Lyobeta 15

The binding of each protein to rGO surface was studied by Raman and Fourier transform infrared spectroscopy. First, 250 µg/mL rGO was suspended either in 50 µg/mL BSA, 200 µg/mL collagen, or 50 µg/mL elastin solution, and agitated at 37 °C for 2 h. The supernatants were collected after spinning down at rpm for 15 min and lyophilized in a Telstar Lyobeta 15 lyophilizer. rGO without protein incubation and proteins without rGO were also studied. At least three samples were studied for each condition.
Raman spectrum was obtained by confocal Raman imaging (Alpha 300 M, Company WITec, Ulm, Germany) with a 532 nm laser (5% laser power, a contact time of the 50 s, and four accumulations). FT-IR, using an attenuated total reflectance (ATR) technique, was performed in a FT-IR Bruker IFS 66/S Spectrometer, with 32 scans at a resolution of 4 cm⁻¹ between the wavelength ranges of 4000–400 cm⁻¹. Air background was applied as a blank. At least three samples were analyzed for each condition.

Chitosan particles were obtained by inducing the gelation of a chitosan solution with TPP, following the method described by Da Silva et al. [16 (link)], with adaptations. Once the LCH or the MCH (1% w/v) were dissolved in the acetic acid solution (1% v/v), marjoram extract was added at different final concentrations (3, 2, 1, 0.5, and 0.25 mg/mL) and the pH value was adjusted to 5.8 with 1 M sodium hydroxide. Then, TPP was dissolved in milli-Q water at 0.1% (w/v) and, in order to study the best ratio for chitosan:TPP, different volumes of the TPP solution were added to the chitosan solution under magnetic stirring at room temperature for 20 min. The ratios of chitosan:TPP assayed were 5:1, 6:1, and 7:1. After that, the suspension was centrifugated (40,000× g, 45 min, T = 25 °C) and the supernatant was discarded to obtain the particles. The particles were frozen, then freeze-dried (LyoBeta 15, Telstar, Spain) and stored at 4 °C until analyses were performed.

For the determination of interactions between the components of the GM-SLN vector, Fourier Transform Infrared Spectroscopy (FT-IR) analysis was performed. SLNs and the GM-SLN vector were lyophilized at −50 °C and 0.2 mbar for 42 h (LyoBeta 15, Telstar, Spain) and the powders were mixed homogeneously with potassium bromide. The blends were compressed using a hydraulic compressor applying a pressure of 10 tons for 5 min. The discs obtained were placed in an FT-IR spectrometer (Thermo Fisher Scientific, Madrid, Spain) and the IR spectrums were recorded in the mid-IR region (4000–400 cm⁻¹). The recorded signals were reported as transmittance percentages.

PLGA (Resomer-RG503; lactide:glycolide ratio of 50:50; MW 40,600; viscosity 0.41 dL/g; Evonik, Germany), PEI (branched form, molecular weight of 25,000 Da), and polyvinyl alcohol (PVA) were purchased from Sigma Aldrich, Darmstadt, Germany.
PEI-coated PLGA NPs were prepared using the solvent extraction–evaporation double emulsion (w/o/w) method, with encapsulation of melanoma patients’ antigens. Briefly, 50 mg of PLGA and 0.65 mg of PEI were dissolved in 1 mL of dichloromethane (DCM) and emulsified with a 1.25% (w/v) peptide solution in acetonitrile (ACN):water (50:50) by sonication (30 s using Branson sonifier 250). This emulsion was then mixed with 5 mL of 5% (w/v) PVA and sonicated for 1 min. The final emulsion was poured into a 2% (v/v) isopropanol solution (20 mL) and stirred for 2 h to allow solvent evaporation. Finally, the resulting nanoparticles were freeze-dried for 42 h with trehalose used as a cryoprotectant (Lyobeta 15, Telstar, Barcelona, Spain).

Selected high-yielding rosemary plants from a germplasm bank were cultivated and harvested in the experimental farm of the Institute of Agricultural and Food Research and Development (IMIDA, Murcia, Spain). RLE was obtained from the leaf by-product that had been previously distilled with water steam and extracted with water to obtain the essential oil and the RWE, respectively [27 (link)]. Then, 3 g of rosemary by-product were mixed with 450 mL acetone:water (1:1, v/v) and kept under constant stirring (30 °C for 90 min). The mix was centrifuged in a Digecen 21 R centrifuge (Orto Alresa, Madrid, Spain) (4560× g and 5 °C for 10 min) and the supernatant was then filtered (Whatman No. 4). The acetone fraction was removed by applying a vacuum at 40 °C in a Syncore Polyvap R-96 evaporation system (Buchi, Flawil, Switzerland), and the water fraction was removed using a lyophilisation system (Lyobeta 15, Telstar, Terrassa, Spain) (100 mbar and −80 °C for 24 h). Lyophilized RLE, a yellow-greenish dry powder with an intense herbal flavour, was stored at −80 °C until further use.