Preparation of immunostimulatory nanoparticles

ISN and empty PLGA NPs (PLGA NPs) were prepared through a modified solvent emulsification-evaporation method based on a previously reported w/o/w double emulsion technique [16, 17]. The first w/o emulsion was created with 20 mg of PLGA 50:50 (Resomer® RG 504 from Sigma-Aldrich, Germany) dissolved in 1 mL of ethyl acetate (EA from Merck). The polymeric solution was homogenized with 100 µL of 2.5 µg/mL human IL-12 (Sigma-Aldrich, Germany) using a Vibra-Cell ultrasonic processor with 14% amplitude for 30s. Empty PLGA NPs were obtained with the same method, but 100 µL of water were added instead of human IL-12. The primary emulsion was added to 4 mL of 2% (w/w) Poly(vinyl alcohol) (PVA, Mowiol® 4–88 31,000 Da from Sigma-Aldrich, Germany) and homogenized using the same conditions. Lastly, the second emulsion w/o/w was poured into 7.5 mL of 2% (w/w) PVA and left in the fume hood under magnetic stirring at 300rpm for 3h for EA evaporation. The purification of ISN and PLGA NPs was done immediately after formulation with three cycles of centrifugation. The centrifugation was performed at 20,000g for 30 min. After the third centrifugation, both formulations were redispersed in water and stored at 20mg/mL at 4 ºC.

Preparation of cationic polymeric nanoparticles

Empty and loaded cationic polymeric nanoparticles (CPN) were prepared according to the previously described method with the same modifications. The organic phase was composed of a mixture of PLGA and DOTAP (Avanti® Polar Lipids). Three different concentrations between 0 and 15% (w/v) of DOTAP were added to the organic phase of the double emulsion. Twenty milligrams of PLGA/DOTAP were dissolved into 1 mL of chloroform due to low DOTAP solubility in EA. The remaining formulation protocol was kept the same and followed the previous protocol for ISN. The obtained CPN were washed three times with ultrapure water by centrifugation at 20,000g for 30 min.

Characterization of nanoparticle formulations

ISN, PLGA NPs, empty CPN, and loaded CPN in suspension were characterized for their average particle size, polydispersity index (PDI), and zeta potential by dynamic light scattering (DLS) using an Anton Paar Litesizer 500 particle analyzer (Anton Paar, Graz, Austria). For the measurement, a set of three replicates with ten measurements were acquired at 90°, 22 C, and 532 nm. The samples were diluted with ultrapure water, NaCl 10 mM, phosphate-buffered solution (PBS) and Simulated Interstitial Fluid (SIF, Biochemazone, Canada) to an appropriate concentration.

The particle morphology was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using Tescan Mira 3 (Tescan, Brno – Kohoutovice, Czech Republic) and FEI Technai Spirit (FEI, Hillsboro, OR, USA) microscopes, respectively. All formulations were purified by centrifugation before SEM and TEM observation to eliminate residual surfactant (PVA). For SEM analysis, a small sample was placed on top of a glass slide mounted on the SEM stab with carbon tape. Before the observation, the SEM stab with the sample was vacuum-coated with a 2 mm layer of gold.

For TEM observations, a drop of 5uL of the sample was deposited onto the grid (200 mesh formvar carbon grid). The excess liquid was blotted after 1 min with filter paper. Immediately after, a 5 ul drop of the staining agent UranyLess EM Stain (EMS, Hatfield, PA 19440) was applied. The excess liquid was blotted after 1 min with filter paper. The grid was allowed to dry under room conditions 2 h before observations with the microscope.

IL-12 encapsulation efficacy

The Encapsulation Efficacy (EE) of IL-12 was calculated by the indirect method after the first washing step of ISN and CPN. The free amount of IL-12 in the supernatant was quantified through a human IL-12 ELISA kit (R&D systems, UK). The supernatant of PLGA NPs and empty CPN were used as a control for the IL-12 quantification.

Cell lines and culture media

Human glioblastoma (U87 MG) and murine macrophage cell line (J774A.1) were purchased from the American Type Cell Culture Collection (ATCC, USA). Human GBM cell line was cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Murine macrophage cell line was cultured and maintained in Roswell Park Memorial Institute 1640 (RPMI-1640, Gibco, Switzerland) supplemented with 10% GBS, 1% penicillin/streptomycin, and 1% glutamine. Both cell lines were kept at 37 ºC, 5% CO2, and 95% relative humidity and passaged twice per week between 80–90% confluency.

In vitro cytotoxic assays

3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in vitro toxicology assay kit (Sigma-Aldrich, Germany) was used to measure the mitochondrial activity after exposure to all nanoformulations for U-87 MG and J774A.1 cells. For both cell lines, cytotoxicity caused by free IL-12, ISN, PLGA NPs, empty CPN, and loaded CPN was evaluated by collecting culture supernatant and determining L-lactate dehydrogenase (LDH) activity using an LDH kit (Roche, Germany). U-87 MG cells were seeded in 96-well plates at 2.5 × 105 cells/mL, while J774A.1 cells were seeded at 1.5 × 105 cells/mL for 24h in the respective cell culture media. After 24h of seeding, both cell lines were exposed to different nanoformulations at concentrations of 20 ng/mL of IL-12 for ISN and PLGA NPs and 5 ng/mL for empty CPN and loaded CPN. 0.2 vol. % Triton X-100 was added to the cell culture medium as a positive control [18]. In clinical trials, patients have received escalating doses of recombinant IL-12 from 30–700 ng/kg, while in vitro studies have shown promising results using IL-12 concentrations between 0.2 and 100 ng/mL[19,20,21]. In our pursuit to mitigate the off-target toxicity associated with IL-12, we selected concentrations of 5ng/mL and 20 ng/mL, falling within the therapeutic range. After 24h of incubation, the supernatant was used for LDH assay while cells were washed before being exposed to 0.5 mg/mL MTT solution. The MTT reaction was ended by the removal of the media and the addition of dimethyl sulfoxide. The final product’s absorbance was measured using a spectrophotometer (Benchmark Microplate reader, Biorad, Cressier, Switzerland) and following each kit’s protocol. All tests were performed with three biological replicates and 8 individual values.

Analysis of cytokine expression

The secretion of immunostimulatory cytokines was studied in human glioblastoma cells and murine macrophages. Both cell lines were seeded in a six-well plate at a density of 2 × 105 cells/mL. After 24h of seeding, cells were treated with 20 ng/mL of free IL-12, ISN, and PLGA NPs. PLGA NPs were incubated with the same dilution as ISN. LPS was used as a positive control at 10 ng/mL. After 24h of exposure, the concentration of released cytokines from both cell lines in the supernatants was measured by ELISA assays (R&D systems, UK) according to the manufacturer’s protocol. For the U-87 MG cell line, the concentration of extracellular IL-6, IL-8, VEGF, and IFN-γ was measured using human IL-6, human IL-8, VEGF, and IFN-γ DuoSet ELISA Kit. For the J774A.1 cell line, mouse IL-6, and TNF DuoSet ELISA kit were used to quantify the extracellular concentration of those cytokines in the supernatant. Absorbance was determined using a spectrophotometer (Benchmark Microplate reader, Biorad, Cressier, Switzerland) at a wavelength of 450nm with the correction wavelength set at 570nm.

Quantitative real-time PCR

U-87 MG cells were seeded in a six-well plate at a density of 1 × 106 cells per well. After 24h of seeding, cells were treated with 20 ng/mL of free IL-12, ISN, and PLGA NPs. LPS was used as a positive control at 10 ng/mL. After 24h of exposure, total RNAs were isolated from the cells. Cell lysis was performed directly in the well, adding 100µL of BL + 1-thioglycerol (TG) buffer (Promega). The total RNA was extracted using ReliaPrep™ RNA Cell Miniprep System (Promega, Madison, WI, USA). Thermo Scientific™ NanoDrop™ 2000 Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the RNA quality and calculate RNA concentration for cDNA. The reverse transcriptase reaction was performed using an Omniscipt RT system kit (Qiagen, Germany) according to the manufacturer’s protocol. Real-time PCR was performed using a 7500-fast real-time PCR system (Applied Biosystems). The threshold cycles (Ct) were calculated, and relative expression levels for each gene of interest were calculated using the Pfall method after normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as housekeeping genes [22]. Primers were purchased from Thermo Fisher Scientific (Zug, Switzerland). Details about the primers are included in Supplementary Information Table S1.

Quantitative immunophenotyping analysis by flow cytometry

U-87 MG cells were seeded in a six-well plate at a density of 1 × 106 cells per well and allowed to attach for 24h. The next day, cells were exposed to 20 ng/mL of free IL-12, ISN, and PLGA NPs. After 24 h and 48 h of treatment, cells were stimulated for 4 h with 2ug/mL of eBioscience™ Cell Stimulation Cocktail (ThermoFisher Scientific Inc., Zug, Switzerland) for intracellular p rotein staining. Cells were then trypsinized, washed, and fixed with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher Scientific Inc., Zug, Switzerland). LIVE/DEAD™ Fixable Violet Dead Cell Stain was used to stain the dead cells (data not shown) and IL-12/IL-23 p40 Monoclonal Antibody, Phycoerythrin (PE) eBioscience™ (Invitrogen, Invitrogen, Thermo Fisher Scientific Inc., Zug, Switzerland) was used for the intracellular staining of IL-12. Data acquisition was performed on a BD LSR FORTESSA (BD Biosciences, San Jose, CA, USA), and flow cytometry data were analyzed using the FlowJo software (Figure S1 shows the used gating strategy).

Statistical analysis

GraphPad Prism Software vs. 9.0 (GraphPad Software Inc.) was used to execute all statistical analyses. Differences between more than two related groups were analyzed using one-way analysis of variance (ANOVA). The one-way analysis of variance (ANOVA) was followed by a post-hoc Tukey test for multiple comparisons. Results are expressed as a mean ± standard deviation from a minimum of three independent experiments. Differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.