Esc peptide-loaded NPs at a theoretical loading of 2% (2 mg of peptide per 100 mg of NPs) were prepared by emulsion/solvent diffusion as previously reported with some modifications.24,38 Briefly, an aqueous solution of the selected Esc peptide (100 µL) was added to a solution of PLGA in methylene chloride (1 mL, 1% w/v) under vortex mixing at a speed of 2,400 rpm (Reax top, Heidolph, Germany). The resulting water-in-oil emulsion was poured in 12.5 mL of ethanol (diffusion phase) to induce polymer precipitation in the form of NPs.
The NP dispersion was diluted with aqueous PVA (12.5 mL, 0.1% w/v), kept under magnetic stirring for 10 min at room temperature and rotary evaporated (Rotavapor®, Heidolph VV 2000, Germany) under vacuum at 30 єC to remove any residual organic solvent. After adjustment to a final volume of 5 mL, the colloidal dispersion was centrifuged at 7,000 rcf for 20 min at 4 °C (Hettich Zentrifugen, Universal 16R) to isolate NPs and the pellet dispersed in ultrapure water. Fluorescently-labelled NPs were employed for in vitro aerosolization studies and to follow NPs diffusion in artificial mucus/bacterial extracellular matrix.
Briefly, NPs containing either Esc(1-21) (i.e., Esc_ Fluo-NPs) or Esc(1-21)-1c (Esc1c_Fluo-NPs) were prepared using PLGA-Rhod in the organic phase at 10% w/w with respect to the total PLGA amount. Control NPs containing Esc(1-21) labelled with rhodamine B (Rho-Esc) were also prepared for peptide diffusion studies in simulated mucus and biofilm (Rho-Esc_NPs). When needed, Esc peptide-loaded NPs were freeze-dried adding trehalose (THR) as cryoprotectant. Just after production, THR was added to the NPs dispersion in ultrapure water (NP/THR1:25 w/w), frozen at -80 °C and freeze-dried for 24 h by a Modulyo freeze-drier (Edwards, UK) operating at 0.01 atm and -60 °C. Nanoparticle characterization. The hydrodynamic diameter (DH), the polydispersity index (PDI) and the zeta potential (¶ potential) of Esc peptide-loaded NPs were determined by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) with a Zetasizer Nano ZS (Malvern Instruments Ltd, UK). For є potential analysis, NP aqueous dispersion was diluted in 10 mM NaCl and analyzed in an electrophoresis cell at a fixed potential of ±150 mV. Results are reported as the mean ± standard deviation (SD) of three measurements performed on three different batches (n=9). The morphology of Esc peptide-loaded NPs was analyzed by transmission electron microscopy (TEM) with a FEI Tecnai G2 200 kV s-Twin microscope equipped with a 4K camera (ThermoFisher Scientific, US). Sample analysis was performed upon air drying of 10 јL nanoparticle dispersions in water (3 mg/mL) mounted on 200 mesh copper grids coated with carbon film (Ted Pella Inc., Nanovision, Italy).Fixed aqueous layer thickness (FALT) measurements were performed by monitoring the influence of ionic strength on particle surface charge. Briefly, different amounts of a stock NaCl solution were added to a NP aqueous dispersion (0.5 mg/mL) and the є potential of the samples was determined. A plot of ln (є) against k (k=3.3 C0.5, where k-1 is the Debye length) gives the thickness of the polymer layer in nm as the slope of a linear regression.39 The actual loading of Esc peptides inside NPs was determined by either an indirect or a direct method as previously reported.35 Just after production, an indirect measurement of the Esc peptide amount encapsulated inside NPs was achieved by RP-HPLC analysis of the supernatant of the NP dispersions after centrifugation. For direct measurement, an aqueous dispersion of Esc peptide-loaded NPs (1 mL, 4 mg/mL) was centrifuged at 7,000 rcf for 20 min at 4 °C (Hettich Zentrifugen, Universal 16R) and the NP pellet freeze-dried for 24 h at -80°C and 0.1 mbar (LyoQuest, Telstar, Italy). Esc peptide content within the NPs was measured by solvent extraction, upon dissolution of freeze-dried NPs in 500 µL of acetonitrile and subsequent dilution with 1 mL of 30 mM sodium sulfate, pH 3. Upon centrifugation (4 °C, 7,000 rcf, 20 min), the samples were analyzed by RP-HPLC as indicated above. Data were obtained from three different batches (n = 6) and reported as actual loading (mg of encapsulated Esc peptide per 100 mg of NPs) and encapsulation efficiency (actual loading/theoretical loading x 100) ± SD.In vitro release kinetics of Esc peptides. These experiments were performed in phosphate buffer at pH 7.2 (120 mM NaCl, 2.7 mM KCl, 10 mM phosphate salts, PBS). Upon appropriate dilution in PBS (4 mg/mL), Esc peptide-loaded NPs were incubated in a horizontal-shaking water bath (ShakeTemp SW 22, Julabo Italia, Italy) operating at 40 rpm and 37 °C. At different time intervals, samples were centrifuged at 7,000 rcf for 20 min at 4 єC to remove the release medium and to isolate NPs. The NPs pellet was freeze-dried for 24 h. Esc peptide content within the NPs was measured by solvent extraction, upon NPs dissolution in 500 µL of acetonitrile and subsequent dilution with 1 mL of 30 mM sodium sulfate, pH 3. Samples were centrifuged (4 °C, 7000 rcf, 20 min) and then analyzed by RP-HPLC as reported above. The amount of Esc peptide released at each time point was calculated as the difference between the total amount encapsulated and the amount detected inside the NPs. Triplicate experiments were performed for each time point of release kinetics and the results are reported as percentage of Esc peptide released from NPs (%) ± SD along the time. In vitro interactions of nanoparticles with mucin and alginates. The interactions of Esc peptide-loaded NPs with the main components of mucus and P. aeruginosa extracellular matrix, that is mucin and alginates respectively, were assessed by turbidimetric measurements. For NP/mucin interactions, experiments were performed by the mucin/particle method.22 Briefly, a saturated aqueous solution of mucin was achieved through dispersion of an excess of the protein in water (0.08% w/v), under stirring overnight, followed by centrifugation and collection of the mucin-containing supernatant. Then, 10 µL of a water dispersion of Esc peptide-loaded NPs (corresponding to 1 mg of NPs) were poured into 1 mL of the mucin dispersion. After vortex mixing (Reax top, Heidolph, Germany), the turbidity of the samples was measured at time 0 and after incubation at 37 °C for different time intervals (30-60 min). In case of NPs/alginate interactions, NPs scattering at 650 nm was assessed in the presence of a non-acetylated polymannuronic acid dispersion in water (1% w/v). The absorbance measurements at 650 nm were carried out by a spectrophotometer (Shimadzu 1204, Shimadzu, Italy) equipped with a 0.1 cm quartz cell (Hellma® Italia, Italy). As controls, the saturated mucin solution (without NPs), the polymannuronic acid dispersion (without NPs) and the Esc peptide-loaded NP dispersions in water were analysed. Triplicate experiments were performed and the results are reported as absorbance at 650 nm ± SD along the time. The size and the ¶ potential of the NP dispersions in mucin were also investigated by DLS and ELS, as described above.