Sialic acid-mediated photochemotherapy enhances infiltration of CD8⁺ T cells from tumor-draining lymph nodes into tumors of immunosenescent mice

Abstract

Immunoscenescence plays a key role in the initiation and development of tumors. Furthermore, immunoscenescence also impacts drug delivery and cancer therapeutic efficacy. To reduce the impact of immunosenescence on anti-tumor therapy, this experimental plan aimed to use neutrophils with tumor tropism properties to deliver sialic acid (SA)-modified liposomes into the tumor, kill tumor cells via SA-mediated photochemotherapy, enhance infiltration of neutrophils into the tumor, induce immunogenic death of tumor cells with chemotherapy, enhance infiltration of CD8⁺ T cells into the tumor-draining lymph nodes and tumors of immunosenescent mice, and achieve SA-mediated photochemotherapy. We found that CD8⁺ T cell and neutrophil levels in 16-month-old mice were significantly lower than those in 2- and 8-month-old mice; 16-month-old mice exhibited immunosenescence. The anti-tumor efficacy of SA-mediated non-photochemotherapy declined in 16-month-old mice, and tumors recurred after scabbing. SA-mediated photochemotherapy enhanced tumor infiltration by CD8⁺ T cells and neutrophils, induced crusting and regression of tumors in 8-month-old mice, inhibited metastasis and recurrence of tumors and eliminated the immunosenescence-induced decline in antitumor therapeutic efficacy in 16-month-old mice via the light-heat-chemical-immunity conversion.

Graphical abstract

Immunosenescence decreased the antitumor therapeutic efficacy in 16-month-old mice. Sialic acid-mediated photochemotherapy enhanced infiltration of CD8⁺ T cells and neutrophils into tumors and eliminated the immunosenescence-mediated decline in antitumor therapeutic efficacy.

1. Introduction

The world population is aging, and the problems associated with old age are becoming increasingly serious¹. After the COVID-19 outbreak, the high disease susceptibility and lower ability to recover of the elderly have resulted in high infection and mortality rates2, 3, 4. The problems associated with treating disease in the elderly have become increasingly prominent, and Nature Publishing Group has established a new sub-journal called Nature Aging to promote the dissemination of research findings in this area⁵. Aging results in the immune system gradually undergoing a series of degenerative changes stemming from the long-term combined action of multiple factors; this is called immunosenescence⁶. T lymphocyte aging is the main manifestation of immunosenescence⁷. A host of clinical data show that the health of the human body is closely related to T lymphocyte function and that T lymphocyte aging is the main cause of disease in the elderly⁸. T lymphocyte functioning is age-dependent, and their ability to recognize and eliminate harmful entities (such as microorganisms or malignant cells) is significantly reduced with age⁹. CD8⁺ T cells aging caused by immunosenescence in tumor-draining lymph nodes (TDLNs) makes it more likely that tumor cells will be able to evade immune recognition and elimination, thereby allowing them to continue to proliferate and increasing the possibility of cancer recurrence after treatment¹⁰. Additionally, innate immune cells (e.g., neutrophils, monocytes, and NK cells) are also affected by immunosenescence (see Scheme 1).

Scheme 1.

Schematic illustration of the construction, neutrophils-mediated drug delivery system, and immunosenescence-mediated antitumor therapeutic efficacy of SL-DOX/SL-ICG/SL-Co. Therapeutic effect of SL-Co could simultaneously enhance the infiltration of CD8⁺ T cells and neutrophils into the tumor, increased the accumulation of drugs at the tumor site, improved the effect of antitumor therapy, and eliminated the decline in anti-tumor therapeutic efficacy in 16-month-old mice caused by immunosenescence.

Most immunosenescence studies to date have focused on the aging of the immune system and the underlying mechanisms¹¹. The impacts of immunosenescence on the therapeutic efficacy of some age-related diseases (especially cancer) have, however, remained largely unexplored. In preclinical antitumor therapy studies, mice that are aged 4–8 weeks (equivalent to humans aged 1–16 years) are used as laboratory animals; however, this age does not match that of clinical patients. Clinical data indicate that the peak incidence of cancer in humans is at 50–70 years of age¹². Immunosenescence greatly affects the distribution of nano-drugs that are closely related to the immune system. The development and application of cancer nano-drugs have, therefore, been stymied by the lack of immunosenescence research to date13, 14, 15. In the past 20 years, our research group has been investigating the close relationship between the immune system and nano-drugs, and we have systematically researched SA-modified nanoparticles (NPs), which can be administered by neutrophils-mediated drug delivery16, 17, 18. Photodynamic/Photothermal therapy (PDT/PTT) increased the accumulation of SA-modified NPs in the tumor by enhancing tumor infiltration by neutrophils¹⁹. SA-modified DOX liposomes prepared in our laboratory-induced the expression of calreticulin (CRT), increased the secretion of HMGB-1, and promoted the release of ATP from tumor cells²⁰. Dendritic cells recognize and phagocytize tumor cells, enhance the ability of CD8⁺ T cells to infiltrate tumors, and attack the tumor cells²¹. Immunogenic cell death (ICD) of tumor cells can transform immunosuppressive or ‘cold’ tumors into immune-infiltrating ‘hot’ tumors, and some mice can even achieve tumor regression²⁰. However, the above research results were all obtained in mice aged 4–8 weeks, without considering the potential impact of immunosenescence on therapeutic efficacy.

SA-modified liposomes achieve antitumor effects by interacting with immune cells (T cells, neutrophils, and monocytes)^20,22,23. Using these liposomes as carriers to deliver drugs can help elucidate the immunosenescence-induced change in the antitumor therapeutic efficacy of phototherapy or chemotherapy. We prepared SA-modified doxorubicin (DOX)-loaded liposomes, SA-modified indocyanine green (ICG)-loaded liposomes, and SA-modified DOX and ICG co-loaded liposomes to treat C57BL/6JNifdc mice with melanoma (B16/F10 cells) that were either 2 months (adolescent, human equivalent of 16 years), 8 months (adult, human equivalent of 33 years), or 16 months (old, human equivalent of 60 years) old²⁴.

We found that 16-month-old mice exhibited immunosenescence, which affected the antitumor therapeutic efficacy of phototherapy and chemotherapy. However, SA-mediated photochemotherapy eliminated the decline in the antitumor therapeutic efficacy of phototherapy or chemotherapy caused by immunosenescence.

2. Materials and methods

2.1. Reagents, animals and cells

B16/F10 and HL-60 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). B16/F10 and HL-60 cells were cultured in RPMI 1640 medium (Procell Life Science&Technology Co., Ltd., WH0221k031, Wuhan, China) containing 10% FBS (Gemini Bio, A24G00J, Calabasas, CA, USA). Female C57BL/6JNifdc mice aged 2, 8, and 16 months were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (No.110011211103615527, No.110011211100204365, Beijing, China). All experimental procedures were executed according to the protocols approved by Shenyang Pharmaceutical University Animal Care and Use Committee.

2.2. Preparation of SA-modified liposomes

SA-modified liposomes were prepared by thin-film evaporation²⁵. Hydrogenated soybean phosphotidylcholine (HSPC, AVT (Shanghai) Pharmaceutical Tech Co., Ltd., C10695, Shanghai, China), dipalmitoyl phosphatidylcholine [DPPC, AVT (Shanghai) Pharmaceutical Tech Co., Ltd., B91150, Shanghai, China], cholesterol (CH, Xinbai Pharmaceutical Co., Ltd., 120502, Nanjing, China), indocyanine green (ICG, Dalian Meilun Biological Co., Ltd., A0807A, Dalian, China), and MT-18 were dissolved in 10% final volume (v/v) of liposomes in anhydrous methanol. A rotary evaporator (Zhengzhou Keda Machinery Instrument Equipment Co., Ltd., RE-501, Zhengzhou, China) was used to remove the organic solvents in a 60 °C water bath for 5 min. A desktop vacuum drying oven (Shanghai Jiecheng Experimental Instrument Co., Ltd., DZF-6020, Shanghai, China) was used to remove the residual methanol at 60 °C for 8 h. An ammonium sulfate solution (200 mmol/L) containing 5% methanol was added, and the mixture was stirred at 60 °C for 20 min. An ultrasonic cell pulverizer (200 W for 2 min, 400 W for 8 min; Ningbo Xinzhi Biotechnology Co., Ltd., Scientz-950E, Ningbo, China) was used to obtain the initial liposome preparations. Initial liposomes samples (100 μL) were added to the top of an anion-cation mixed-fiber column to remove free drug (1: 1, v/v, Guilin Zhenghan Science and Technology Development Co., Ltd., Guilin, China); the mixture was allowed to stand for 2 min, and a centrifuge (Xiangyi Centrifuge Instrument Co., Ltd., L 530, Changsha, China) was used to centrifuge the mixture at 500×g for 4 min. Then, 50 μL of sterile water was added for injection, centrifuged at 500×g for 4 min, and elution was repeated twice with water. After being passed through 0.80-, 0.45-, and 0.22-μm microporous membranes, the resulting product was mixed with 1 mL of DOX (Beijing HVSF United Chemical Material Co., Ltd., HF190916, Beijing, China) solution (10 mg/mL) and incubated in a 60 °C water bath for 20 min. The above process was repeated, and an anion-cation mixed-fiber column was used to remove the free drug. SA-modified ICG and DOX co-loaded liposomes (SL-Co) were obtained. Before use, 1 mL of the prepared liposomes was placed in a dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd., 10 kDa, Shanghai, China) and dialyzed against 500 mL of 5% sucrose solution for 4 h, with a change of the dialysate every 2 h. SL-Co was mixed with 10 mg/mL DiR (Dalian Meilun Biological Co., Ltd., D122OA, Dalian, China) absolute ethanol solution and then stirred at 60 °C for 20 min to prepare SA-modified triple-loaded liposomes (0.2 mg/mL DiR, SL-DiR).

The hydration medium was replaced with a 200 mmol/L ammonium sulfate solution and the above steps were repeated to prepare SA-modified DOX-loaded liposomes (SL-DOX). Similarly, the hydration medium was replaced with a 5% methanol solution and the above steps were repeated to prepare SA-modified ICG-loaded liposomes (SL-ICG).

2.3. Characterization of SA-modified liposomes

A Nicomp-380 dynamic light scattering particle sizer (PSS, Nicomp-380, Pleasanton, CA USA) was used to measure the particle size and Zeta potential of the diluent after SL-DOX, SL-ICG, and SL-Co were diluted 200 times with sterile water for injection.

Transmission electron microscopy (TEM) images of the liposomes were acquired using the microscope (JEOL Corporation, JEM-2100, Tokyo, Japan). Then, 10 μL of liposomes were placed on a 200-mesh copper grid and allowed to stand for 5 min. Subsequently, 2% phosphotungstic acid solution was added and allowed to react for 3 min for staining.

The photothermal effect of liposomes was recorded using a thermal imaging camera (FLIR Systems, Inc., FLIR TG167, Portland, OR, USA). Then, 200 μL aliquots of SL-DOX, SL-ICG, or SL-Co were added to a 0.5 mL EP tube and irradiated (2 W/cm²) with an 808 nm laser (Changchun New Industries, MDL-H-808-4W, Changchun, China) for 3 min, and the temperature variation of the solution in the EP tube was observed.

An ultraviolet–visible spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd., UV1801, Beijing, China) was used to determine the encapsulation efficiency. Then, 200 μL aliquots of SL-DOX, SL-ICG, or SL-Co without free drug were added separately to 10 mL volumetric flasks. Next, 100 μL of liposomes with free drug and 100 μL of sterile water for injection were added separately to 10 mL volumetric flasks. After constant volume demulsification of methanol, the absorbance (A₀) of liposomes with free drug and the absorbance (A₁) of liposomes without free drug (DOX: 480 nm, ICG: 784 nm) were measured and the encapsulation rate (EE) was calculated using Eq. (1):

$$\text{EE }(\text{\%})=A_{\text{0}}\text{/}{A}_1\text{ × }100$$ (1)

The ultraviolet–visible spectrophotometer (Beijing Ruili Analytical Instrument Co., Ltd.) was used to determine the cumulative release rate. Then, 2.0 mL aliquots of SL-DOX, SL-ICG, SL-Co, or SL-DOX after 808 nm laser irradiation for 3 min (La-DOX, 2 W/cm²; La-ICG, 2 W/cm²; La-Co, 2 W/cm²) were added separately to 10 kDa dialysis bags, which were placed in a dissolution cup containing 198 mL 5% sucrose solution. The mixture was stirred at a constant speed of 75 rpm at 37 ± 1 °C, and 4 mL of the dialysate was removed at 0, 1, 3, 6, 9, 12, 24, and 48 h. The same amount of the release medium was added at 37 ± 1 °C. The ultraviolet–visible spectrophotometer was used to determine the DOX concentration and cumulative release rate (n = 3). We repeated the above release experiment. Then, 100 μL of liposomes from the dialysis bag was removed, and the amount of ICG released from the liposomes was determined by measurement of the encapsulation efficiency of ICG.

2.4. Cytotoxicity and wound-healing assay

CCK-8 reagent (Dalian Meilun Biological Co., Ltd., MAO218-2-Jul-07G, Dalian, China) was used to determine the cytotoxicity toward B16/F10 cells. B16/F10 cells in logarithmic growth phase were added to a serum-free medium to prepare cell suspensions (1 × 10⁶ cells/mL). In addition to the blank and preparation wells, 100 μL of cell suspension (1 × 10⁶ cells/mL) was added to a 96-well plate and incubated for 12 h at 37 °C in a 5% CO₂ cell incubator. Serum-free medium containing various concentrations of liposomes was added and incubated for 48 h. CCK-8 reagent was added and the absorbance was measured 3 h later at 450 nm using a microplate reader (Bio-Rad, Model 680, Hercules, CA, USA). We calculated the Cell viability (%), IC₅₀ values, and combination index (CI), as shown in Eqs. (2), (3).

$$\text{Cell viability }(\text{\%})=({\text{OD}}_{\text{sample}}{\text{–OD}}_{\text{preparation}})\text{/}({\text{OD}}_{\text{control}}{\text{–OD}}_{\text{blank}})\text{ × }100$$ (2)

$$\text{CI }={[\text{CA}]}_{50}{\text{/}[\text{A}]}_{50}+{[\text{CB}]}_{50}{\text{/}[\text{B}]}_{50}$$ (3)

A cell-free area was generated in the B16/F10 cells monolayer to determine their migration rate. Briefly, 1 mL of cell suspension (1 × 10⁶ cells/mL) was added to a 6-well plate. After being cultured in a 37 °C, 5% CO₂ cell incubator for 12 h, a 10 μL pipette tip was used to generate cell-free areas in the monolayer, and serum-free medium containing liposomes (DOX final concentration: 50 μg/mL, ICG final concentration: 20 μg/mL), which has been subjected to 808 nm laser irradiation for 3 min (2 W/cm²), was added. The width of the cell-free areas was determined using an inverted microscope at 0, 6, 12, and 24 h.

2.5. Isolation of T lymphocytes and neutrophils

2.5.1. Isolation of T lymphocytes

The grind-sieve method was used to isolate CD8⁺ T cells from the TDLNs (from the lateral fold to the thigh muscle tissue, close to the deep iliac circumflex branch, n = 3) of mice of different ages²⁶. The TDLNs were dissected and placed in a centrifuge tube containing 1 mL PBS. The thymus tissue was disintegrated by repeated scraping over a metal grid, tissue debris was removed through a mesh cell screen (5 cm × 5 cm); the filtrate was mixed with an equal volume of red blood cell lysate and paced at 4 °C for 5 min. It was then centrifuged at 1000×g for 4 min to collect the cells, and subsequently, 200 μL of PBS containing 1 μL PE-conjugated anti-mouse CD8 antibody (Bio-Legend, B266455, San Diego, CA, USA) and 1 μL FITC-conjugated anti-mouse CD3 antibody (Bio-Legend, B274724, San Diego, CA, USA) were added and the sample incubated for 30 min. The total number of isolated cells in the TDLNs was counted using a hemocytometer, and the variation in CD8⁺ T cells in the isolated cell samples was determined by flow cytometry.

The grind-sieve method was used to isolate T lymphocytes from the thymus of 2-, 8-, and 16-month-old mice (n = 3). Using the above isolation method for T lymphocytes, CD3⁺ T cells in the thymus were labeled and measured.

2.5.2. Isolation of neutrophils

Density gradient centrifugation was used to isolate neutrophils from the tibia and femur of 2-, 8-, and 16-month-old mice (n = 3). An 1 mL PBS solution containing bone marrow collected from the tibia and femur was prepared. Tissue debris was removed using a mesh cell screen (5 cm × 5 cm). The separation solution was prepared according to the kit specifications (Tianjin Hao Yang Biological Manufacture Co., Ltd., LZS1100, Tianjin, China); then, 3 mL of 100% cell separation solution, 1.5 mL of 80% cell separation solution, and 1 mL of sample solution were added to the centrifuge tube, which was centrifuged at 1000×g for 30 min. After centrifugation, the cells between the 100% and 80% cell separation solutions were removed. Then, the mixture was centrifuged at 1000×g for 4 min to remove the supernatant, and 200 μL PBS containing 1 μL PE-conjugated anti-mouse Ly6G antibody (Bio-Legend, B281017, San Diego, CA, USA) was added and incubated for 30 min. The total number of cells isolated from the tibia and femur was determined using a hemocytometer, and the variation in neutrophils in the isolated cell samples was determined by flow cytometry (Becton, Dickinson and Company, FACSCanto II, Franklin Lakes, NJ, USA).

2.6. Uptake of SA-modified liposomes

The uptake of SA-modified liposomes by B16/F10 cells, neutrophils, and HL-60 cells was determined using CLSM (ZEISS, ZEISS LSM 800, Oberkochen, Germany) and flow cytometry (Becton, Dickinson and Company). Cells were incubated with SA solution for 12 h, and 1 mL of SA-presaturated HL-60 cells or normal HL-60 and B16/F10 cells suspension (1 × 10⁶ cells/mL) was added to 6-well plates containing cell slides and incubated for 4 h at 37 °C in a 5% CO₂ cell incubator. 1 mL of serum-free medium containing SL-Co was added (DOX final concentration: 50 μg/mL; ICG final concentration: 20 μg/mL, 808 nm laser: 3 min) and the cell suspension was further incubated. The medium was removed from the 6-well plates used for the ingestion experiment after 2 h. Then, 500 mL of 4% paraformaldehyde was separately added to the 6-well plate and the cells were stained with DAPI (Dalian Meilun Biological Co., Ltd., MA0128-Jan-14H, Dalian, China) for 10 min (50 μg/mL). The cell slides were then removed and placed upside down on a glass slide with 10 μL of mounting medium containing anti-fluorescence quenching reagent and observed.

The same processes, for 12 h and 30 min, of adding B16/F10 cells, preparations, laser irradiation, and 200 μL of PBS containing 1 μL FITC-conjugated anti-mouse CRT antibody (eBiosciences, San Diego, CA, USA) to 6-well plates were used for the ICD experiment. The proportion of CRT⁺/PI^- cells was determined by flow cytometry. Then, 1 mL of cell medium supernatant was removed, centrifuged at 1000×g for 20 min, and the related cytokines were determined using the ATP Test Kit (Quanzhou Ruixin Biological Technology Co., Ltd., Shenzhen, China)and HMGB-1 ELISA Kit (R&D Systems, Minneapolis, MN, USA).

2.7. Tissue distribution of SA-modified liposomes

The skin on the back of 2-, 8-, and 16-month-old mice were depilated, disinfected, and injected with 200 μL of a B16/F10 cell suspension (10⁷ cells/mL). When the tumor volumes reached approximately 1000 mm³, the tumor-bearing mice were divided into three groups: 2-month-old mice injected with SL-DiR (2M-DiR) group, 8-month-old mice injected with SL-DiR (8M-DiR) group, and 16-month-old mice injected with SL-DiR (16M-DiR) group via the tail vein (DiR: 1 mg/kg). At 2, 6, 12, and 24 h after the injection, fluorescence images of the 2-, 8-, and 16-month-old mice (n = 3) were obtained using a live imaging device. The brain, heart, lung, tumor, spleen, kidney, and liver of all of the mice were dissected and images of the fluorescence were captured.

2.8. Anti-tumor therapy with SA-modified liposomes

The skin of the back of the legs of 2-, 8-, and 16-month-old mice (n = 24) was depilated, disinfected, and injected with 200 μL of a B16/F10 cell suspension (10⁷ cells/mL). When the tumor volume reached 100 mm³, the tumor-bearing mice were randomly divided into 12 groups (n = 6): 2-month-old mice injected with 5% glucose (2M-Con) group, 2-month-old mice injected with SL-DOX (2M-DOX) group, 2-month-old mice injected with SL-ICG (2M-ICG) group, 2-month-old mice injected with SL-Co (2M-Co) group, 8-month-old mice injected with 5% glucose (8M-Con) group, 8-month-old mice injected with SL-DOX (8M-DOX) group, 8-month-old mice injected with SL-ICG (8M-ICG) group, 8-month-old mice injected with SL-Co (8M-Co) group, 16-month-old mice injected with 5% glucose (16M-Con) group, 16-month-old mice injected with SL-DOX (16M-DOX) group, 16-month-old mice injected with SL-ICG (16M-ICG) group, and 16-month-old mice injected with SL-Co (16M-Co) group. Liposomes were injected into the tail vein 1, 4, and 7 days after grouping (DOX: 5 mg/kg, ICG: 2 mg/kg). At 6 h after administration, the tumor site of each mouse was subjected to 808 nm laser irradiation for 3 min (2 W/cm², Zoletil® 50: 50 mg/kg, Beijing You Pet Biotechnology Co., Ltd., Beijing, China), and the temperature variation at the tumor site was observed. The measurements of the body weight and the long and short diameters of the tumors were performed to calculate the tumor volume.

The TDLNs of 2-month-old mice were removed to eliminate ICD induced by photochemotherapy. Mice were randomly divided into three groups (n = 6): 2-month-old mice injected with 5% glucose (2M-Sham), 2-month-old mice injected with SL-Co (2M-LN), and 2-month-old mice (no TDLN) injected with SL-Co (2M-No-LN). Consistent with the above administration method, the long and short diameters of the tumors were measured to calculate the tumor volumes.

2.9. Immunofluorescence assays

Immunofluorescence was used to detect enhanced CD8⁺ T cell infiltration in the tumors. On the 25th day, TDLNs, heart, kidney, thymus, skin, lung, and liver were placed in 4% polymethylene fixation solution and then embedded in paraffin. Ultra-thin sections (5 μm) were generated and stained with H&E (Wuhan Servicebio Biotechnology Co., Ltd., Wuhan, China). The sections were observed under an inverted microscope. Tumors were homogenized, and cytokines in tumor supernatants were assayed by using Enzyme-linked immunosorbent assay (ELISA) kits for interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), IL-10, and IL-2 (eBiosciences, 16MSTE1QPQ, 62SBL6ULPC, E-EL-R0016C, BIJLXURBFW, San Diego, CA, USA). In addition, paraffin sections of the embedded tumors were subjected to TUNEL and immunofluorescence staining (CD11b⁺ dendritic cells; CD4⁺ T cells; Neutrophils; CD8⁺ T cells, Wuhan Servicebio Biotechnology Co., Ltd., Wuhan, China). Paraffin sections of the embedded TDLNs were stained with immunofluorescence probes (CD4⁺ T cells and CD8⁺ T cells). All fluorescence-labeled sections were observed under a fluorescence microscope (NIKON CORPORATION, Nikon Eclipse Ti-Sr, Tokyo, Japan). T cells in TDLNs from mice of different ages were separated according to the process described in Subsection 2.5.1, and the variation in CD8⁺ T cells in TDLNs was determined using flow cytometry (CD3⁺ T cells, CD8⁺ T cells).

2.10. Statistical analysis

Data are presented as means ± standard deviation. The IC₅₀ and P value were determined using SPSS 23.0 software (IBM Corp., IBM SPSS Statistics 23.0, Armonk, NY, USA). One-way ANOVA followed by a post hoc test was used for multiple group comparisons. Independent Samples t-test was used for comparisons between two groups. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. ns, not significant.

3. Results

3.1. SA-modified DOX and ICG co-loaded liposomes

Immune cells have received widespread attention as drug delivery carriers because of their excellent anti-tumor therapeutic efficacy. SA can combine to the L-selectin on the surface of neutrophils, but SA cannot be modified on the surface of liposomes. Thus, we designed MT-18 with a molecular weight of 561 Da (Supporting Information). We found that the molecular docking energy and interacting amino acid residues of MT-18 were the same as those of SA by Autodock Vina; MT-18 retained the ability of SA to target L-selectin on neutrophils and could be modified on the surface of the liposome using octadecyl (Supporting Information)^27,28. To obtain SA-modified photosensitive liposomes with uniform particle size and good stability, we prepared SL-DOX, SL-ICG, and SL-Co by thin-film evaporation. The physical appearance of SL-DOX, SL-ICG, and SL-Co did not change significantly at 4 °C after 20 d (Fig. 1A). The particle sizes of SL-DOX, SL-ICG, and SL-Co were 110–125 nm, and the Zeta potential ranged from −10 to −15 mV (Table 1). The results of TEM are consistent with the results of Nicomp-380 dynamic light scattering particle sizer. DOX could interact with sulfate to form an insoluble polymer inside SL-Co and SL-DOX (Fig. 2 B and C). After 808 nm laser irradiation for 3 min, the temperatures of SL-Co and SL-ICG were above 60 °C, but the temperature of SL-DOX did not exceed 30 °C, indicating that SL-DOX had no thermal effect (Fig. 2D). The release of DOX and ICG from SL-Co within 48 h was not significantly different from the release from SL-DOX and SL-ICG (DOX: P value = 0.224, ICG: P value = 0.460). After 808 nm laser irradiation for 3 min, the release of DOX and ICG from La-Co within 48 h was significantly increased (DOX: P value < 0.001, ICG: P value < 0.001). SL-ICG and SL-Co not only had uniform particle sizes and good stability under non-illuminated conditions but also accelerated the release of drugs under 808 nm laser irradiation.

Figure 1.

(A) Image of 1 mL of SL-DOX, SL-ICG, and SL-Co placed separately in 2 mL penicillin bottles at 4 °C for 20 days. (B) TEM of SL-DOX, SL-ICG, and SL-Co, scale bar = 200 nm. (C) An insoluble precipitate formed by the interaction of DOX and sulfate in the aqueous phase of SL-Co, scale bar = 50 nm. (D) Thermal images of 200 μL of SL-DOX, SL-ICG, and SL-Co irradiated vertically by 808 nm laser for 3 min (2 W/cm²), and temperature change curve of the liposomes. (E) Cumulative release curves of SL-DOX, SL-ICG, SL-Co, La-DOX, La-ICG, and La-Co (DOX: 1 mg/mL; ICG: 0.4 mg/mL, n = 3). Data are presented as mean ± SD. ∗∗P < 0.01. ns, not significant.

Table 1.

Prescription composition, particle size, PDI, zeta potential, and EE of liposomes. Data are presented as mean ± SD (n = 3).

Liposomes	Drug	Drug/lipid (w/w)	Composition (n/n/n)	Particle size (nm)	PDI	Zeta potential (mV)	EE (%)
SA-ICG	ICG	3: 20	MT-18/DPPC/HSPC/CH = 5/30/25/40	109.2 ± 1.4	0.112 ± 0.010	‒13.1 ± 1.2	25.6 ± 1.8
SA-DOX	DOX	1: 10	MT-18/DPPC/HSPC/CH = 5/30/25/40	108.1 ± 1.3	0.109 ± 0.009	‒11.2 ± 1.2	94.8 ± 2.1
SA-Co	ICG, DOX	3: 20, 1: 10	MT-18/DPPC/HSPC/CH = 5/30/25/40	107.4 ± 1.6	0.123 ± 0.011	‒12.6 ± 1.3	24.5 ± 1.6, 87.7 ± 2.6

PDI, polydispersity index; EE, encapsulation efficiency; ICG, indocyanine green; DOX, doxorubicin; SL-ICG, SA-modified ICG-loaded liposomes; SL-DOX, SA-modified DOX-loaded liposomes; SL-Co, SA-modified ICG and DOX co-loaded liposomes; MT-18, methyl sialic acid-octadecanoic acid; DPPC, dipalmitoyl phosphatidylcholine; HSPC, hydrogenated soybean phosphotidylcholine; CH, cholesterol.

Figure 2.

SA-mediated photochemotherapy eliminates the decline of antitumor therapeutic efficacy caused by immunosenescence. Tail vein injection of mice of different ages, (A) tumor volume of mice injected with 5% Glu, (B) tumor volume of mice injected with SL-DOX, (C) tumor volume of mice injected with SL-ICG, (D) tumor volume of mice injected with SL-Co, (E) percent survival after 59 d of treatment (n = 6, death, the shortest tumor dimension reached 1.5 cm, and the volume reached more than 2000 mm³, treatment was judged to be ineffective). (F) 2-, 8-, and 16-month-old mice were injected with liposomes after 6 h (DOX: 5 mg/kg, ICG: 2 mg/kg), and the highest temperature of tumor site for the indicated durations of 808 nm laser irradiation are specified. (G) TUNEL fluorescence section of tumor site in each treated group (apoptotic cells: green). (H) Fluorescence intensity of TUNEL in 200 μm × 200 μm image was measured using ImageJ (n = 3, scale bar = 20 μm). (I) The tumors of mice in the 8M-Co group became crusted (red arrow) and regressed (red triangle). Data are presented as mean ± SD. ∗∗∗P < 0.001. ns, not significant.

3.2. SA-mediated photochemotherapy eliminated the immunosenescence-mediated decline in antitumor therapeutic efficacy in 16-month-old mice

There is an inevitable which influences each other between aging, immunity, and tumors. To explore the relationship between immunosenescence and the antitumor therapeutic efficacy of SA-mediated photochemotherapy, we examined anti-tumor-treated efficacies in 2-, 8-, and 16-month-old tumor-bearing mice. For the SA-mediated non-photochemotherapy group, the tumor volume of 16-month-old mice was significantly larger than that of 8-month-old mice (Glucose: P value < 0.001, DOX: P value < 0.001, ZOL: P value < 0.001), there was not a significant difference in tumor volumes between 2- and 8-month-old mice during anti-tumor treatment (Glucose: P value = 0.151, DOX: P value = 0.911, ZOL: P value = 0.126), indicating that immunosenescence could influence the antitumor therapeutic efficacy in 16-month-old mice (Fig. 2A‒C). Mice aged 2-, 8-, and 16-month that were injected with SL-Co exhibited effective inhibition of tumor growth, and the reduction of the anti-tumor therapeutic effect seen previously in 16-month-old mice was not discernible in the 16M-Co group (Fig. 2D). After 59 days, the total survival time of the 16-month-old mice injected with SL-ICG and SL-DOX significantly decreased compared to that of the 8-month-old mice. The total survival times of the 2-, 8-, and 16-month-old mice injected with SL-Co were the same (Fig. 2E). During the first phototherapy treatment, the temperatures of the tumor sites of the 2-, 8-, and 16-month-old mice injected with SL-ICG and SL-Co could reach 48–50 °C, but it took relatively longer for the temperature of the tumor sites in the 16-month-old mice to reach the maximum temperature (Fig. 2F). The TUNEL fluorescence intensity in 8M-DOX was 3.9-times that in 16M-DOX and 1.7-times that in 2M-DOX. The TUNEL fluorescence intensity in 8M-Co was 1.2-times that in 16M-Co and 1.1-times that in 2M-Co, indicating non-significant differences between them (Fig. 2G and H). The results of the TUNEL stained sections were consistent with the tumor volume growth curves, indicating that SA-mediated photochemotherapy eliminated the decline in anti-tumor therapeutic efficacy in the 16-month-old mice. Fifty percent of the tumors in the 8M-Co group became scabbed and regressed by SA-mediated photochemotherapy, and there were no signs of recurrence within 59 days (Fig. 2I).

3.3. Decline in CD8⁺ T cells functioning in 16-month-old mice is caused by immunosenescence

Immunosenescence is due to degeneration of the thymus^7,29. Degeneration of the thymus influences the phenotype and function of T cells, which can influence the body's immune system⁹. To document the relationship between aging and immunity, immune tissue sections, and T lymphocytes of 2-, 8-, and 16-month-old mice were analyzed. Functional atrophy of the thymus occurred in 16-month-old mice (Fig. 3A), and the cell density in the lymph nodes of 8-month-old mice was significantly higher than that of 2- and 16-month-old mice (Fig. 3B). The variation in the number of T lymphocytes in the thymus and lymph nodes accurately reflects the variation in the immune system of 2-, 8-, and 16-month-old mice. The total cell number and the ratio of CD3⁺ T cells in the thymus of 16-month-old mice were lower than those of 2- and 8-month-old mice (Fig. 3 C and D). The number of CD3⁺ T cells in the thymus of 8-month-old mice was 6.38-times that of 16-month-old mice (Fig. 3E). The total cell number and the ratio of CD3⁺CD8⁺ T cells in the lymph nodes of 16-month-old mice were lower than those of the 8-month-old mice (2.89-times, Fig. 3F and G). The numbers of T cells CD8⁺ and CD3⁺ T cells in the lymph nodes of 8-month-old mice were 4.22- and 3.86-times that of 16-month-old mice, respectively (Fig. 3 H and I). The numbers of T cells in the thymus and lymph nodes were, however, the same. Thus, it can be concluded that immunosenescence occurred in 16-month-old mice.

Figure 3.

Aging of CD8⁺ T cells of 16-month-old mice caused by immunosenescence. (A) Thymus, and (B) TDLNs of 2-, 8-, and 16-month-old mice, scale bar = 200 μm. (C) Total cell number, (D) ratio of CD3⁺ T cells, (E) and a total number of CD3⁺ T cells in thymus, n = 3. (F) Total cell number, (G) ratio of CD3⁺CD8⁺ T cells, (H) a total number of CD8⁺ T cells, (I) and a total number of CD3⁺ T cells in TDLNs (CD8⁺ T cells were labeled with PE-CD8 antibody; CD3⁺ T cells were labeled with FITC-CD3 antibody, n = 3). Data are presented as mean ± SD.

3.4. Neutrophils-mediated delivery of SA-dependent photochemotherapy to target tumors

Neutrophils also undergo a series of degenerative changes in their function and number, and their self-regulatory, phagocytic, and antigen-presenting capabilities are also significantly weakened⁷. The ratio of neutrophils in the tibia and femur of 16-month-old mice was lower than that in 2- and 8-month-old mice (Fig. 4A). The total cell number and the number of neutrophils in the tibia and femur of 8-month-old mice were 1.77-, and 2.71-times, respectively, that of 16-month-old mice (Fig. 4B and C). The anti-tumor effects of SA-modified liposomes involve three stages: phagocytosis, transport, and effectuation¹⁷. We used SA-presaturated HL-60 cells and SA-modified liposomes to explore the relationship between the neutrophils drug delivery system and immunosenescence. After neutrophils and HL-60 cells were incubated with SA (10 mol/L) for 12 h, we found that the uptake capability of SA-presaturated cells was lower than that of normal cells. The binding of free SA to the L-selectin of neutrophils inhibits the uptake of SL-Co, and it can be concluded that tumor delivery of drugs by liposomes is mediated by neutrophils (Fig. 4D). After injection with SL-DiR, the fluorescence intensity of DiR in the tumors of 2-, 8-, and 16-month-old mice reached the highest value at 6 h. The fluorescence intensity of DiR in the brain, heart, lung, spleen, kidney, and tumors of 2-, 8-, and 16-month-old mice was not different at 2, 6, 12, and 24 h. DiR accumulation in the tumors of 16-month-old mice was significantly lower than that in 8-month-old mice at 6 and 12 h (6 h: P value < 0.001, 12 h: P value < 0.001), and the amount of DiR accumulation in the liver of 16-month-old mice was significantly higher than that in 8-month-old mice at 24 h (P value < 0.001). Furthermore, the amount of DiR accumulation in tumors and livers of 8- and 2-month-old mice was not different at 2, 6, 12, and 24 h (Fig. 4E‒G). The above results indicate that neutrophils delivered the SL-DiR to the tumor sites, and neutrophil senescence in the 16-month-old mice affected liposome accumulation in the tumors.

Figure 4.

Neutrophil-mediated drug delivery by SA-modified liposomes to target tumors. (A) Purity of neutrophils, (B) total cell number, and (C) total number of neutrophils in the femur and tibia (neutrophils were labeled with PE-Ly6G antibody, n = 3). (D) SA-presaturated HL-60 cells and normal HL-60 cells were incubated with SL-Co for 2 h; images of intracellular DOX (red) and ICG (green) fluorescence are shown (scale bar = 10 μm). (E) Tail vein injection of SL-DiR in 2-, 8-,16-month-old mice (DiR: 1 mg/kg, n = 3); fluorescence distribution image was shown. (F) Fluorescence intensity of DiR in liver and (G) tumor of the mice at 2, 6, 12, and 24 h. Data are presented as mean ± SD. ∗∗∗P < 0.001.

3.5. SA-mediated photochemotherapy enhanced immunogenic death of B16/F10 cells

Immunogenic death of tumor cells generated a series of damage-associated molecular patterns (DAMPs) (Fig. 5A)³⁰. To verify that SA-mediated photochemotherapy enhanced immunogenic death of B16/F10 cells, surgical removal of TDLNs from 2-month-old mice was performed to generate a no-immune-response mouse model. For the SL-Co group, the tumor volume of the mice with no TDLNs was significantly larger than that of normal mice (P value < 0.001), indicating that SA-mediated photochemotherapy enhanced the infiltration of immune cells from TDLNs into the tumor (Fig. 5B). In the CCK8 test, the IC₅₀ value of La-Co was lower than those of La-ICG and La-DOX groups, and CI < 1 (Table 2). There was a greater accumulation of DOX in B16/F10 cells in the La-Co group than that in the SL-Co group, and there was no difference in ICG uptake between the SL-Co and the SL-ICG groups (Fig. 5C). In the in vitro ICD experiment, the ratio of CRT⁺/PI^-, release of ATP, and secretion of HMGB-1 in the La-Co group were significantly higher than those in the other groups (Fig. 5D). For non-photochemotherapy, the number of dendritic cells in the tumors of the 16-month-old mice was lower than that in the 2- and 8-month-old mice. SA-mediated photochemotherapy enhanced dendritic cell infiltration into the tumors of the 16-month-old mice (Fig. 5E). SA-mediated photochemotherapy converted light energy into heat energy to accelerate the release of DOX, thereby increasing the toxicity of DOX and enhancing B16/F10 cell ICD.

Figure 5.

SA-mediated photochemotherapy enhanced immunogenic death of B16/F10 cells. (A) ICD of tumor cells. (B) Changes in tumor volume of mice without TDLNs and normal mice during the 21 d of anti-tumor treatment. (C) B16/F10 cells were incubated with the liposomes for 2 h; confocal fluorescence images of non-irradiated and irradiated B16/F10 cells are shown (Red: DOX; Green: ICG, La-Co: 808 nm laser irradiated for 3 min, scale bar = 30 μm). (D) B16/F10 cells were incubated with liposomes for 12 h, confocal fluorescence image of B16/F10 cells (red: CRT, blue: DAPI) was taken and ratio of CRT⁺/PI^‒ in B16/F10 cells (n = 3, scale bar = 30 μm) was measure. Changes in the levels of ATP and HMGB-1 were determined by ATP and HMGB-1 kits. (E) Fluorescence images of dendritic cells (green) in tumor of mice in different treated groups (scale bar = 40 μm). Data are presented as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Table 2.

IC₅₀ value and CI after 48 h of incubation of B16/F10 cells with liposomes. Data are presented as mean ± SD (n = 6).

Parameter	SL-ICG	SL-DOX	SL-Co
IC50 (μg/mL)	26.9 ± 0.5	15.9 ± 0.3	6.4 ± 0.3
CI	–	–	0.7 ± 0.1

CI, combination index; IC₅₀, half maximal inhibitory concentration; SL-ICG, SA-modified ICG-loaded liposomes; SL-DOX, SA-modified DOX-loaded liposomes; SL-Co, SA-modified ICG and DOX co-loaded liposomes; ‒, not applicable.

3.6. SA-mediated photochemotherapy enhanced the CD8⁺ T cells tumor infiltration in 16-month-old mice

The ultimate goal of anti-tumor therapy is to eliminate cancer cells, and enhancing the infiltration of immune cells into the tumor is key to achieving this^31,32. Cytokines (TNF-α, IFN-γ, IL-2, and IL-10) activate immune cells, enhance their infiltration into tumors, and regulate the immune response. The concentrations of TNF-α, IFN-γ, IL-2, and IL-10 in the tumors of 2-, 8-, and 16-month-old mice injected with SL-ICG and 5% glucose were significantly lower than those in mice injected with SL-DOX or SL-Co. The concentrations of TNF-α, IFN-γ, IL-2, and IL-10 in the tumors of 8-month-old mice were higher than those of 16-month-old mice, and SL-Co could narrow differences in the concentration of cytokines between 2-, 8-, and 16-month-old mice (Fig. 6A and B). The proportion of CD3⁺CD8⁺ T cells in TDLNs of 2-, 8-, and 16-month-old mice injected with SL-ICG and 5% glucose was lower compared to those injected with SL-DOX or SL-Co (Fig. 6C). The total cell number, the number of CD3⁺ T cells, and the number of CD3⁺CD8⁺ T cells in the TDLNs of the 8-month-old mice were higher than those of the 16-month-old mice (Fig. 6D‒F). There was not a significant difference in the number of CD4⁺ T cells in the TDLNs of 2-, 8-, and 16-month-old mice injected with the same preparation (Fig. 7A and B). The number of CD8⁺ T cells in the TDLNs of the 8M-DOX group was 2.7-times that of the 16M-DOX group, and the number of CD8⁺ T cells in the TDLNs of the 8M-Co group was 1.4-times that of the 16M-Co group. The number of CD8⁺ T cells in the TDLNs of the 8-month-old mice was significantly higher than that of the 16-month-old mice (Fig. 7C). The change in the trend of CD4⁺ and CD8⁺ T cells in the thymus was the same as that in the TDLNs (Fig. 7D‒F). Therefore, it would appear that photochemotherapy can increase the release of cytokines that induce T cell activation in the tumor and enhance tumor infiltration of CD8⁺ T cells in the TDLNs of immunosenescent mice.

Figure 6.

Changes in the levels of cytokines in tumors and the number of T cells in TDLNs of mice in each treated group. (A, B) The changes in cytokine levels in the tumors (1 cm × 1 cm × 1 cm) in each treated group were determined by IFN-γ, TNF-α, IL-10, and IL-2 ELISA kits. Taking the concentration of each cytokine in the tumors of 16M-Con as the control group, the changes in the concentration for the other treated groups were calculated (n = 3). (C) The changes in the number of CD3⁺CD8⁺ T cells in TDLNs in each treated group. (D) Total cell number, (E) total CD3⁺ T cell number, and (F) total CD3⁺CD8⁺ T cell number in lymph nodes (n = 3). Data are presented as mean ± SD.

Figure 7.

Fluorescence images of sections of TDLNs and tumors in mice in the different treated groups. (A) Fluorescence images of TDLNs of mice in different treated groups; ImageJ was used to measure the fluorescence intensity of (B) CD4⁺ T cells and (C) CD8⁺ T cells in 200 μm × 200 μm pictures (red: CD8, green: CD4, blue: DAPI, n = 3, scale bar = 20 μm). (D) Fluorescence images of tumors in the different treated groups. ImageJ was used to measure the fluorescence intensity of (E) CD4⁺ T cells and (F) CD8⁺ T cells in 200 μm × 200 μm pictures (red: CD8, green: CD4, pink: Gr-1, blue: DAPI, n = 3, scale bar = 20 μm). Data are presented as mean ± SD.

3.7. SA-mediated photochemotherapy reduced tumor recurrence and metastasis

As long as tumor cells are still present, recurrence and metastasis will remain the main factors that threaten patient survival. In the wound-healing assay, there was not a significant difference in the migration ability of tumor cells in the control versus La-ICG groups (12 h: P value = 0.074, 24 h: P value = 0.158), and the migration ability of B16/F10 cells in the La-Co group was significantly less than that in the La-ICG group at 12 and 24 h (12 h: P value < 0.001, 24 h: P value < 0.001, Fig. 8A and B). DOX effectively reduced the migration ability of tumor cells, and its inhibitory effect on tumor cell migration was greater than that of ICG. Only the 16-month-old mice injected with 5% glucose and SL-ICG exhibited lung and liver metastases of the tumor cells (Fig. 8C). The tumor sites of mice formed scabs in 2M-ICG, 8M-ICG, and 16M-ICG groups during the treatment. We found that the tumor recurrence in situ after the administration was stopped, and the tumor recurrence was the most serious in 16-month-old mice (Fig. 8D). During treatment, T cells senescence enhanced the metastasis and recurrence of tumors, and the decrease in the number of T cells in the 16-month-old mice led to a decrease in the therapeutic effect in the SL-ICG group, leading to metastasis of tumors in mice in the 16M-ICG group. There was no obvious pathological damage to the heart, thymus, kidney, skin, or TDLNs in each treated group, indicating that the SA-modified liposomes did not damage non-cancerous tissues (Fig. 8E).

Figure 8.

(A) Changes in migration ability of cancer cells; B16/F10 cells were incubated with serum-free medium containing laser irradiated liposomes (DOX final concentration: 50 μg/mL; ICG final concentration: 20 μg/mL, 2 W/cm²), and was imaged using an inverted microscope at 0, 6, 12, and 24 h. (B) The width of the cell-free area was measured by ImageJ. (C, D) The 25th day after tail vein injection of 2-, 8-, and 16-month-old mice with the liposomes (DOX: 5 mg/mL, ICG: 2 mg/mL, 3 injections), the tumor recurrence of mice in situ and metastasis (red). Scale bar = 50 μm. (E) The dissected heart, thymus, kidney, skin, and TDLNs were paraffin-embedded and sectioned, scale bar = 50 μm. Data are presented as mean ± SD. ∗∗∗P < 0.001. ns, not significant.

4. Discussion

In this study, SA-mediated photochemotherapy successfully reversed the immunosenescence-mediated decline in anti-tumor therapeutic effects in 16-month-old mice caused by the light-heat-chemical-immunity conversion. Aging causes immune organs to gradually become functionally degraded, and the number of immune cells in the human body decreases, thereby increasing the risk of disease and the difficulty of treating disease (especially immune-related diseases)³³. The aging of the thymus, which is the main organ involved in T cell maturation, directly affects the number of mature T cells in the body and indirectly affects the type and quantity of T cells in TDLNs⁹. The thymus and lymph nodes of 2-, 8-, and 16-month-old mice were used to probe for the occurrence of immunosenescence in 16-month-old mice. Neutrophils undergo a series of degenerative changes in their function and number. The number of neutrophils in the 16-month-old mice decreased significantly, and neutrophils-mediated drug delivery was also affected by immunosenescence. However, SA-mediated photochemotherapy increased the infiltration of neutrophils into tumors.

TDLNs have recently been shown to be the key to generating immune responses and enhancing T cell infiltration into tumors. Continuous induction of effective anticancer immune responses is related to the production of activated T cells in TDLNs^10,34. Studies have shown that the solid stress of the tumor prevents T lymphocytes from entering the tumor, and the number of T cells in the lymph nodes is not affected by the tumor^35,36. Atrophy of the thymus in 16-month-old mice results in a decrease in the number and tumor infiltration capacity of T cells in TDLNs. Phototherapy reduces the number of tumor cells, changes the solid pressure of the tumor in the TDLNs, and enhances neutrophil infiltration into the tumor. However, phototherapy could not charge the number of T cells and improve the anti-tumor therapy effect in 16-month-old mice, leading to different degrees of recurrence and metastasis of tumors in 16-month-old mice. Chemotherapy regulates tumor-associated immune cells, downregulates tumor-associated macrophage-mediated immunosuppression, induces immunogenic death of tumor cells, enhances CD8⁺ T cells infiltration into tumors, and transforms immunosuppressive cold tumors into immune-infiltrating hot tumors37, 38, 39. However, chemotherapy could not improve the efficacy of neutrophil-mediated drug delivery in 16-month-old mice. Unilateral enhancement of drug delivery or improvement of anti-tumor therapy cannot solve the problem of reduced therapeutic effects caused by immunosenescence⁴⁰.

As the understanding of tumor cells, tumor microenvironment, and their interaction with the host immune system continues to deepen, the research and development of strategies for anti-tumor therapy continue to change, and the effect of combined administration of an increasing number of new drugs in the early stages of tumor development is now being explored^22,41. Compared with a single anti-tumor-treated strategy, the combined use of multiple administration strategies can break down the barriers of tumor immunosuppression by non-overlapping mechanisms, increase the number of T lymphocytes infiltrating tumors, and improve anti-tumor therapeutic efficacy. It can reduce the side effects of chemotherapy, prevent the accumulation of drugs in non-cancerous tissues, improve the survival status of patients, and achieve the therapeutic effect of “1 + 1 > 2"^42,43. During treatment, the accumulation of preparations in the tumor cannot be increased. With increasing age, immunosenescence and changes in the physiological environment of the body profoundly affect the treatment of traditional photochemotherapy¹⁵. For SA-mediated Photochemotherapy, SA-modified liposomes were targeted to tumors through a neutrophil drug delivery system, and the delivery efficiency of SA-modified liposomes was determined by neutrophils. Photochemotherapy inhibited the growth and migration ability of tumor cells, promoted the presentation of tumor cell antigens, and enhanced neutrophils and T cells infiltration into the tumor. The mutual reinforcement of SA and photochemotherapy have been used to cure tumors.

5. Conclusions

The immune system is important for the distribution of NPs in vivo; thus, immunosenescence can have a substantial influence on the anti-tumor therapeutic efficacy of NPs. This study showed that the number of CD8⁺ T cells and neutrophils in 16-month-old mice significantly decreased, 16-month-old mice exhibited immunosenescence, and the immune system of 8-month-old mice was stronger than that of 2-month-old mice. SL-ICG enhanced neutrophils tumor infiltration and specific cytotoxic cells at the tumor site (even scab formation). However, aging of T cells leads to extensive metastasis and the recurrence of tumors, and we noted a significant difference in the treated efficacy in mice of different ages. Regarding the administration of SL-DOX, chemotherapy with this drug-induced ICD in tumor cells and enhanced tumor infiltration by CD8⁺ T cells. However, neutrophil aging significantly reduced the efficacy of antitumor therapy in 16-month-old mice. Regarding the administration of SL-Co, its therapeutic effect could simultaneously enhance the infiltration of CD8⁺ T cells and neutrophils into the tumor, increase the accumulation of drugs at the tumor site, improve the effect of antitumor therapy, and eliminate the decline in anti-tumor therapeutic efficacy in 16-month-old mice caused by immunosenescence.

Author contributions

Dezhi Sui contributed equally in the design of the study and writing of this manuscript. Yanzhi Song started the project and was joined later with Changzhi Li in the design and execution of majority of the experiments. Yihui Deng assisted in the design and performance of membrane fusion studies and with Xueying Tang. Junqiang Ding assisted with experimental design. Qiongfen Yang, and Zhaowei Qi assisted with rearing mice. Xinrong Liu provided reagents. All authors read and approved the manuscript. Dezhi Sui and Yanzhi Song is involved in overall project design and manuscript preparation. All of the authors have read and approved the final manuscript.

Conflicts of interest

The authors have no conflicts of interest to declare.

Appendix A. Supplementary data

The following is the Supplementary data to this article: