Nanoliposomes co-encapsulating Ce6 and SB3CT against the proliferation and metastasis of melanoma with the integration of photodynamic therapy and NKG2D-related immunotherapy on A375 cells

DPPC, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethyleneglycol)-2000] (DSPE-PEG2000), DOTAP, and cholesterol were purchased from Avanti Polar Lipids (USA), Ce6 was purchased from Luminescence Technology (China), and SB-3CT was obtained from TargetMol, Boston, MA (USA).

Lip-SCs were synthesized using a film dispersion and extrusion method according to our previous study [29]. Briefly, a mixture of DPPC, DSPE-PEG2000, DOTAP, and cholesterol in a mass ratio of 15:3:3:4 (total mass, 12.5 mg) was dissolved in chloroform and incubated for 5 min CHCl₃ was removed under nitrogen protection to form a thin phospholipid film. Phospholipid films were hydrated with 1 ml phosphate-buffered saline (PBS) at 48 °C for 20 min. For Ce6 and/or SB-3CT encapsulation, 5 mg ml⁻¹ Ce6 or 3 mg ml⁻¹ S⁻¹ B⁻¹ 3CT in acetone was mixed with chloroform solution to form thin lipid films, followed by hydration. After hydration, liposomes with uniform particle sizes were obtained by extrusion 11 times through 100 nm polycarbonate membranes using a mini-extruder system (Avanti Polar Lipids, Inc., AL, USA). The morphology of the liposomes was characterized using cryogenic transmission electron microscopy (Cryo-TEM) (Talos F200C, FEI). The size and polydispersity index (PDI) were characterized using dynamic light scattering (DLS) (Mastersizer 3000, Malvern Instruments, UK).

The encapsulation efficiency was measured using fluorescence spectroscopy (F-4500, Hitachi, Japan) for Ce6 and ultraviolet–visible spectrophotometry for SB-3CT.

$$\begin{eqnarray*}&&\begin{array}{l}{\rm{EE}}\left( \% \right)\\ =\,{\rm{mass}}\,{\rm{of}}\,{\rm{loaded}}\,\mathrm{drug}/\mathrm{mass}\,{\rm{of}}\,{\rm{added}}\,{\rm{drug}}\times 100 \% .\end{array}\end{eqnarray*}$$

The PDT-triggered Ce6 release of liposomes was measured via dialysis. Briefly, 1 ml Lip-SC solution was injected into dialysis devices with a cutoff of 100 kDa (Float-A-Lyser, Spectrum Labs) in 2 l PBS at 37 °C for 48 h. PDT irradiation was performed using a 660 nm diode continuous laser (50 mW cm⁻², 5 min) 2 h after the beginning of dialysis. The concentration of Ce6 remaining in the dialysis devices at different time points was measured using fluorescence spectroscopy.

A375 (human skin melanoma cell line) and NK-92MI (NK cells from patients with human malignant non-Hodgkin's lymphoma) cells were purchased from American Type Culture Collection (Virginia, USA). A375 cells were incubated in DMEM (Hyclone, GE Healthcare Bio-Sciences Corp., Massachusetts, USA) containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Massachusetts, USA) and 1% penicillin-streptomycin (10 000 U ml⁻¹) in a 37 °C/5% CO₂ humidified chamber. NK92-MI cells were grown in minimum essential medium alpha (Gibco, Thermo Fisher Scientific, Massachusetts, USA) with 0.2 mM Inositol, 0.1 mM β-mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum, and 12.5% FBS.

Singlet oxygen (¹O₂) production was measured using Singlet Oxygen Sensor Green (SOSG), a fluorescent ¹O₂ probe (Invitrogen, USA). SOSG (final concentration, 10 μM) was mixed with 1 μg ml⁻¹ Ce6 or Lip-SC (containing 1 μg ml⁻¹ Ce6) and exposed to 660 nm laser irradiation (50 mW cm⁻²) for 1, 2, and 5 min. The fluorescence intensity was measured using fluorescence spectroscopy (Ex/Em = 504/525 nm).

Cellular uptake was characterized using fluorescence spectroscopy and fluorescence microscopy. To quantitatively measure the cellular uptake of Ce6-loading liposomes, A375 cells were seeded in 6-well plates with 1 × 10⁵ cells ml⁻¹ and incubated with Lip-SC at 37 °C. Then, the cells were collected at different time points (4 h, 6 h, 8 h, 10 h, and 12 h) for fluorescence spectrum analysis. For fluorescence microscope detection, A375 cells were seeded in confocal dishes and incubated with Lip-SC for 8 h. The nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, Invitrogen, USA) for 5 min. Then, the cells were washed with PBS to be observed under a fluorescence microscope (ECLIPSE Ti, Nikon Corporation, Japan).

Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect intracellular ROS generation in A375 cells, which was characterized using fluorescence microscopy. The A375 cells were seeded in confocal dishes at a density of 1 × 10⁵ cells ml⁻¹ and incubated with 1 μg ml⁻¹ Ce6, Lip-Ce6, and Lip-SC (containing 1 μg ml⁻¹ Ce6) for 4 h. Untreated cells were considered controls. After incubation, the cells were washed with PBS and then treated with 660 nm laser irradiation (50 mW cm⁻²) for 5 min. Fluorescence images were captured using a fluorescence microscope under fluorescein isothiocyanate (FITC) channels.

In vitro cytotoxicity was measured using the Cell Counting Kit-8 (CCK-8) assay. A375 cells were seeded in a 96-well plate and treated with DMEM containing Lip-S, Lip-Ce6, and Lip-SC, with different concentrations, for 8 h. The cells were divided into two groups: one group was irradiated with 660 nm laser for 5 min and further incubated for 24 h. The other group was not irradiated and was incubated for 24 h. For viability tests, CCK-8 was added to each well, which was subsequently incubated for 1 h. The absorbance at 450 nm was measured on a microplate reader (Multiskan FC, Thermo Fisher Scientific, USA). Cell viability was calculated as follows:

$$\begin{eqnarray*}\begin{array}{lll}{\rm{Cell}}\,{\rm{viability}}\left( \% \right) & = & \left(\left[{\rm{A}}\right]{\rm{sample}}-\left[{\rm{A}}\right]{\rm{blank}}\right)/\\ & & \left(\left[{\rm{A}}\right]{\rm{con}}-\left[{\rm{A}}\right]{\rm{blank}}\right)\times 100 \% \end{array},\end{eqnarray*}$$

where [A]sample refers to OD450 values of the cells under different treatments, [A]con refers to OD450 values of the cells only, and [A]blank refers to OD450 values of the CCK-8 solution.

For live–dead cell staining, A375 cells were seeded in 35 mm diameter confocal dishes and incubated with Lip-S, Lip-Ce6, and Lip-SC for 8 h. Then, the cells were treated in the dark or with irradiation (660 nm, 50 mW cm⁻²), followed by incubation for another 24 h. After that, co-staining of calcein-acetoxymethyl ester (AM) and propidium iodide (PI) was performed. Fluorescence images were captured using fluorescence microscopy.

The enhanced PDT effects of Lip-SC on A375 cells were estimated using flow cytometry. A375 cells (1 × 10⁶ cells ml⁻¹) were seeded on 24-well plates overnight and treated with blank liposomes, Lip-SB3CT, Lip-Ce6, or Lip-SC for 8 h and exposed to a 660 nm laser (50 mW cm⁻², 5 min), followed by incubation for another 12 h. Then, the cells were harvested and stained using the Annexin V-FITC/PI Apoptosis Detection Kit (V13241, Thermo Fisher Scientific, Massachusetts, USA). The treated cells were analyzed using the FACScan system (BD Biosciences, NJ, USA), an automated flow cytometer.

500 μl blood sample was obtained from mouse and collected into a test tube containing heparin sodium. Then centrifugation at 3000 rpm for 10 min to precipitated the erythrocytes and wash with PBS twice. The precipitate of erythrocyte cells was resuspended by 100 μl PBS. Next, 10 μl of erythrocyte cells were treated with 1 ml PBS containing various concentration of Lip-SC (4, 2, 1, 0.5 and 0.25 μg ml⁻¹). After incubation at 37 °C for 4 h, the mixtures in tubes were centrifuged at 3000 rpm for 10 min and then taken photos. The absorbance at 540 nm of the supernatant was measured. The absorbance is based on hemoglobin solubilized from the erythrocytes. Thus, the absorbance corresponds to the number of hemolyzed erythrocytes. Hemolysis induced with ddH₂O and PBS was taken 100% and 0%, respectively.

A375 cells were seeded in 6-well plates and treated with Lip-S, Lip-Ce6, and Lip-SC. After 48 h, the cells were washed twice with PBS, and the cells in each group were lysed with the radioimmunoprecipitation (RAPI) assay buffer containing 1 mM phenylmethanesulfonylfluoride (Solarbio, China). Protein samples (20 μg) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels. Then, the proteins were transferred onto a nitrocellulose filter (NC) membrane. After preincubation in blocking solution (5% milk) at room temperature for 1 h, the NC membrane was incubated with rabbit anti-human monoclonal antibody of MICA, MICB, ULBP1, ULBP2, MMP2, and MMP9 (1:1 000) (Abcam, UK) and rabbit anti-human β-actin (Cell Signaling Technology Inc., USA) at 4 °C overnight. After being washed thrice for 10 min with Tris-buffered saline containing 0.1% Tween-20 (TBST) (Solarbio, China), the membrane was incubated with a secondary antibody linked to horseradish peroxidase (goat anti-rabbit immunoglobulin G; Jackson Immunoresearch Laboratories Inc., USA) for 1 h at room temperature. After washing with TBST thrice for 10 min, the membranes were incubated with the substrate of chemiluminescence kit (Pierce ECL, Thermo Fisher Scientific, Massachusetts, USA). Images were captured using ClinxChemiScope (Clinx Science Instruments Co. Ltd, Shanghai, China). The grayscale band value was measured using Image-Pro Plus (Media Cybernetics, Inc., Maryland, USA).

The cell culture supernatant was collected before or after 48 h exposure to the various treatments. Soluble major histocompatibility complex class I-related chain A and B (MICA and MICB) were measured using Human MICA ELISA Kit and Human MICB ELISA Kit (Solarbio, China) according to the manufacturer's instructions. Plates were read at OD₄₅₀ nm.

The inhibition of tumor cell migration was determined using adhesion and invasion assays. A375 cells were incubated with liposomes and treated with 660 nm laser irradiation (50 mW cm⁻²) for 5 min After being treated for 48 h, the cells were collected and counted.

For adhesion assay, the cells (1 × 10⁵ cells/well) were added to 96-well plates, incubated for 30 min, and washed with PBS to remove nonadherent cells. The remaining cells were stained with 0.1% crystal violet for 5 min and then dissolved using 30% acetic acid. Absorption was obtained at 590 nm. The percentage of inhibition was expressed using 100% being the control.

Cells (5 × 10⁵ cells/well) treated with serum-free medium were seeded into transwell upper chambers (Corning, Inc., NY, USA) with 8 μm pore membranes coated with Matrigel, and 600 μl medium with 20% FBS was added to the bottom chamber. The cells were allowed to invade for 9–12 h at 37 °C. The invaded cells were stained with 0.1% crystal violet and photographed.

NK cell-mediated cytotoxicity was measured using lactate dehydrogenase (LDH) release assay. Briefly, the A375 cells were incubated with Lip-SB3CT, Lip-Ce6, and Lip-SC in a 96-well plate and then treated in dark or with 660 nm laser irradiation. NK cells were incubated with treated target cells for 4 h at a 4:1 E–T ratio. After 4 h of incubation, 100 μl supernatant was transferred to a new 96-well plate. LDH released in the supernatant was detected using the LDH Detection Kit according to the manufacturer's instructions.

BALB/c nude mice (4−6 weeks old, 17 ± 4 g, male and female in half) were purchased from Xi'an Keaoke Biological Technology Co., Ltd (Xi'an, China) for animal studies. All animal procedures were approved by the Animal Ethics Committee of Xi'an Jiaotong University. A total of 1 × 10⁶ A375 cells in 200 μl of PBS medium were hypodermically injected into the subcutaneous tissue of all mice.

In vivo fluorescence imaging of mice was initiated as the tumor volume increased to approximately 200 mm³. Ce6 and lip-Ce6 (100 μl, 200 μmol l⁻¹) were injected intravenously into tumor-bearing mice. The mice were imaged by an IVIS Spectrum (Perkin Elmer) at different post-injection times (0, 2, 8, 12, 24 h). In addition, 24 h after injection, the mice were dissected, and tumor tissues and main organs (i.e. heart, liver, spleen, lung, and kidney) were imaged ex vivo. During the imaging process, the excitation wavelength was 645 nm and the collected emission wavelength was 680–720 nm.

The results were statistically analyzed using GraphPad Prism 9 (GraphPad Software, Inc., California, USA). The results are expressed as mean ± standard deviation. Comparisons between multiple groups were conducted using one-way analysis of variance. Statistical significance was defined as p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

The liposomes were prepared using a film dispersion and extrusion method. The chemical structures and molecular weights of lipids, Ce6, and SB-3CT are shown in figure S1 (available online at stacks.iop.org/NANO/32/455102/mmedia). The Cryo-TEM images showed that the liposomes could keep regular morphologies (figure 2(a)). Meanwhile, no aggregates were observed in the Cryo-TEM images. As seen in the DLS results (figure 2(b)), the sizes of the liposomes were approximately 140 nm with PDIs of approximately 0.05 (table 1). Figure S2a implies the successful loading of Ce6 and the synthesis of Lip-SC. The encapsulation of Ce6 by liposome was further investigated by FT-IR spectra (figure S2b). Compared to the spectra of Ce6 and the mixture (blank liposome and Ce6), the characteristic vibrations band of Ce6 at 1600 cm⁻¹ disappeared in that of lip-Ce6 due to the embedding, confirming successful encapsulation of Ce6. Liposomes had high drug-encapsulating efficiency for both SB-3CT and Ce6 (table 1 and table S1). When Ce6 and SB-3CT were mixed with the phospholipid at drug/lipid (D:L) ratios (mass/mass) of 1:20 and 1:10, the drug loading efficiency could reach 87.28% and 66%, respectively. However, when co-encapsulated, the encapsulation rates slightly decreased to 71.3% for Ce6 and 60.01% for SB-3CT.

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To evaluate the stability of liposomes, we investigated changes in sizes after 2 weeks. After being stored at 4 °C for 14 d, the particle sizes did not change significantly, indicating that the prepared liposomes had good stability (figure 2(c)). Additionally, figure S2c proves the long-term (50 h) release of Ce6 from Lip-Ce6 with or without irradiation. The release of Ce6 was slow without irradiation; approximately 15.2% of Ce6 had been released from liposomes after shaking for 5 h in a dialysis bag and up to 22.3% after 10 h. However, the release efficiency increased sharply from 15.3% to 43.7% after irradiation for 5 min and reached the maximum efficiency of 81.4% at 30 h.

Due to low solubility, the hydrophobic Ce6 was encapsulated into the lipid bilayer of the liposomes, and the presence of Ce6 in the lipid bilayer makes the lipid shell of liposomes susceptible to disruption by laser irradiation, allowing the encapsulated drugs to effectively escape from the liposomes. To test the release of liposomes, they were incubated with PBS at 37 ℃ with different stimuli. Under the 660 nm laser irradiation, a drastic increase in Ce6 was observed in the surrounding medium. The Ce6 released was approximately 86.73% at 12 h after irradiation with 660 nm laser, whereas it was approximately 38.26% without irradiation (figure 2(d)). Additionally, the fluorescence spectra of the released Ce6 from Lip-Ce6 were also examined to prove the release of Ce6 under different conditions (figure S2d).

The generation of SOSG in liposomes under 660 nm irradiation was studied. The fluorescence intensity of Lip-Ce6 and Lip-SC were higher than that of free Ce6, and both exhibited the same ROS generation ability (figure 2(e)). Meanwhile, the fluorescence intensity of Lip-SB3CT remained extremely low after irradiation. Afterward, to confirm the antitumor effect of liposomes, the ROS levels generated by free Ce6, Lip-Ce6, and Lip-SC in A375 cells after laser irradiation were detected using fluorescence microscopy. Lip-Ce6 and Lip-SC generated higher ROS levels in A375 cells than free Ce6 after 660 nm laser irradiation (50 mW cm⁻², 5 min), as represented by the fluorescence signal (figure 2(f)).

The effective uptake of nanoliposomes by tumor cells is important for tumor treatment. To examine the cellular uptake efficiency of Lip-SC, fluorescence spectroscopy was used to detect the fluorescence intensity of Ce6 contained in the A375 cells after incubation for 4, 6, 8, 10, and 12 h. The fluorescence intensity of Ce6 gradually increased in a time-dependent manner within 4–8 h and reached a maximum intensity at 8 h (figure 3(a)). Then, the fluorescence intensity decreased with the extension of incubation time. These data indicated that Lip-SC is effectively taken up by A375 cells. Simultaneously, the distribution of Lip-SC in A375 cells after 8 h incubation was evaluated using fluorescence microscopy. An obvious fluorescent signal of Ce6 can be observed from the cells (figure 3(b)), and the results were in line with those of fluorescence spectroscopy. Moreover, Ce6 was mainly distributed in the cell membrane and cytoplasm after release.

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To assess the toxicity of nanoliposomes to A375 cells, cell viability was measured using the CCK-8 assay. In the absence of laser irradiation, blank liposomes show non-cytotoxicity to A375 cells, with cell viability as high as 92%, indicating that blank liposomes had good biocompatibility and no obvious cytotoxicity to A375 cells. Meanwhile, SB-3CT, as an MMP inhibitor, displayed dose-dependent toxicity (figure 4(a)). Comparatively, lower cytotoxicity effects were observed of Ce6 with a concentration lower than 1 μg ml⁻¹. To examine the effects of PDT, cells with different treatments were exposed to laser irradiation. Blank liposomes were non-cytotoxic to cells (figure 4(b)). Lip-SC-treated cells showed the highest cell death rates compared with those treated with Lip-Ce6 at the same concentration. Lip-Ce6 and Lip-SC showed a dose-dependent cytotoxicity, and the cell survival rates dropped to 42.6% ± 0.7% and 46.4% ± 3.7%, respectively, at a dose of 0.1 μg ml⁻¹. Meanwhile, the rates decreased to 10.3% ± 1.0% and 4.2% ± 0.5% for Lip-Ce6 and Lip-SC, respectively, at a concentration of 0.4 μg ml⁻¹.

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Subsequently, the cell-killing effect of liposomes was examined using live–dead cell co-staining. For live–dead cell co-staining, PI stains dead cells and emits red fluorescence, whereas calcein-AM stains live cells and emits green fluorescence. A large percentage of cells treated with blank liposomes were stained with calcein-AM, indicating that blank liposomes did not cause significant cell death (figure 4(c)). Compared with control cells, the number of cells treated with Lip-SB3CT stained with calcein-AM decreased, indicating that SB-3CT inhibited cell proliferation rather than killing effect. Once the 660 nm laser was applied, in the Lip-SC group, most cells were stained with PI, which showed greater significant toxicity than any single treatment (PDT or SB-3CT alone). These results proved that Lip-SC plus irradiation treatment had the most effective cancer-killing efficacy, which were consistent with the CCK-8 results.

Moreover, the anticancer efficacy was also studied using Annexin V-FITC/PI staining using flow cytometry. Blank liposomes induced little apoptosis (6.7%) in A375 cells after irradiation (figure 4(d)). Lip-SB3CT caused apoptosis regardless of the presence (54.4%) or absence (55.2%) of laser irradiation, and most apoptotic cells were in early apoptosis (38.9% and 43.0% with or without irradiation, respectively). However, compared with cells treated with Lip-SC without irradiation, cells that underwent laser irradiation had a sharp decrease in early apoptosis (from 46.8% to 30.3%) and moved to late apoptosis (from 3.63% to 25.2%), indicating significant cell apoptosis.

In addition, we performed the hemolysis test to assess biocompatibility of the Lip-SC nanoliposomes. The percentage of hemolysis induced by Lip-SC nanoliposomes was lower than 2% even at the concentration of 4 μg ml⁻¹ (figure S3). This indicated the low hemolytic and excellent biocompatibility of Lip-SC.

The escape of tumor cells from the primary tumor to the metastatic site is a complex multistep process, with reduced cell adhesion and increased migration and invasion capabilities. To determine the effects of Lip-SC treatment on melanoma metastasis, cell adhesion and invasion assays were performed. Lip-SB3CT can significantly inhibit the cell adhesion (44.27% ± 0.96%) and invasion (75.67% ± 6.49%) abilities of A375 melanoma cells (figures 5 and S4). Before light was applied, Lip-Ce6 had no effect on cell adhesion (91.48% ± 6.11%) and invasion (97.81% ± 5.71%). After exposure to 660 nm laser for 5 min, the cell adhesion (71.39% ± 3.49%) and invasion (64.78% ± 1.65%) abilities of the Lip-Ce6 group were significantly reduced. Additionally, the cell adhesion and invasion abilities of the Lip-SC group after light exposure were also lower than those without light, although no significant difference was observed. Therefore, combining PDT with MMP inhibitor treatment could efficiently inhibit cancer cell adhesion and invasion.

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Several mechanisms have demonstrated that high concentrations of soluble NKG2DLs derived from tumor cells may inhibit tumor immunity and NK cell-mediated target cell lysis by downregulating the expression of NKG2DLs or proteolysis of MICA or MICB, thereby contributing to tumor immune escape. MMP participates in the shedding of soluble NKG2DLs, making it an immunosuppressive molecule that mediates the escape of malignant tumors. SB-3CT can inhibit the production of soluble NKG2DLs by inhibiting the synthesis of MMP-2 (figure S5a). Therefore, to understand the immunomodulatory effects of SB-3CT, the relationship between NKG2DL expression and MMP activity in A375 cells was examined after Lip-SC treatment. The upregulation of MMP activity can induce the downregulation of NKG2DL expression in A375 cells, decreasing sensitivity to NK cells and promoting tumor cells to escape. SB-3CT can significantly inhibit the expression of MMP-2, thereby inducing the expression of NKG2DL (figure S5b), while reducing the production of soluble NKG2DLs (figure S5c); thus, NK cells can better recognize and kill tumor cells.

Lip-SB3CT and Lip-SC can inhibit the expression of MMP-2 (figure 6(a)). Simultaneously, Lip-SB3CT and Lip-SC can restore the expression of MICB and ULBP2. Before light exposure, Lip-Ce6 did not affect the expression of MMP-2 and MMP-9 but can induce the expression of MICB and ULBP2; meanwhile, after 660 nm laser irradiation, Lip-Ce6 can inhibit the expression of MMP-2, and the specific mechanism has been further explored. Additionally, Lip-Ce6 can significantly induce the expression of ULBP2 after laser exposure. Before light exposure, Lip-SB3CT and Lip-SC can inhibit the production of soluble MICA, and the production of MICB changed significantly (figure 6(b)). After exposure to 660 nm laser for 5 min, Lip-Ce6 can also inhibit the production of soluble MICA (figure 6(c)), which is consistent with the suppression effects of MMP-2 expression on Western blotting. Similarly, the generation of soluble MICB is not affected by light. Thus, drug-loaded liposomes (i.e. Lip-SB3CT, Lip-Ce6, and Lip-SC) can inhibit the expression of MMP-2, increase the expression of ULBP2, and, simultaneously, inhibit the production of soluble MICA.

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Though Lip-SC improved NKG2DL expression on the surface of A375 cells, NK cell-mediated cytotoxicity assays were performed to directly verify the enhancement of NK-related immunotherapy by Lip-SC. When A375 cells were treated with Lip-SB3CT and Lip-SC, significant enhancements of NK cell-mediated killing were observed, whereas for cells treated with Lip-Ce6 without irradiation, no enhancements of NK cell-mediated killing were observed (figure 7), which is in line with the ULBP-2 and MICA/MICB data in figure 5. However, cells exposed to 660 nm laser were significantly more sensitive to NK cell lysis (figure 7(b)). Approximately 85.67% ± 2.46% of cells treated with Lip-SC plus irradiation were killed by NK cells, whereas only 62.24% ± 3.43% of cells without treatment were killed. Together, these results suggest that Lip-SC sensitizes A375 cells that are targets of NK cell-mediated killing.

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In order to investigate the anticancer feasibility of Lip-SC in vivo, we explored the biodistribution of nanoliposomes by tumor imaging using A375 tumor-bearing nude mice as animal models. As shown in figure 8, distinctly fluorescent signals were observed in tumor areas after an intravenous injection. Lip-SC nanoparticles were accumulated in tumor 8 h post-injection and sustained inside tumor for 24 h (figure 8(a)). As to free Ce6 treated mice, little to no tumor accumulation was observed. It is clearly shown that there are still strong fluorescence signals at the time of 24 h, which suggested that the Lip-SC could be retained for a long time after accumulation in tumor areas.

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24 h after intravenous injection, the mice were removed and the biodistribution of Lip-SC in the tumor and other tissues was studies. The main fluorescence was observed in the tumor, while some fluorescence existed in the liver and lung. On the contrary, there is little fluorescence in the heart, spleen, and kidneys. The mean fluorescent signal of Lip-SC in tumor tissue and other organs is demonstrated in figure 8(b). From the results, Lip-SC treated mice indicated higher tumor uptake than free drugs. Other signals were observed mainly in liver due to drug metabolism. In a word, lip-SC were able to efficiently accumulate and be retained in tumor areas after intravenous injection, whose outcome is beneficial for anticancer applications in vivo.