Materials
Ivermectin (I141334), IR780 (207399-07-3), 6-aminohexanoic acid (A306000), triethylamine (T103285), 4-dimethylaminopyridine (DMAP) (D109207), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) (E106172) and 1,3-Diphenylisobenzofuran (DPBF) were purchased from Aladdin Industrial Corporation. Hydroxychloroquine (BD134189) was purchased. Hyaluronic acid (MB7264) was purchased from Meilunbio. Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640, was purchased from Thermo Fisher Science. Dimethyl sulfoxide (DMSO) crystal violet was purchased from Micros Sigma. Fetal bovine serum albumin (FBS) was provided by Biowest. Annexin V-FITC/PI cell apoptosis detection kit was purchased from Yeasen Biotechnology (Shanghai) Co., Ltd. LDH release detection kit was provided by Beyotime. 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich.
Synthesis of IR780-COOH
Briefly, IR780 (320 mg) was dissolved in 10 ml of anhydrous DMF, followed by the addition of 6-aminohexanoic acid (263.5 mg) and 278 µL of triethylamine. The reaction mixture was mixed under nitrogen protection at 85℃ under nitrogen protection at 85℃, avoiding light and stirring for 4 h. The solvent was removed by rotary evaporation and then purified on a silica gel column using methanol: dichloromethane (1:100, v/v) as the eluent to obtain IR780-COOH.
Synthesis of H780
A mixture of IR780-COOH (100 mg), EDCI (40.6 mg), and NHS (123.0 mg) was stirred in anhydrous DMF (20 mL) under nitrogen protection at 0 °C for 30 min. After the reaction, HCQ (107.1 mg) and DMAP (26.0 mg) were added to the mixture after the solvent returned to room temperature. The reaction was stirred under nitrogen protection for 48 h, and the solvent was removed by rotary evaporation. The crude product was purified on silica gel column using methanol: dichloromethane (1:70, v/v) as the eluent to obtain H780.
Preparation of HA/H-I NPs
In order to improve the stability of nanosystem, hyaluronic acid (HA) was used as a carrier to load H780 and IVM through self-assembly to prepare nanoparticles. Firstly, 7.5 mg of H780 and 6.0 mg of IVM were mixed with 1 mL of methanol solution and sonicated for 2 min. Then, the mixture was slowly added dropwise 10 mL of deionized water and stirred for 1 h. After that, the solvent in the methanol solution was evaporated by rotary evaporation at 35℃ for 10 min, resulting in a solution of H-I NPs. Subsequently, using ultrasonication, H-I NPs were slowly added to the HA solution and sonicated for 10 min to obtain the HA/H-I NPs solution.
Characterization of HA/H-I NPs
The topography, particle size, and zeta potential of HA/H-I NPs were characterized using a JEM-200CX transmission electron microscope (TEM), a Brookhaven BI-200SM dynamic light scattering (DLS) instrument, and a Malvern laser particle size analyzer.
Stability experiments of HA/H-I NPs
The prepared HA/H-I NPs and H-I NPs were stored in a refrigerator at 4 °C and the changes in their physical state were observed for 7 days, recorded and photographed. In addition, the prepared HA/H-I NPs solutions were diluted to appropriate concentrations and stored at 4 °C. The absorbance changes at the maximum absorption wavelength and the particle size changes of HA/H-I NPs were measured separately for 7 days.
Single-linear state oxygen detection
1,3-Diphenylisobenzofuran (DPBF) was employed as a basis to detect the generation of reactive ROS under NIR laser irradiation of photosensitizers. We diluted IR780, H780, and HA/H-I NPs to 10 µM and added 30 µ L DPBF (dissolved in DMF, 1 mg·mL− 1) to 2 mL of water, IR780, H780, and HA/H-I NPs solutions, respectively. IR780 was irradiated with 808 nm NIR laser (1 W·cm− 2) for 5 min, and H780 and HA/H-I NPs were irradiated with 660 nm NIR laser (1 W·cm− 2) for 5 min. The absorbance of the solutions at 510 nm was recorded at 0 s, 30 s, 60 s, 90 s, 120 s, 180 s, 240 s, and 300 s after the NIR laser irradiation.
In vitro release and encapsulation efficiency
This passage describes a method for releasing H780 and IVM from HA/H-I NPs using dialysis. The HA/H-I NPs solution is placed in various dialysis bags (MWCO = 3500 Da), with each bag containing 1 mL of solution. After sealing, all bags are immersed in 25 mL of phosphate buffer solution (PBS; pH 7.4 or 5.0) containing 5% (v/v) Tween-80 at 37 °C and shaken at a speed of 100 rpm. Then, 1 mL of the release medium is taken out and replaced with fresh release medium. The released amount of H780 is determined by UV-visible spectrophotometry (UV-8000 S), and the concentration is calculated based on the standard curve. The released amount of IVM is detected by high-performance liquid chromatography (Wooking HPLC K2025), and the concentration is calculated based on the standard curve. The encapsulation efficiency of HA/H-I NPs is also tested.
Cell culture
RKO and HCT116 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics, and CT26 cells were cultured in RPMI 1640 medium. These cells were maintained in a cell culture chamber at 37 °C in 5% humidified CO2.
In vitro cell uptake
To perform cell uptake experiments, RKO and HCT116 cells were seeded onto 6-well plates at a density of 5 × 104 cells per well and cultured for 1 day. After discarding the initial culture medium, fresh culture medium containing 10µM HA/H-I NPs was added into a 6-well plate at 0, 2, 4, 6, and 8 h, respectively. For qualitative analysis experiments, cells were washed three times with pre-chilled PBS, fixed with 4% paraformaldehyde for 15 min, and fluorescence pictures were taken by fluorescence inverted microscopy. For quantitative experiments, cells were washed 3 times with pre-chilled PBS and collected after trypsin digestion. 0 h group was used as a blank control and the cell fluorescence signal was recorded using a flow cytometer (BD Facsaria III).
In vitro cell viability assay
RKO and HCT116 cells were seeded onto 96-well plates at a density of 3 × 104 cells per well and cultured for 1 day. Then, the original medium was replaced with 200 µL medium that contained different concentrations of IVM, HCQ, IR780, H780, or HA/H-I NPs, and further cultured for 6 h. The cells were rinsed with pre-chilled PBS three times, and fresh medium was added. IR780 was irradiated with 808 nm laser for 3 min (1 W·cm− 2). H780 and HA/H-I NPs were irradiated with 660 nm laser for 3 min (1 W·cm− 2). After further incubation for 24 h, cell viability was detected by standard MTT assays. The cell survival rate was calculated according to the following equation:
$$\text{Cell}\,\text{Survival}\,\text{Rate}\,(\%) = \frac{As-Ab}{Ac-Ab} \times 100\% $$
Where As is the absorbance of cells after treatment with medium of different samples plus drugs, Ac is the absorbance of the cells in the standard medium and Ab is the absorbance of the blank.
Intracellular ROS assay
RKO and HCT116 cells were seeded onto 6-well plates at a density of 5 × 104 cells per well and cultured for 1 day. When the attached cells reached a density of approximately 80%, the original medium was replaced with fresh medium containing PBS, IVM, H780, and HA/H-I NPs at a concentration of 10 µM and further cultured for 6 h. This was followed by three washes with prechilled PBS. Then, H780 and HA/H-I NPs were irradiated with 660 nm laser (1 W·cm− 2) for 3 min and incubated with serum-free culture containing DCFH-DA for 1 h. For qualitative analysis experiments, cells were washed 3 times with pre-chilled PBS, fixed in 4% paraformaldehyde for 15 min, and fluorescence photographs were taken by fluorescence inverted microscopy. For quantitative experiments, cells were washed 3 times with pre-chilled PBS and collected after trypsin digestion. The 0 h group served as a blank control, and the intracellular fluorescence signal was recorded by flow cytometry (BD Facsaria III).
Clone formation analysis
Clone formation assays are used to assess the long-term effects of cell proliferation. RKO and HCT116 cells were seeded onto 12-well plates at a density of 5 × 103 cells per well and cultured for 2 days. Then, cells were treated with the same concentrations of PBS, IVM, H780 and HA/H-I NPs for 6 h. After 3 washes with precooled PBS, H780 and HA/H-I NPs were irradiated with 660 nm laser (1 W·cm-2) for 3 min, and incubation was continued for 7 days. Then, they were fixed with 4% paraformaldehyde for 30 min and stained with crystalline violet overnight. A digital camera was used to take pictures and record.
LDH analysis
RKO and HCT116 cells were seeded onto 96-well plates at a density of 5 × 104 cells per well and cultured for 2 days. Then, the original medium was replaced with 200 µL medium containing different concentrations of IVM, H780 or HA/H-I NPs and further incubated for 6 h. The cells were rinsed three times with pre-chilled PBS, and medium without serum and double antibodies was added. H780 and HA/H-I NPs were irradiated with 660 nm laser for 3 min (1 W·cm− 2). Next, the cells were cultured overnight. Subsequently, the 96-well cell culture plates were placed in a multi-well centrifuge for 5 min at 400 g, and 120 µL of supernatant was carefully transferred from each well to a new 96-well cell culture plate using a pipette. Then, 60 µL of LDH working solution was added to each well of the new 96-well cell culture plate and incubated for 30 min at room temperature in a shaker using tin foil protected from light. Finally, the absorbance at 490 nm was measured using an enzyme marker to assess the effect of different drug treatment conditions on the cells.
Apoptosis assay
The Annexin V-FITC Apoptosis Detection Kit was utilized to analyze the percentage of cells undergoing apoptosis. Briefly, cells were seeded into 6-well plates at a density of 5 × 104 cells per well and cultured for 1 day. Cells were treated with the indicated concentrations of IVM, H780 and HA/H-I NPs for 6 h. After 3 washes with precooled PBS, H780 and HA/H-I NPs were irradiated with 660 nm laser (1 W·cm− 2) for 3 min, incubation was continued for 1 day. Then, cells was stained with Annexin V-FITC and PI combination for 15 min. Apoptosis was detected by flow cytometry according to the manufacturer’s protocol.
Immunoblotting assay
Briefly, cells were inoculated into 6-well plates at a density of 5 × 104 cells per well and incubated overnight. When the attached cells reached 75%, the original medium was replaced with fresh medium containing PBS, IVM, H780, and HA/H-I NPs at a concentration of 10 µM and further incubated for 6 h. Subsequently, the medium was washed three times with pre-cooled PBS and replaced with a fresh complete medium. Then, H780 and HA/H-I NPs were irradiated either with or without the 660 nm laser (1 W·cm-2) for 3 min, respectively, and the cells were incubated for 24 h. Cells were then collected, washed with pre-chilled PBS, and then lysed with radioimmunoprecipitation assay (RIPA) buffer (1% sodium deoxycholate, 1% Triton X-100, 10% SDS, supplemented with phosphatase inhibitors and protease inhibitors). Total lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride membranes and blocked with skim milk for 2 h at room temperature. Secondary antibodies were applied for 2 h after gentle overnight shaking at 4 °C with the indicated primary antibodies. Protein expression was detected by chemiluminescence.
Animal model
Healthy BALB/c mice aged 6–8 weeks were selected as experimental subjects, and all in vivo experiments were performed by the standards of the Chengdu University Animal Use and Care System. Before completing the experimental manipulations, the mice were placed in a suitable environment for 2 weeks to help them adapt to their new environment. Then, we collected CT26 cells at the logarithmic growth stage and injected approximately 5 × 106 CT26 cells into the right hind limb of each mouse by subcutaneous injection, thus establishing a tumor-bearing mouse model. Throughout the process, we strictly controlled the experimental conditions to ensure the safety and welfare of the mice (SYXK, sichuan, 2018 − 185; 2,022,312).
In vivo imaging
When the tumor volume approached 100 mm3, 100 µL of H780 and HA/H-I NPs were slowly injected into the mice through the tail vein, respectively. The dose was equivalent to 3 mg/kg of H780 component. Images were taken at 1, 2, 4, 6, 8, and 24 h after injection using the in vivo imaging system (Xenogen IVIS Kinetic system). The BALB/c mice were sacrificed at 24 h after injection. Then the organs including the heart, liver, spleen, lung, kidney, brain, and tumor were collected for imaging biodistribution analysis by the imaging system.
In vivo infrared imaging
When the tumor volume approached 100 mm3, 100 µL of saline, H780, and HA/H-I NPs were administered intravenously at a dose equivalent to 3 mg/kg of H780. After 4 h, the tumors of H780 and HA/H-I NPs treated mice were directly irradiated with 660 nm NIR laser. The temperature of the tumor site in mice was photographed and recorded using the NIR photothermal therapy imaging camera at 1-min intervals for a total of 5 min during NIR laser irradiation.
Evaluation of in vivo anti-tumor effect
The tumor-bearing mice were randomly divided into 6 groups (n = 5 per group) receiving either physiological saline, IVM, H780, or HA/H-I NPs. Each group of mice received an equal dose of 3 mg/kg IVM, H780, or HA/H-I NPs via tail vein injection every 2 days. Four h after administration, the mice were subjected to direct irradiation at the tumor site with a wavelength of 660 nm for 3 min. During the treatment period, tumor volume and mouse weight were measured every two days.
The tumor volume was calculated as follows:
$$\text{T}\text{u}\text{m}\text{o}\text{r} \, \text{v}\text{o}\text{l}\text{u}\text{m}\text{e}=\frac{a{b}^{2}}{2}$$
Where the longest and shortest tumor diameters are a and b, respectively.
On the 15th day, the mice were anesthetized and the tumors and organs were removed. Finally, the tumor tissue was photographed and weighed.
The tumor suppression rate was calculated according to the following equation:
$$\text{T}\text{u}\text{m}\text{o}\text{r} \, \text{s}\text{u}\text{p}\text{p}\text{r}\text{e}\text{s}\text{s}\text{i}\text{o}\text{n} \, \text{r}\text{a}\text{t}\text{e}=\frac{Wc-Wt}{Wt}\times 100\%$$
Where Wc is the mean tumor weight in the NS group and Wt is the mean tumor weight in the treatment group.
Histological examination
At the end of treatment, the relevant organs and tumors were separated and incubated in 4% paraformaldehyde solution. The associated tissues were embedded in paraffin, sectioned onto slides, and stained with hematoxylin and eosin (H&E). Associated tumors will also be frozen sectioned and fixed onto slides for further LC3B staining. Finally, tissue sections were observed with a light microscope (Nikon Eclipse Ci) and photographed.
Statistical analyses
Statistical analysis was conducted using GraphPad Prism 8.0 software, and all data are presented as means ± standard error of the mean (SEM), unless otherwise specified. To compare different groups, Student’s t-test, ANOVA, or non-parametric ANOVA was used as appropriate and as indicated in the figure legends (*P < 0.05, **P < 0.01, ***P < 0.001).