Materials
Lipoic acid (LA), p-toluenesulfonic acid (PTSA), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-Hydroxysuccinimide (NHS) were purchased from Sigma (USA). 2, 2-dimethoxypropane (DMP) was obtained from Alfa Aesar Co. LtD (China). Docetaxel (DTX), 2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorofluoresceindiacetate (DCFDA), Calcein-AM/PI Double Staining Kit, 4′,6-diamidino-phenylindole (DAPI) were provided by Beyotime Biotechnology (China). Resiquimod (RESQ), cRGD peptide were gained from MedChemExpress (USA). BUV395 anti-mouse CD45, FITC anti-mouse CD80, BV605 anti-mouse CD86, FITC anti-mouse CD3, PE anti-mouse CD4, BUV496 anti-mouse CD8a were supplied by BD Biosciences.
Cell lines
Mouse Hepa1-6 and H22 tagged with luciferase HCC cells were purchased from ATCC. These cells were cultivated in DMEM high glucose medium supplemented with 10% FBS, 100 U mL− 1 penicillin, and 100 µg mL− 1 streptomycin and in the incubator containing 5% CO2 under 37 °C.
Mice
BLAB/c mice (15 ~ 18 g) were purchased from laboratory animal center of Zhejiang Academy of Medical Sciences. All animal experiments strictly followed the laboratory animal guidelines formulated by the National Institutes of Health Laboratory Animal Care and Use.
Preparation of TPs
The ROS/GSH-responsive thioketal polymers (TPs) were synthesized via a condensation reaction between dihydrolipoic acid and 2,2-dimethylpropane (DMP). Briefly, 5 g of lipoic acid was dissolved in 50 mL of sodium bicarbonate aqueous solution and mixed with 0.2 M sodium borohydride. The mixture was stirred in an ice bath for 30 min. The resulting solution was extracted 3 times with 40 mL of chloroform, and the organic phase was dried overnight using anhydrous magnesium sulfate. Subsequently, 200 mg of the dried product and 100 mg of DMP were dissolved in 50 mL of distilled benzene and transferred to a three-necked flask. Methane byproducts were removed, and the system was purged with argon gas to ensure a deoxygenated environment. The mixture was heated to 95 °C under continuous stirring, followed by the addition of a recrystallized p-toluenesulfonic acid (PTSA) solution (3 µM) in distilled ethyl acetate. The reaction was maintained under an argon atmosphere for 1 h, after which distilled DMP (1 mM) was added at a rate of 8.67 mg/h. The mixture was then stirred overnight and finally precipitated in cold water.
Preparation of LET-TPs@DTX NPs
A solution containing 5 mg of LET-Br and 5 mg of TPs was prepared in 10 mL of an aqueous solution with 0.1% sulfuric acid. The mixture was heated and condensed for 2 h to facilitate dehydration and ester formation. For effective loading of DTX, an organic solvent-based volatilization method was employed. Specifically, 100 mg of LET-TPs and 5 mg of DTX were dissolved in 5 mL of chloroform. The resulting solution was stirred for 10 min and then sonicated for 1 h to ensure homogeneity. Following this, the organic solvents were removed using rotary evaporation. The unloaded drugs were separated by centrifugation for 10 min (12000 rpm/min, 25 ℃). This process successfully yielded LET-TPs@DTX nanoparticles. Calculate the encapsulation rate and loading capacity of the drug based on the unloaded drug, and the calculation formula is as follows: encapsulation rate (EE%) = (total drug – no packaged drug)/total drug; the loading capacity (LC%) = the total drug load/total nanoparticle weight.
Synthesis of LET-TPs@DTX@R/c NPs
A total of 100 mg of LET-TPs@DTX NPs were incubated with 5 mg of EDC and 5 mg of NHS, and the mixture was gently shaken at 120 rpm for 12 h to activate the carboxyl groups. Subsequently, 5 mg of RESQ and 5 mg of cRGD were added to the mixture, followed by continuous stirring for an additional 24 h to facilitate conjugation. To remove any unbound RESQ and cRGD, the solution was dialyzed using dialysis bags with a molecular weight cutoff (MWCO) of 30 kDa at 5000 rpm. Finally, the purified LET-TPs@DTX@R/c NPs were obtained and stored at 4 °C for subsequent experiments.
Characterizations
High-resolution transmission electron microscopy (HRTEM) images were acquired using a transmission electron microscope (TEM; HT7700, Japan) to monitor the synthesis process. The hydrodynamic size distribution and zeta potential of the nanoparticles were analyzed using dynamic light scattering (DLS; ZS90, Malvern) under conditions simulating the tumor microenvironment, specifically in response to 10 mM γ-glutathione (γ-GSH) and weakly acidic microenvironment (pH = 5.5). Fluorescent imaging was performed using a confocal laser scanning microscope (CLSM, Zeiss LSM710). Flow cytometry analysis was conducted on a flow cytometer (BD, C6) to evaluate cellular uptake and other biological interactions. Ultraviolet-visible (UV-Vis) absorption spectra were recorded using a spectrophotometer (Shimadzu, UV3600). Additionally, bioluminescence imaging was performed using an IVIS Spectrum imaging system to assess in vivo distribution and therapeutic efficacy.
The generation of 1O2 was further confirmed using electron spin resonance (ESR) spectroscopy with the radical scavenger TEMPO. Briefly, a solution of LET-TPs@DTX@R/c nanoparticles (5 mg/mL) containing TEMPO was exposed to laser irradiation (0.2 W cm⁻²) for 5 min. After centrifugation, the supernatant was collected, and its ESR spectra were analyzed using an optical spectrum instrument (Bruker EMX ESR spectrometer). This method provided direct evidence of 1O2 generation under photodynamic conditions.
The UV-vis absorption spectra of LET-TPs@DTX@R/c NPs were measured using a spectrophotometer (UV2550, Shimadzu, Japan) across a range of pH buffers (pH = 5.5, 6.5, 7.5, 8.5, 9.5). Additionally, the fluorescence spectra of LET-TPs@DTX@R/c NPs were acquired using a fluorescence spectrophotometer (F7000, Hitachi) under the same pH conditions. Stability is a critical factor influencing the therapeutic efficacy of nanoparticles, and the stability of LET-TPs@DTX@R/c NPs was assessed by monitoring their size distribution over a period of 7 days at 37 °C using dynamic light scattering (DLS). This evaluation ensured the robustness of the nanoparticles under physiological conditions.
In vitro photothermal property of LET-TPs@DTX@R/c
Infrared thermal imaging was employed to monitor the temperature changes of LET-TPs@DTX@R/c NPs at varying concentrations (0, 2, 5, and 10 µg/mL) over different time intervals (0, 2, 3, 4, and 5 min) under 808 nm laser irradiation (2 W/cm²). Specifically, a 200 µL solution of LET-TPs@DTX@R/c NPs containing 4 µg/mL of LET-Br was irradiated with an 808 nm laser (2 W/cm²) for 5 min, followed by a 5-minute natural cooling period after the laser was turned off. Temperature changes were recorded using an infrared thermal camera. To evaluate photostability, the nanoparticles underwent five consecutive cycles of heating and cooling treatments, ensuring their robustness under repeated photothermal conditions.
Drug release rate analysis
The release behavior of DTX from LET-TPs@DTX@R/c NPs was evaluated under conditions mimicking the tumor microenvironment and normal physiological environment. Specifically, 10 mL of LET-TPs@DTX@R/c NPs was dialyzed against 100 mL of PBS containing 10 mM GSH at two different pH levels (pH = 5.5 and pH = 7.5) using a dialysis bag with a molecular weight cutoff of 3.5 kDa. At predetermined time intervals (2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h), 1 mL of the buffer was sampled and replaced with an equal volume of fresh buffer to maintain sink conditions. The concentration of released DTX was quantified using a microplate reader, enabling the assessment of the pH- and GSH-responsive drug release kinetics of the nanoparticles.
ROS generation ability of LET-TPs@DTX@R/c
The generation of intracellular ROS was detected using DCFH-DA as a fluorescent probe. Hepa1-6 cells were seeded into 6-well plates at a density of 3 × 10⁵ cells per well and allowed to adhere overnight. The next day, the cells were treated with LET-TPs@DTX, LET-TPs@DTX@R, or LET-TPs@DTX@R/c (2 mg/mL DTX equivalent) and incubated for 4 h. Prior to irradiation, the culture medium in each well was replaced with 2 mL of fresh medium containing 10 µM DCFH-DA. The cells were then exposed to 808 nm laser irradiation (2 W/cm²) for 5 min and further incubated at 37 °C for 1 h. After thorough washing with PBS, the cells were either imaged using a fluorescence microscope or harvested for quantitative analysis via flow cytometry to assess ROS levels.
Cell uptake assay
To evaluate the cellular uptake efficiency of LET-TPs@DTX@R/c NPs, Hepa1-6 cells were seeded into a 12-well culture plate at a density of 1.0 × 10⁵ cells per well and allowed to adhere for 12 h. The cells were then incubated with LET-TPs@DTX, LET-TPs@DTX@R, and LET-TPs@DTX@R/c NPs (2 mg/mL DTX equivalent) for 2 h, with untreated cells serving as the control group. After incubation, the cells were washed thoroughly to remove uninternalized nanoparticles. Cellular uptake was assessed using fluorescent confocal microscopy to visualize nanoparticle internalization and flow cytometry to quantify the uptake rate. This approach provided both qualitative and quantitative insights into the efficiency of nanoparticle uptake by Hepa1-6 cells.
In vitro cytotoxicity assay
Briefly, cells were seeded into 96-well plates at a density of 1.0 × 10⁴ cells per well and cultured overnight at 37 °C. Serial dilutions of free DTX, LET-TPs, LET-TPs@DTX, and LET-TPs@DTX@R/c NPs (0.4 mg/mL DTX equivalent) were prepared in fresh culture medium and added to the wells. After incubation, the drug-containing medium was replaced with fresh medium, and the cells were exposed to 808 nm laser irradiation for 5 min. Untreated cells served as the control group. Following an additional incubation period of 12 h and 24 h, 10 µL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated based on the absorbance values. This method allowed for the assessment of the cytotoxic effects of the NPs under photothermal conditions.
Therapeutic anti-HCC effect of LET-TPs@DTX@R/c
To establish an orthotopic HCC mouse model, male BALB/c mice aged 6 ~ 8 weeks were injected directly into the liver with 1 × 10⁶ H22-Luc cells. Seven days post-inoculation, the mice were randomly divided into five groups (n = 8) and administered the following treatments every two to three days starting from day 8: saline (control), free DTX, LET-TPs@DTX, LET-TPs@DTX@R, and LET-TPs@DTX@R/c NPs at a dosage of 5 mg/kg in a volume of 100 µL. 8 h after intravenous administration, the treatment groups were exposed to 808 nm laser irradiation (0.5 W/cm², 5 min). Tumor progression was monitored using an IVIS imaging system at various time points (days 8, 13, 17, and 21) by measuring the luminescence intensity of the H22-Luc signal. Additionally, survival rates and body weight were recorded every other day to assess treatment tolerability. At the endpoint of the study, liver tissues were harvested from all mice and photographed for macroscopic evaluation of tumor size. To further verify the anti-tumor effects, liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and processed for histopathological and immunohistochemical analysis. Antigen retrieval was performed using a citrate buffer solution (pH = 6.0) under high-temperature conditions in a pressure cooker. Liver sections were stained with P53 and Ki67 mouse monoclonal antibodies to assess tumor proliferation and apoptosis. This comprehensive approach allowed for the evaluation of therapeutic efficacy and mechanistic insights into the anti-tumor effects of the treatment regimens.
Pharmacokinetics, biodistribution and metabolism studies in vivo
For blood pharmacokinetics analysis, mice (n = 5) were intravenously injected with 100 µL of free DTX, LET-TPs@DTX, or LET-TPs@DTX@R/c NPs, all at the same therapeutic dose (5 mg/kg DTX equivalent). Blood samples (10 µL) were collected by gently nicking and milking the tail vein at various time points (5 min, 10 min, 2 h, 4 h, 8 h, 16 h, 32 h, and 24 h). The concentration of DTX in the blood was quantified using high-performance liquid chromatography (HPLC) to determine the blood circulation half-life of DTX in vivo. Chromatographic separation was performed on a COSMOSIL 5 C18-MS-II column (5 μm, 250 mm × 4.6 mm) using a mobile phase consisting of 0.1% phosphoric acid aqueous solution and acetonitrile. The flow rate was set to 1.0 mL/min, the column temperature was maintained at 30 °C, and the detection wavelength was 230 nm. Additionally, the biodistribution of DTX, LET-TPs@DTX, and LET-TPs@DTX@R/c was evaluated. At the end of the experimental period, major organs (liver, spleen, kidneys, heart, and lungs) were dissected, rinsed with PBS, weighed, and homogenized. The tissues were then digested with aqua regia solution, and the DTX content was quantified using HPLC to assess the distribution of the nanoparticles in different organs. This comprehensive approach provided insights into the pharmacokinetic profile and tissue distribution of the therapeutic agents.
Immune responses effect
To evaluate immune responses, orthotopic HCC-bearing mice treated with various regimens were sacrificed on day 15 (n = 5). To analyze T cell activation in the spleen, T cells were isolated from mice subjected to different treatments, as described in previously published protocols. Briefly, splenic T cells were harvested and analyzed by flow cytometry using the following antibodies: anti-CD45-BV421, anti-CD3-APC, anti-CD4-FITC, and anti-CD8-PE. Additionally, to assess the activation of tumor-infiltrating mature dendritic cells (DCs) and cytotoxic T lymphocytes (CTLs), tumors were excised from mice receiving different treatments, and single-cell suspensions were prepared based on established methods. The cells were then stained with anti-CD80-PC7, anti-CD86-BV605, anti-CD3-APC, anti-CD8-PE, anti-CD4-FITC, anti-CD44-PE-Cy7, and anti-CD62L-BV421 antibodies for flow cytometry analysis. Furthermore, the levels of cytokines (TNF-α and IFN-γ) in tumor tissues were quantified using mouse ELISA kits according to the manufacturer’s instructions. This comprehensive analysis provided insights into the systemic and tumor-specific immune responses elicited by the different treatments.
Toxicity studies in vivo
To assess the in vivo toxicity of LET-TPs@DTX@R/c NPs, BALB/c mice were divided into two groups: a PBS control group and a treatment group. The treatment group received intravenous injections of LET-TPs@DTX@R/c NPs at a dose of 5 mg/kg DTX, administered twice weekly for three consecutive weeks. At the end of the treatment period, blood samples and major organs (heart, liver, spleen, lungs, and kidneys) were collected for toxicity evaluation. Blood samples were processed to analyze biochemical indices of liver and kidney function, including mean corpuscular volume (MCV), blood urea nitrogen (BUN), creatinine (CRE), and mean corpuscular hemoglobin (MCH), as well as routine blood parameters such as white blood cell count (WBC), hemoglobin (HGB), red blood cell count (RBC), and platelet count (PLT). Additionally, tissue sections from the harvested organs were stained with hematoxylin and eosin (H&E) to enable histopathological examination and identify potential lesions. This comprehensive approach provided a detailed assessment of the systemic and organ-specific toxicity of LET-TPs@DTX@R/c NPs.
Statistics
All experimental results are expressed as means ± standard deviation (S.D.). Statistical analyses were performed using GraphPad Prism (version 9.0). The log-rank test was used to analyze survival time, while one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference (HSD) post hoc test was applied for multiple comparisons. Differences between experimental groups and control groups were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.