Materials
Len was purchased from Shanghai Rhawn Co., Ltd. (Shanghai, China). γ-Benzyl-L-glutamate-N-carboxyanhydride (BLG-NCA) was provided by Shanghai Yixin Biological Technology Co., Ltd. (Shanghai, China). N, N’-Diisopropylcarbodiimide (DIC), 1-hexylamine, 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N-(Boc) ethanolamine were obtained from Aladdin Reagent Co., Ltd. (Beijing, China). Linoleic acid (LA), stearic acid (SA), trifluoroacetic acid (TFA) and triethylamine (TEA) were bought from Shanghai Rhawn Co., Ltd. (Shanghai, China). Poly (ethylene glycol) monomethyl ether (mPEG, Mn = 5000) was bought from Sigma-Aldrich (St. Louis, MO, USA). RPMI 1640 and fetal bovine serum (FBS) were provided by Gibco BRL Life Technology (Grand Island, NY, USA). Penicillin and streptomycin were obtained from Huabei Pharmaceutical Co. Ltd. (Shijiazhuang, China). The antibodies used for flow staining were sourced from Biolegend (San Diego, CA, USA) and eBioscience (San Diego, CA, USA). Other reagents and solvents were provided by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Methods
Synthesis of PLA-g-mPEG and PSA-g-mPEG
Poly(L-glutamic acid) (PLG) was prepared via the ring-opening polymerisation of BLG-NCA using 1-hexylamine as the initiator. The ratio of BLG-NCA monomer to 1-hexylamine was 180:1. Following a 72-h reaction at 30 °C, the mixture was precipitated, filtered and redissolved with DMF. PLG was obtained after dialysis (MWCO = 5000 Da) and freeze-drying. PLG-g-mPEG was prepared by Steglich esterification between PLG and mPEG-OH. PLG and dried mPEG were dissolved in DMF at 40 °C, and DIC and DMAP were added after cooling to room temperature. The reaction mixture was precipitated with ether, filtered and dried in a vacuum after the reaction at room temperature for 72 h. After the white solid was dissolved in water and underwent dialysis and freeze-drying, white solid PLG-g-mPEG was obtained. The mass ratio of PLG to mPEG is 1:4. PLG-NH(Boc)-g-mPEG was obtained by the esterification of PLG-g-mPEG with N-(Boc) ethanolamine. Subsequently, Boc protection groups were removed using TFA to obtain PLG-NH2-g-mPEG.
Next, SA (28 mg) was dissolved in DMF (3 mL), and the solution was dehydrated and deoxygenated; EDC (40 mg) and NHS (30 mg) were dissolved in DMF (2 mL) and added into the SA solution for activation. Subsequently, PLG-NH2-g-mPEG (68 mg) and TEA (40 µL) were dissolved in DMF (6 mL) and added to the mixture. The reaction was conducted at room temperature for 24 h. The product was dialysed in DMF and pure water and then lyophilised to generate PSA-g-PEG. PLA-g-PEG was synthesised under the same experimental conditions, except that the LA was replaced with SA.
Synthesis of Len-RNPs and Len-NRNPs
Len-RNPs were prepared via the thin film hydration method. First, Len and PLA-g-mPEG were co-dissolved in CH2Cl2 at a mass ratio of 10%. The solvent was dried using a rotary evaporator at 40 °C. Deionised water was added to the mixture to form micelles through self-assembly. The unassembled drug aggregates were removed via filtration through a 0.22-µm membrane and then freeze-dried.
We set a control Len nanoparticle without ROS-responsive capability (Len-NRNPs). The Len-NRNPs were synthesised following the same synthesis method and experimental conditions, except that PLA-g-mPEG was replaced with PSA-g-mPEG.
The drug loading content values of Len was determined using a high-performance liquid chromatography (HPLC) system comprising a Waters 2414 Refractive Index Detector, Waters 515 HPLC pump and reverse-phase C-18 column. The resulting nanoparticles exhibited a hydrodynamic diameter of ~ 165.9 nm. Dynamic light scattering (DLS) was used to characterise the nanoparticle sizes.
In vitro drug release
The in vitro release of Len from the samples was evaluated under different conditions at 37 °C (n = 3). Len-RNPs or Len-NRNPs (containing 1.0 mg Len and dissolved in 5 mL buffer) were added to a dialysis bag (MW 3500 Da) and then immersed in 45 mL buffer with continuous shaking (100 rpm) at 37 °C under three conditions: without US treatment (US: 1.0 MHz, 2.0 W/cm2, 50% duty cycle, 2 min) and with US treatment for 1 and 3 min. At specified intervals, 5 mL of the medium was withdrawn and replaced with an equal amount of fresh medium. The amount of released Len was measured via HPLC, with a mobile phase comprising water and acetonitrile (20/80, V/V), at a flow rate of 1.0 mL/min and wavelength of 300 nm.
Cell lines and animals
Murine H22 hepatic cancer cell line was obtained from the BeNa Culture Collection (Beijing, China). Healthy BALB/c mice (female, 8 weeks old, 18–20 g), Kunming mice (female, 8 weeks old, 20–25 g) and Sprague–Dawley (SD) rats (female, 7 weeks old, 200–220 g) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and all procedures were approved by the Animal Care and Use Committee of Jilin University.
To establish a subcutaneous cancer model, the H22 cells were washed with normal phosphate-buffered saline (PBS) twice and diluted with normal PBS to a concentration of 2 × 107 cells/mL. H22 cells (2 × 106 cells, 100 µL) were injected into the right flank region of the BALB/c mice.
Pharmacokinetics study
Female SD rats were randomly assigned into two groups (n = 3). Len (10 mg/kg, dissolved in the 8:1:1 mixture of PBS, ethanol, and Cremophor EL) or Len-RNPs (10 mg Len/kg, dissolved in PBS) were administered via the tail vein. Blood samples were collected from the orbital cavity through a capillary tube at specific intervals (5 min and 0.5, 1, 2, 4, 8, 10 and 24 h) and mixed with heparin sodium. Serum was obtained via centrifugation and treated with acetonitrile: H2O (80:20, v/v). Finally, the obtained plasma solutions were assessed via HPLC. The half-life (t1/2) and area under the concentration–time curve of the drug from 0 to 24 h in plasma (AUC0 − t) were calculated using PKSolver.
Biodistribution study
Female BALB/c mice were subcutaneously inoculated with H22 cells (2 × 106) in the right flank region. When the tumour size reached a size of ~ 300 mm3, free Len or Len-RNPs were intravenously injected into the mice at the same dose of Len (10 mg/kg; n = 3). The US conditions were as follows: 1.0 MHz, 2.0 W/cm2, 50% duty cycle and 2 min. The mice were euthanised at each time point, and organs and tumours were harvested at 6, 24 and 48 h, accurately weighed and ground into pieces. The tissue debris was treated with a mixture of acetonitrile: H2O (80:20, v/v). The final concentration was 100 mg/mL, and the supernatant was separated via centrifugation (1000 ×g at 4 °C for 20 min). Finally, the obtained supernatant was assessed via HPLC.
In vivo anti-tumour efficiency
H22 cells (2 × 106) were subcutaneously injected into the right flank region of female BALB/c mice. When the tumour volumes reached approximately 120 mm3, the mice were randomly assigned into 8 groups (n = 5) and subjected to treatment with PBS, Len (10 mg/kg, dissolved in the 8:1:1 mixture of PBS and ethanol, Cremophor EL), US (1.0 MHz, 2.0 W/cm2, 50% duty cycle, 2 min), Len + US, Len-NRNPs (10 mg/kg at Len), Len-RNPs (10 mg/kg at Len), Len-NRNPs + US or Len-RNPs + US. Mice were intravenously injected with different formulations every 2 day and received three treatments in total. Body weights and tumour volumes were recorded every day. At the end of the experiments, mice were euthanized by using carbon dioxide in accordance with the NIH Guidelines for the euthanasia of animals.
The tumour volume was measured using calipers and calculated as follows:
Tumour volume (V) = a × b2/2
where a is the major axis and b is the minor axis of the tumour
The tumour suppression rate (TSR) was calculated as follows:
TSR (%) = [(Vc − Vx)/Vc] × 100%
where Vc and Vx represent the mean tumour volumes of the PBS and treatment groups, respectively.
Immunohistochemical analyses
At the end of the treatments, the tumours and main organs from each group were collected and fixed in 4% (W/V) PBS-buffered paraformaldehyde over 48 h, and then embedded in paraffin. The paraffin-embedded tumours and organs were cut into 5 μm–thick slices that were stained with hematoxylin and eosin (H&E) to evaluate the histological alterations of tumours and the major organs.
Flow cytometry analysis
To analyze the immune cells in tumour tissues, the obtained tumours were digested in a tumour dissociation buffer. Single-cell suspensions were obtained after filtration using nylon mesh and incubated with antibodies against the immune cells. Flow cytometry was conducted using a BD FACS Celesta flow cytometer. The results were analysed using FlowJo software. The cells were stained with the following antibodies: FITC-conjugated anti-CD3, APC-conjugated anti-CD8a and PE/Cy7-conjugated anti-CD4 antibodies for T cells; PE-conjugated anti-CD49b antibodies for natural killer (NK) cells; PE-conjugated anti-CD11c, APC-conjugated anti-CD80 and APC/Cy7-conjugated anti-MHC II antibodies for activated DCs; APC/Cy7-conjugated anti-CD11b and PE-conjugated anti-Gr-1 antibodies for MDSCs; PE/Cy7-conjugated anti-F4/80 and FITC-conjugated anti-CD206 antibodies for M2-like macrophages; PE/Cy7-conjugated anti-F4/80 and APC-conjugated anti-CD80 antibodies for M1-like macrophages. The gating strategy for all samples was configured to ensure the removal of cell debris, dead cells, and large clumps or cell aggregates.
Analyses of inflammatory cytokines
To analyze inflammatory cytokines in the serum, peripheral blood was collected from mice at the end of the experiment. Cytokines in the serum, including interferon-gamma (IFN-γ), interleukin 6 (IL-6), interleukin 10 (IL-10) and tumour necrosis factor alpha (TNF-α) were detected using enzyme-linked immunosorbent assay kits. All tests were conducted following the manufacturer’s protocols.
Safety evaluation of nanoparticles in anticancer therapy
Blood biochemical and routine analyses were performed to evaluate biosafety in vivo after anticancer treatment. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were quantified to assess liver function in treated mice. Kidney function was determined by measuring the serum blood urea nitrogen (BUN) and creatinine (CRE) levels. The organs (heart, liver, spleen, lung, and kidney) were obtained from mice in each group and sliced and stained via H&E staining.
Statistical analysis
All experiments were performed at least three times and results were expressed as means ± standard deviation (SD). Data from two groups were compared via a two-tailed unpaired Student’s t-test, whereas multiple groups were compared using one-way analysis of variance and Tukey’s multiple comparison test. Differences were considered significant at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
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- Source: https://www.nature.com/articles/s41598-024-79069-9