A minimalist multifunctional nano-prodrug for drug resistance reverse and integration with PD-L1 mAb for enhanced immunotherapy of hepatocellular carcinoma | Journal of Nanobiotechnology

Materials

Dichloromethane (DCM), Tetrahydrofuran (THF), Ethyl acetate, Methanol (MeOH) were supplied by Hengyang Hongjin Chemical Company. Sodium sulfate (anhydrous) was obtained from Tianjin Beilian Fine Chemicals Development Co., Ltd. 5-FU, IND, Trifluoroacetic acid (TFA), Dicyclohexylcarbodiimide (DCC), Bromoacetic acid, 4-dimethylaminopyridine (DMAP), 2,2’-Dithiodiethanol, Di-tert-butyl dicarbonate ((BOC)₂O), Potassium hydroxide (KOH) and C6 were obtained from Macklin Co., Ltd. GSH (Reduced) and Sodium bicarbonate (NaHCO₃) were supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. 0.25% trypsin-EDTA, penicillin-streptomycin, RIPA Lysis Buffered Solution, Fetal bovine serum (FBS) and DMEM medium were obtained from MeilunBio Co., Ltd. MTT was supplied by Coolaber. GSH and oxidized glutathione (GSSG) Assay Kit, Annexin V-FITC Assay Kit, ROS Assay Kit and Hoechst 33,342 were obtained from Beyotime Biotechnology Co., Ltd.

C57BL/6j mice were supplied from Hunan Slack Jingda Experimental Animal Co., Ltd. (Hunan, China). All animals were kept in the Laboratory Animal Centre in a specific pathogen-free (SPF) condition.

Synthesis of FUA

First, the 5-FU derivative FUA was synthesized according to previous report. Briefly, KOH (1.32 g) was mixed with deionized water (5 mL), followed by mixing with 5-FU (1.046 g). Stirring at 60 °C continued until a transparent solution was formed. At 60℃, bromoacetic acid (1.146 g) was introduced and stirred for 6 h. After the solution was cooled, the pH was adjusted to 5, and impurities were removed by suction filtration after refrigeration for 6–8 h. The pH was then lowered to 2, and the mixture was refrigerated until the product separated out. The crystallized compound FUA was separated and dried under vacuum.

Synthesis of bOC-IND

A mixture of equal volumes of THF and deionized water (35 mL) was dissolved with NaHCO₃ (740 mg), (BOC)₂O (630 mg), and IND (520 mg). The solution was stirred with ice cooling for 10 min, then transferred to room temperature, where it was allowed to react for 24 h. Following this, THF was removed, and the remaining liquid was neutralized to pH 1.0 using 1 M hydrochloric acid, followed by overnight refrigeration. Next, the residue was extracted with ethyl acetate, and after evaporating the ethyl acetate, a yellow solid was obtained. The final products were characterized using ¹H NMR.

Synthesis of bOC-IND-SS-OH

BOC-IND (300 mg), DCC (466.7 mg) and 2,2′Dithiobisethanol were combined in DCM (10 mL) and kept reaction at 30℃ for 24 h. Following filtration and rotary evaporation, the target compound was separated and purified using chromatographic separation, eluting with MeOH/DCM (v/v, 1:200-1:100). The final products were characterized using ¹H NMR.

Synthesis of FU-SS-IND-BOC

The BOC-IND-SS-OH was stirred in DCM (10 mL) at 25℃, and DMAP, DCC, FUA were added to kept reaction for 24 h. Following filtration and rotary evaporation, the target compound was separated and purified using chromatographic separation, eluting with MeOH/DCM (v/v, 1:80 − 1:30). The final products were characterized using ¹H NMR.

Deprotection of bOC groups of FU-SS-IND

FU-SS-IND-BOC was combined with a mixture of DCM and TFA for 2 h at 25℃. Following filtration and rotary evaporation, the target compound was separated and purified using chromatographic separation, eluting with MeOH/DCM (v/v, 1:80 − 1:25). The final products were characterized using ¹H NMR.

Preparation of the FU-SS-IND NPs

The FU-SS-IND NPs were synthesized using the nanoprecipitation method. Briefly, 50 µL methanol with dissolved FU-SS-IND (10 mg mL^− 1) was gradually introduced into 1.2 mL water, followed by stirring for half an hour. FU-SS-IND NPs loaded with C6 or IR780 were prepared employing a comparable nanoprecipitation technique. The morphology and particle size of nano-prodrug were assessed by TEM and Malvern ZS90 DLS instrument, respectively.

Drug release profile of nano-prodrug in vitro

By employing a dialysis method and UV-visible spectrophotometer, the release characteristics of FU-SS-IND NPs in vitro were investigated. In this investigation, FU-SS-IND NPs (1 mg mL^− 1, 0.5 mL) were enclosed in a dialysis bag (MWCO = 3500 Da) and placed into a 30 mL aqueous solution with varying GSH concentrations (0, 1, 5, 10 mM). The system was then kept at 37℃ for 48 h with agitation. Samples were collected at specific time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36 and 48 h), the release of 5-FU in FU-SS-IND NPs was determined using UV-vis spectroscopy.

Cell culture

Hepa1-6 (a mouse HCC cell line) was supplied from the Cell Bank of the Chinese Academy of Sciences. At 37℃ in a 5% CO₂ atmosphere, the cells were cultured in DMEM high glucose medium containing 10% FBS and 1% penicillin/streptomycin.

To establish 5-FU resistance in Hepal-6 cells, Hepal-6 cells were continuously cultured in escalating doses of 5-FU (5–30 µM) for at least 6 months, until the cells could be expanded in medium containing 30 µM 5-FU.

In vitro cellular uptake of FU-SS-IND NPs

We added C6 to construct C6@FU-SS-IND NPs to evaluate Hepa1-6/FU uptake behavior. Hepa1-6/FU cells were cultured in 12-well plates or 24-well plates. Subsequently, free C6 or C6@FU-SS-IND NPs were introduced into the culture medium. After 0 h, 0.5 h, 3 h, 6 h incubation, 12-well plates cells were harvested and suspended in 500 µL PBS for detecting by FCM. For 24-well plates, cells were immobilized with 4% paraformaldehyde, subsequently stained with Hoechst 33,342 for 15 min. The resulting cells were imaged using a FIM to obtain fluorescent images.

In vitro cytotoxicity assessment

The cytotoxicity assessment used 96-well plates and the seeding number of Hepa1-6/FU cells was 3 × 10⁵ cells per well, followed by treated with the drug/nano-prodrug for 48 h. Furthermore, to assess the cytotoxicity of FU-SS-IND NPs after GSH treatment, Hepa1-6 cells were exposed to varying concentrations of GSH (0, 1, 5 and 10 mM) for 12 h prior to dosing, followed by 48 h with FU-SS-IND NPs, with the separate GSH group serving as a control. Then, MTT solution (5 mg/mL) was applied and treated for 4 h. The crystals that resulted were dissolved in 100 µL SDS solution, and a microplate read recorded their absorbance at OD 562 nm. Cell viability was calculated as:

In vitro apoptosis analysis

The apoptosis experiment used 12-well plates and the seeding number of Hepa1-6/FU cells was 1 × 10⁵ cells per well. Then, PBS, 5-FU, IND, 5-FU + IND and nano-prodrug FU-SS-IND NPs were diluted with DMEM medium containing 5% FBS to treat the cells respectively. After 48 h, cells were treated by the prescribed procedure of the Annexin V-FITC/PI Assay Kit. Quantitative detection of apoptotic rate in Hepa1-6/FU cells was performed using FCM.

Cysteine content detection assay

To exploit cysteine levels, Hepa1-6/FU cells were exposed to either PBS, 5-FU, IND, 5-FU + IND or FU-SS-IND NPs. 48 h post treatment, cells were harvested. The levels of cysteine were then measured by the Cysteine Assay Kits.

ICD induction

The HMGB1 release and CRT exposure were evaluated using FIM. The seeding number of Hepa1-6/FU cells was 8 × 10⁴ cells/well on 24-well plates. Following 24 h incubation, the cells were then exposed to either 5-FU, IND, 5-FU + IND or FU-SS-IND NPs. 48 h post incubation, cells were fixed with ice-cold methanol or 4% paraformaldehyde, blocked by 1% bovine serum albumin. Following 1 h treatment, then the cells were treated with either anti-CRT antibody or anti-HMGB1 antibody overnight at 4℃. After PBS washing, secondary antibodies were treated with cells for 2 h at room temperature. Afterward, cells were stained for 15 min with Hoechst 33,342. The resulting cells were imaged using a FIM to obtain fluorescent images.

The ATP assay Kit was employed to assess the released ATP levels. Hepa1-6/FU cells were treated with either 5-FU, IND, 5-FU + IND or FU-SS-IND NPs. The culture media was gathered for analysis after 48 h of incubation.

Intracellular GSH and ROS content

The intracellular GSH levels between Hepa1-6 and Hepa1-6/FU cells and the intracellular GSH content of Hepa1-6/FU incubated with various preparations were assessed by GSH and GSSG Assay Kit. The cells were incubated with PBS, 5-FU, IND, 5-FU + IND, FU-SS-IND NPs. After 48 h treatment, cells were harvested by centrifugation. The levels of GSH were then assessed by the GSH and GSSG Assay Kits.

The intracellular ROS levels in Hepa1-6/FU exposed to various conditions were investigated using the ROS fluorescence probe (DCFH-DA). In brief, Hepa1-6/FU cells were incubated in 24-well plates. After 24 h incubation, Hepa1-6/FU cells were expose to 5-FU, IND, 5-FU + IND and FU-SS-IND NPs for 48 h. Subsequently, serum-free medium supplemented with DCFH-DA was utilized to replace the medium and treated for a further 30 min at 37℃. The cellular ROS generation was imaged by FIM and quantitatively analyzed by FCM.

Hemolysis evaluation

Hemolysis test was used to evaluate the blood biocompatibility and feasibility of intravenous administration of FU-SS-IND NPs. Fresh Sprague-Dawley rat blood was centrifuged at 3000 rpm for 5 min, the pellet was collected, and red blood cells were extracted by washing 3 times with PBS. Add PBS (pH7.4) to prepare a 2% (v/v) red blood cell suspension. Mix different concentrations of FU-SS-IND NPs solution (80, 400, and 800 μg/mL) with an equal volume of red blood cell suspension and add to the centrifuge tube while incubating at 37℃ for 3 h. PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively. Then, the mixture was centrifuged at 1200×g for 10 min at 4℃, the supernatant was transferred to a new 96-well plate, and absorbance was measured at OD 560 nm using a microscope. The hemolysis rate was calculated according to the following equation:

In vivo biodistribution

In the right lower buttocks of the mice, tumor-bearing mouse models were established by inoculating a total of 5 × 10⁶ Hepa1-6/FU cells/mouse. The mice were partitioned into two groups at random once their tumor volume reached nearly 400 mm³. The one group was injected with IR780@FU-SS-IND NPs intravenously, the other one injected with free IR780 was used as its control. All the mice were imaged at 0.5, 6, 9, 12, 24, 36, 48 h after injection, using a small animal optical molecular imaging system for acquiring the body fluorescence images.

In vivo antitumor performance

The mice were allocated into 5 groups (n = 5) after their tumor volume reached nearly 100 mm³.The mice received treatments of saline, 5-FU, IND, 5-FU + IND, FU-SS-IND NPs once every 2 days for 4 doses, at a dose of 10 mg kg^− 1 5-FU. The formula for calculating tumor volume: volume = width² × length × 0.5.

The mice were allocated into 4 groups (n = 5) after their tumor volume reached nearly 100 mm³. The mice received treatments of saline, FU-SS-IND NPs, PD-L1 mAb and FU-SS-IND NPs + PD-L1 mAb once every 2 days for 4 doses (FU-SS-IND NPs were injected intravenously on days 1, 3, 5, and 7, respectively; intraperitoneal injection of 10 mg kg^− 1 of PD-L1 mAb on days 2, 4, 6, and 8, respectively). The mice’s body weight and tumor volume were recorded bidaily, and the survival rate of the mice in each group was monitored within 60 days.

Tumors and main organs of the mice were harvested the day the treatment experiment was concluded, immobilized with 4% (v/v) paraformaldehyde, processed for paraffin embedding for subsequent H&E staining or immunofluorescence assays.

In vivo immune response experiments

Tumor-infiltrating lymphocytes or spleen cells with various treatments were assessed through FCM. Lymph nodes, tumors and spleens from each group of tumor-bearing mice were harvested on day 9 after treatment, dispersed through a filter screen into cell suspension, stained with the fitting antibodies, and analyzed by FCM. Tcm cells were detected by CD3, CD8a, CD62L, and CD44 antibodies, CD8⁺ T cells were assessed with CD3, CD8a, and CD4 antibodies, Treg cells were identified using CD3, CD4, CD25 and Foxp3 antibodies, the maturation of DCs were assessed with CD11c, CD86 and CD80 antibodies, and PD-L1 level in tumor cells was detected by PD-L1 antibody.

Whole blood was harvested from mice to extract serum on the day the experiment ended, and the levels of IFN-γ and TNF-α were determined through ELISA kit.

Statistical analysis

The results were presented as mean ± standard deviation (SD). The statistical analysis utilized one-way analysis of variance (ANOVA) and Student’s t-test in GraphPad Prism version 8 software. Statistical significance between the datasets was defined as follows based on the p-values: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.