86% with 50:1 target ratio (spleen cells:CT26 cells). The cell inhibition rate of 0.1 Gy NLF–MC540 group had significant differences from those in negative control group under the target ratio of 5:1 (P0 Gy control vs. 0.1 Gy NLF–MC540 = 0.00003) and 10:1 (P0 Gy control vs. 0.1 Gy NLF–MC540 = 0.00106).
PDT-based immunotherapy has been widely studied [32–33]. The low dose X-PDT treatment effectively destroyed the primary tumor and concurrently stimulated a noteworthy cytokine production, thereby activating an antitumor immune response. The ELISA assay was utilized to discover the mechanism of immune activation and cytokine production in combating the tumor. Specifically, the serum levels of two immune-relevant cytokines, IFN-γ and TNF-α, were measured via ELISA assay to assess the nonspecific immune response to the tumor. The NLF–MC540 with 0.1 Gy X-ray irradiation group significantly upregulated the level of two cytokines (IFN-γ and TNF-α) while other groups did not present significant influence on these cytokine expressions (Fig. 7(e)–(f)). After low dose X-PDT, IFN-γ and TNF-α in serum were upregulated 7 and 6 times than negative control, respectively. Subsequently, the ELISPOT experiments were conducted to verify the presence of specific antitumor immunity after low dose X-PDT. The NLF–MC540 with 0.1 Gy X-ray irradiation group significantly activated the specific antitumor immunity in IFN-γ secreting immune cells. The number of spots attributable to the IFN-γ levels in the NLF–MC540 with 0.1 Gy X-ray irradiation group was (279 ± 73) spots·well–1, which is 14 times greater than that in the negative control group (20 ± 12) spots·well–1. Other treatment groups showed no significant difference in specific IFN-γ secreting compared with the control group (Fig. 7(g)). The NLF-MC540 with 0.1 Gy X-ray irradiation group also had the ability to activate the specific immune cells which secrets TNF-α at the level of (187 ± 37) spots·well–1, which is 6 times greater than that in the negative control group (28 ± 17) spots·well–1, as shown in Fig. 7(h). The same as the IFN-γ secreting, the level of TNF-α secreting in other groups showed no significant difference compared with the control group.
A variety of cytokines are important biomarkers for different immune responses. TNF-α and IFN-γ are typical markers of cellular immunity and have critical roles in tumor immunotherapy [18]. Furthermore, the use of low dose X-PDT promoted the maturation of DCs which upregulates the release of TNF-α and IFN-γ. Both innate immunity and adaptive immunity were activated by the crosstalk between TNF-α and IFN-γ [34]. Thus, the low dose X-PDT could activate potent anti-tumor immunity by immunotherapy pathway. The tumor cell fragments generated by X-PDT and the local inflammatory response were taken up by DCs as tumor associated antigens, induced the maturation of DCs, and the activation of CD8+ T cells, which inhibited the growth of distant tumors [35–36]. Due to the infiltration of DCs and CTLs in tumor microenvironment and the upregulation of the level of TNF-α and IFN-γ, we proposed the mechanism of low dose X-PDT induced immunotherapy (Fig. 1). XLNPs were as X-ray irradiation receptor and internal illuminant to excite the coupled photosensitizer to produce singlet oxygen and eliminate tumor cells, the cell fragments were generated. The neutrophils also infiltrated to play a role of cytotoxic and phagocytosis [37]. The cell fragment also led infiltration of DCs. Then DCs took up the cell fragment as antigens and became matured. Then DCs presented antigen and secretes cytokines to activate CD8+ T cells, treating distant tumors. Subsequent investigations have been conducted to elucidate the specific functions of DCs in the activation of anti-tumor immunity induced by low dose X-PDT, and the findings will be expounded upon in the forthcoming study.
This work established a novel X-PDT system NLF–MC540. In vitro and in vivo, the new system NLF–MC540 demonstrated significant tumor inhibition efficacy and biosafety when irradiated by 0.1 Gy X-ray irradiation. Furthermore, it preliminarily elucidated the mechanism of low dose X-PDT was linked to anti-tumor immunity and tumor immune microenvironment. This study provided new strategies to manage colorectal cancer, focused on optimizing treatment efficacy, minimizing safety issues, and enhancing patient outcomes.
All of the BALB/c mice were purchased from the Animal Experiment Center of the Fourth Military Medical University. All animal experiments complied with the National Research Council's Guide for the Care and Use of Laboratory Animals, and the animal experiments were approved by the Animal Care and Ethic Committee of Fourth Military Medical University (Approval NO. KY20213144-1).
This research was funded by the National Natural Science Foundation of China (Nos. 81771972, 52171243, and 52371256) and the National Key Research and Development Program of China (No. 2017YFC0107405). We also acknowledge the assistance of Jianru Chen in cell experiments.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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