A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes | Journal of Nanobiotechnology

Materials

GA was bought from PureChem (China). PLGA (RESOMER® RG 502 H) was obtained from Evonik (China). Fluorescein sodium salt was purchased from Sigma (USA). BSA and trypsin (Mw, ∼ 23.8 kDa; activity, ≥ 250 U/mg) were purchased from Biofroxx (Germany). DAPI and DiD were obtained from Bioscience Technology (China). Transwell® (1 μm pore size) was purchased from Jet Bio-Filtration (China). Gelatin (250 g Bloom) was supplied by Macklin Biochemical (China). AgNO₃, phosphatidylcholine (PC), cholesterol, ethanol absolute, dichloromethane, trichloromethane, dimethyl sulfoxide (DMSO), and other chemical reagents were purchased from Kelong Chemical (China) .

RIPA, PMSF, and phosphatase inhibitor cocktail were bought from Servicebio (China). β-tublin and PD-L1 antibodies were acquired from Abmar (USA). CD31 and CD44 antibodies were purchased from ImmunoWay (USA). YF®488-conjugated goat anti-rabbit IgG was obtained from US Everbright (China). FITC-conjugated anti-CD44 was bought from 4 A Biotech (China).

Cell culture

Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Corning (USA). Fetal bovine serum (FBS) was purchased from Excell Bio (China). The mouse cell lines 4T1, bEnd.3, and 4T1-Luc were obtained from ATCC. Cells were cultured in DMEM or RPMI 1640 medium containing 1% antibiotics (penicillin-streptomycin, 10,000 U/mL) and 10% FBS in an atmosphere of 5% CO₂ at 37 °C.

Structure of trypsin

Trypsin’s crystal structure was acquired from the Protein Data Bank (PDB ID: 1MCT) and depicted with rainbow ribbons on a white background. The pink stick-and-surface model reveals the structure of cysteines (Cys), while the orange ball model reveals the structure of calcium ion.

Preparation and characterization of GA nanoformulations

GA-loaded Lip, BSA NP, and PLGA NP were prepared based previously reported method [33,34,35]. To prepare GA-loaded Lip, PC, cholesterol, and GA were dissolved in trichloromethane, and the solution was rotary evaporated to form a thin film, followed by adding PBS to hydrate. The sonication was performed in an ice-water bath for 10 min to obtain GA-Lip. To prepare GA-BSA NP, GA was dissolved in the dichloromethane and ethanol, then added to BSA solution, sonicated, and rotated evaporation to remove the organic solvent. To prepare GA-PLGA NP, PLGA and GA were dissolved in dichloromethane, and then added to 3% (w/v) PVA solution, emulsified using a vortex and sonicated. The mixture was rotary evaporated until the organic solvent was removed. The suspension was filtered and washed by centrifugation.

The particle size, polydispersity index (PDI), and zeta potential of these formulations were diluted with RO water and measured by a Litesizer™ 500 (Anton Paar, Austria). Drug loading and encapsulation efficiency of GA were detected using high-performance liquid chromatography (HPLC) (Shimadzu, Japan) with the chromatographic condition of 90% (v/v) acetonitrile and 10% (v/v) 0.2% phosphoric acid solution.

Internalization of nanoformulations after trypsin treatment

Nanoformulations were labeled with Coumarin 6 (C6) to measure the effect of trypsin on cell uptake and internalization. 4T1 breast cancer cells were seeded in 12-well plate at a density of 2 × 10⁵ cells/well and incubated for 24 h. After treatment with trypsin for 2 min, cells were incubated with C6 nanoformulations (2 µg/mL C6) for 2 h and collected. Cells were transferred to climbing slices, fixed after 24 h’s recovery, and stained by DAPI. The internalizations of C6 nanoformulations were observed through an FV1200 scanning confocal microscope (Olympus, Japan). Cell samples for flow cytometry were washed, resuspended in phosphate-buffered saline (PBS), and analyzed through FACSVerse™ (BD, USA).

To investigate trypsin’s effect on cell membranes, 4T1 cells were collected after treatment with trypsin for 2 min and 30 min. Cell membranes and nucleus were stained with DiD and DAPI for visualization using confocal microscopy. Meanwhile, cells were treated with 0.5% trypsin for 30 min, washed with PBS, and fixed by electron microscope fixative at 4 ℃ followed by SEM observation.

Measurement of total and membrane proteins after trypsin treatment

The 4T1 cells were seeded in 6-well plate at a density of 1 × 10⁶ cells/well and incubated for 24 h. After treatment with different concentrations (0.125-1%, w/v) of trypsin, 4T1 cells were washed with PBS and lysed in RIPA buffer containing PMSF and phosphatase inhibitor cocktail to extract the total proteins. The membrane proteins were extracted by a membrane extraction kit (Sangon Biotech, China). Total and membrane proteins with or without trypsin treatment were measured by the Bradford protein determination assay, followed by separation with SDS-PAGE gel. The obtained gel was stained with the Coomassie Blue superfast staining solution (Beyotime) for gel imaging. To detect CD44 and PD-L1 expression on trypsin-treated 4T1 cells, a western blot and flow cytometry analysis was performed. For western blot, total proteins were transferred onto the PVDF membrane. The membranes were soaked in a blocking solution, followed by incubation with the primary antibodies at 4 ℃ and the secondary antibody at room temperature. CD44 and PD-L1 were visualized by an OI 600 automatic chemiluminescence imaging system (BIO-OI, China). Tumor cells for flow cytometry analysis were treated with FITC-conjugated anti-CD44 or primary antibody against PD-L1, followed by incubating with YF^® 488-conjugated goat anti-rabbit IgG secondary antibody.

Endothelial permeability of nanoformulations after trypsin treatment

To evaluate the permeability of nanoformulations through the tight junction of endothelial cells, we developed an in vitro tight junction model with the immortalized mouse brain endothelial cell line, bEnd3, in a Transwell® system. bEnd.3 cells were cultured on Transwell® membranes (24-well type) coated with 2% gelatin for 5 d to form a confluent cell layer at an initial density of 3 × 10⁴ cells/well. A 4 h leaking test and fluorescein sodium permeability assay confirmed the tight junction formation [36, 37]. The in vitro tight junction model was treated with trypsin-containing serum-free medium for 30 min. Trypsin was removed and washed with PBS. C6 nanoformulations were added to the endothelial layer made by bEnd.3 cells. At the indicated time point (0 min, 10 min, 30 min, and 60 min), 100 µL of media in the lower chamber was collected and replenished with an equal amount of culture medium. The permeability rate of C6 nanoformulations was calculated by measuring the fluorescence intensity of C6 in the collected medium using a SpectraMax® i3 microplate reader (Molecular Devices, USA).

In vitro cell viability and apoptosis analysis

The viability of 4T1 cells treated with GA and GA nanoformulations was assessed by an MTT assay. 4T1 cells were seeded in a 96-well plate at a density of 5 × 10³ cells/well and incubated with free GA or GA nanoformulations for 48 h. The MTT solution subsequently replaced the culture medium. The MTT solution was discarded after 4 h’s incubation. Then, the formazan crystals were dissolved by adding DMSO. The absorbance of the formazan crystals was measured at 490 nm using a SpectraMax® i3 microplate reader. The viability of 4T1 cells treated with trypsin was assessed by an CCK-8 assay with the same procedure mentioned above. Each well was added with 10 µL of CCK-8 solution and incubated for 2 h. Formazan was measured at 450 nm by the microplate reader.

Apoptosis of 4T1 cells was analyzed after sequential treatment with trypsin and GA nanoformulations in the Transwell® system mentioned above. bEnd.3 cells in a Transwell® system were treated with different concentrations (0.125-1%, w/v) of trypsin in a serum-free medium for 30 min. The Transwell® insert was moved to a 24-well plate with 4T1 cells grown on the lower chamber at an initial density of 3 × 10⁵ cells/well. The whole system was treated with GA nanoformulations for 1 h, followed by the removal of the upper Transwell®. Finally, 4T1 cells were collected after incubation with GA nanoformulations for another 48 h. The cells were stained using Annexin V-fluorescein isothiocyanate (Annexin-V-FITC)/propidium iodide (PI) kit and analyzed by flow cytometry.

The viability of 4T1 cells was tested after concurrent treatment with trypsin and GA nanoformulations on 4T1 cells and bEnd.3 co-cultured model. The model was established by 4T1 cells seeded to a 24-well plate with bEnd.3 culturing on the upper Transwell®. The whole system was treated with GA nanoformulations and trypsin for 48 h. The MTT assay was performed to measure the viability of 4T1 cells. The absorbance was measured at 570 nm on a microplate reader.

Proteomics analysis

The label-free quantitative proteomic assessment was conducted by PTM BIO (China). In brief, 4T1 cells post-treatment with 0.5% trypsin (0.2 µmol/L) for 30 min were sonicated three times on ice using a high-intensity ultrasonic processor (Scientz, China) in lysis buffer (8 mol/L urea, 1% protease inhibitor cocktail). The remaining debris was removed by centrifugation. The supernatant was collected, and the protein concentration was calculated with a BCA kit according to the manufacturer’s instructions. The protein solution was reduced with 5 mmol/L dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was diluted by adding 100 mmol/L TEAB to a urea concentration of less than 2 mol/L. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for overnight digestion.

The tryptic peptides were dissolved in solvent A (0.1% formic acid, 2% acetonitrile/in water) and directly loaded onto a homemade reversed-phase analytical column. Peptides were separated with a nanoElute^® UHPLC system (Bruker Daltonics). The peptides were subjected to a capillary source followed by the timsTOF Pro (Bruker Daltonics) mass spectrometry. The electrospray voltage applied was 1.60 kV. Precursors and fragments were analyzed at the TOF detector, with an MS/MS scan range from 100 to 1700 m/z. The timsTOF Pro was operated in parallel accumulation serial fragmentation (PASEF) mode. Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEF-MS/MS scans were acquired per cycle. The dynamic exclusion was set to 30 s.

Construction and characterization of hydrogels

Trypsin@PSA Gel was prepared by mixing 4-arm PEG-SH (20 kDa, Yare Biotech, China), AgNO₃, and trypsin. Briefly, 20 mg of 4-arm PEG-SH was weighted and dissolved in 100 µL of 45 mg/mL trypsin in deionized water (solution A). The solution A was mixed with 200 µL of AgNO₃ solution in different concentrations. Unloaded PSA was prepared by replacing trypsin with deionized water.

Trypsin@PSA Gel with trypan blue was injected into water through a 26 G needle to investigate its injectability. PSA and Trypsin@PSA Gel were freeze-dried to observe the morphology by scanning electron microscope (SEM) (Zeiss, Germany). The viscoelasticity of PSA and Trypsin@PSA Gel was measured by a MARS™ rheometer (Thermo Fisher, USA). The storage modulus (G′) and loss modulus (G″) were assessed at 6.28 rad/s, with the shear strain amplitude ranges from 0.1–100%. Raman spectra were measured by an Alpha300R Raman spectrometer (WiTech, Germany) using an argon laser with a wavelength of 488 nm as the excitation source. The swelling properties of hydrogels were studied by the gravimetric method. Trypsin@PSA Gel and PSA were weighed before and after the immersion with PBS for 3 d.

Activity, stability, and release profiles of trypsin in Trypsin@PSA Gel

Trypsin activity in Trypsin@PSA Gel was measured by a trypsin activity assay kit (Jining, China) at pH 7.4. The Bradford protein assay kit (Beyotime, China) was used to investigate trypsin’s stability and release profiles in Trypsin@PSA Gel at pH 6.5 and 7.4. Before measuring the activity and stability, Trypsin@PSA Gel were diluted in water to make the solid hydrogel into a colloidal sol. To measure the release profile of trypsin, Trypsin@PSA Gel was freeze-dried and added to 5 mL media at pH 6.5 and 7.4. Samples were shaken at 37 ℃ at 100 rpm/min. At certain time points, 50 µL of the release medium was collected to measure the amount of released trypsin.

Biocompatibility of unloaded PSA

BALB/c mice were randomly divided into three groups to explore the biocompatibility of the hydrogel. Mice were injected subcutaneously with 100 µL of PBS or PSA and sacrificed on day 7. Tissue around the injection site was collected and fixed with 4% formaldehyde for H&E staining.

Biodistribution of DiD-labeled lip after intratumoral injection of Trypsin@PSA Gel

The animal experiments were approved by the ethics committee of the Chengdu University of Traditional Chinese Medicine (2022-45), and all animal experiments were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The subcutaneous breast cancer model was established by injecting 1 × 10⁶ 4T1 cells into the lower right abdomen of female BALB/c mice (18–20 g). DiD-labeled Lip was prepared for in vivo visualization. When tumor volume reached 200–300 mm³, mice were randomly divided into five groups (n = 3): Blank, DiD-Lip, PSA + DiD-Lip, trypsin + DiD-Lip, and Trypsin@PSA Gel + DiD-Lip. DiD-Lip was injected 24 h after intratumoral injection of PBS, PSA, trypsin, or Trypsin@PSA Gel. The administrated doses included: DiD (0.2 mg/kg) and trypsin (0.3 mg per tumor). At 4 h, 8 h, and 24 h after DiD-Lip injection, mice were anesthetized with isoflurane and photographed by an IVIS^® spectrum imaging system (PerkinElmer, USA). Then, mice were sacrificed at 48 h. Tumors were collected, photographed, and placed at -80 ℃. For immunofluorescence staining, tumors were treated with primary antibodies against CD31 at 37 °C, washed three times with PBS, and incubated with YF^® 488-conjugated goat anti-rabbit IgG for 30 min.

In vivo antitumor efficacy

The subcutaneous breast cancer model was developed by injecting 3 × 10⁶ 4T1-Luc cells into the right flank of BALB/c mice. One week later, mice were randomly divided into four groups (n = 8): PBS, GA-Lip, Trypsin@PSA Gel, and Trypsin@PSA Gel + GA-Lip. Specifically, PBS or Trypsin@PSA Gel were injected intratumorally on days 1, 3, 5, 7, and 9. GA-Lip was injected intravenously on days 2, 4, 6, 8, and 10. The administrated doses included: GA (12 mg/kg) and trypsin (0.3 mg per tumor). The body weight and tumor volume of treated mice were monitored every other day. On day 16, all mice were sacrificed, and the tumor weight was measured. Tumors were fixed with 4% formaldehyde for H&E, TUNEL, and immunofluorescence-stained with FITC-conjugated anti-CD44. SEM images of tumor obtained from PBS and the Trypsin@PSA + GA-Lip group were captured to illustrate the tumor vasculature.