Synthesis of PNA*.
The PNA oligomer (Table 1) was synthesized using the Fmoc solid-phase strategy according to the procedure used in our previous studies5,6,8,13. 50 mg of 4-methylbenzhydrylamine (MBHA) resin (0.5 mmol/g) was swelled in CH2Cl2 for 30 min and washed with dimethylformamide (DMF) (×5). Then, the resin was treated with a solution of 20% piperidine solution in DMF for 10 min. After washings in DMF (×5), the resin was treated with 5 equiv. Fmoc-Gly-OH dissolved in N-methyl-2-pyrrolidone (NMP) 0.2 M, 5 equiv. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) dissolved in DMF 0.2 M and 5 equiv. N,N-diisopropylethylamine (DIPEA)/7.5 equiv. lutidine for 40 min at RT. The coupling of two Fmoc-L-Ser[PO(OBzl)OH]-OH was achieved using the following conditions: 8 equiv. Fmoc-L-Ser[PO(OBzl)OH]-OH monomer dissolved in NMP 0.4 M, 8 equiv. HATU dissolved in DMF (0.4 M), and 8 equiv. DIPEA/12 equiv. lutidine for 15 h at RT. After the serine couplings, a glycine residue was attached on the N-terminal of the serine tract following the previously described coupling with the glycine monomer. PNA monomers were coupled using the following conditions: 5 equiv. monomer building block in NMP (0.2 M), 5 equiv. HATU dissolved in DMF (0.2 M) and 5 equiv. DIPEA/7.5 equiv. lutidine, 40 min at RT. After each coupling step, capping with Ac2O in the presence of pyridine was performed for 20 min at RT. Fmoc groups were removed by two treatments with a 5% solution of 1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU) in DMF solution (5 min). After the assembly of the PNA tract, two 2-[2-(Fmoc-amino)ethoxy]ethoxyacetic acid linkers (Fmoc-AEEA-OH; Sigma) were coupled to the terminal amino group of PNA using the following conditions: 8 equiv. Fmoc-AEEA-OH dissolved in NMP (0.4 M), 8 equiv. HATU dissolved in DMF (0.4 M), and 8 equiv. DIPEA/12 equiv. lutidine. After the removal of the Fmoc group, 5 equiv. of fluorescein isothiocyanate (FITC) 0.2 M were dissolved in DMF/DIPEA (2.5:97.5 v/v), and the solution was added to the resin, which was gently shaken in the dark for 15 h. At the end of synthetic cycles, PNA* was cleaved from the solid support by treatment with trifluoroacetic acid (TFA)/anisole/ethanedithiol (9:1:1; v/v/v) for 4 h, and the product was precipitated with cold diethyl ether. Once recovered by centrifugation, the precipitate was washed twice with diethyl ether, dissolved in water, and finally lyophilized.
PNA* was obtained with a 48–50% overall yield (94–95% average yield for each coupling step as estimated by Fmoc spectrophotometric measurements after detachment of Fmoc groups from the resin). The crude sample was purified by semipreparative HPLC analyses, and purifications were carried out on a Jasco (Easton, MD, USA) PU-2089 Plus pump equipped with a Jasco UV-2075 Plus UV detector using a 10 × 250 mm C-18 reverse-phase (RP) column (particle size 5 µm) eluted with a linear gradient of CH3CN containing 0.1% (v/v) TFA in H2O (from 0 to 100% in 45 min, flow 1.2 mL/min). The collected fractions were lyophilized, and the final pure product was characterized by ESI-MS (positive mode): ESI-MS (m/z) calcd. for PNA* [M + 2H]+2 1495.49, found 1495.4; calcd. for [M + 3H]3+ 997.33, found 997.3; calcd. for [M + 4H]4+ 748.24, found 748.2 (Fig. S1). The amount of PNA* sample dissolved in pure water was estimated by spectrofluorimetric analysis at λex 476 nm/λem 500-560 nm (GloMax Explorer, Promega, Italy). The response's linearity was evaluated at the concentration range of 0.01−5 nmol/mL (r2 ≥ 0.999).
Production and characterization of PNA*-hNPs.
PNA*-hNPs were prepared by an emulsion/solvent diffusion technique as previously reported27. Briefly, 100 µL of a water solution of PNA* (50 nmol/mL) were added to 1 mL of methylene chloride containing PLGA 502H (10 mg; 1% w/v)29 under vortex mixing (2400 rpm, Heidolph, Germany) and DPPC (0.5 mg/mL), achieving a water-in-oil emulsion (w/o). Just after mixing, the w/o emulsion was added to 12.5 mL of ethanol 96% (v/v) under moderate magnetic stirring, leading to the immediate formation of hNPs. The obtained formulation was diluted with 12.5 mL of Milli-Q water and maintained under stirring for 10 min. Afterward, the residual organic solvent was removed by rotary evaporation under vacuum at 30 °C (Heidolph VV 2000, Germany) to a hNP dispersion final volume of 5 mL. The obtained hNPs were isolated by centrifugation at 7000 rcf for 20 minutes at 4 °C (Hettich Zentrifugen, Germany) and dispersed in water to the desired concentration.
To evaluate PNA release kinetics and follow the intracellular distribution of PNA and hNPs, we labeled with a fluorescent tag both the PNA and the hNPs. The PNA was labeled using the FITC fluorophore as described above, while the hNPs were labeled with Rhodamine B by adding Rhod-labelled PLGA (PLGA-Rhod) (PolySciTech, U.S.A.) in the organic phase at 10% w/w with respect to the total PLGA amount.
Determination of size and zeta potential of hNPs.
The hydrodynamic diameter and the polydispersity index of hNPs were determined by dynamic light scattering (DLS), while the z potential was measured by electrophoretic light scattering (ELS) (Zetasizer Nano ZS, Malvern Instruments Ltd, UK). Results are expressed as mean value ± standard deviation (SD) of triplicate measurements on different batches.
PNA actual loading.
To assess the actual loading of PNA* in the hNPs, we used both indirect and direct methods, whose results were compared for uniformity. In the indirect method, we detected the amount of non-encapsulated PNA*. Immediately after their production, the PNA*-hNPs were collected by centrifugation (7000 rcf for 20 minutes), and the supernatant was analyzed for PNA* content by spectrofluorimetric analysis, as described above. For the direct method, just after production, PNA*-hNPs were collected by centrifugation (7000 rcf for 20 minutes) and resuspended in water at the concentration of 1 mg hNPs/100 µL. 100 µL of the hNPs dispersion were then diluted with 900 µL of NaOH 0.5 N and incubated at RT for 2 h under magnetic stirring to achieve the polymer's complete degradation. The obtained solution was analyzed in triplicate by fluorescence spectroscopy, as described above, to quantify the amount of encapsulated PNA*. The two methods gave consistent results, reported in Table 2 as actual loading (nmol of encapsulated PNA* per mg of hNPs) and encapsulation efficiency (actual loading/theoretical loading x 100).
PNA in vitro release kinetics from hNPs.
The in vitro release kinetics of PNA* from PNA*-hNPs was first evaluated in phosphate buffer (120 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4) at pH 7.2 (PBS). Release studies were performed upon dilution of PNA*-hNP dispersions in PBS to a final hNP concentration of 5 mg/mL. The obtained hNPs dispersions were incubated in a horizontal-shaking water bath at 37 °C (ShakeTemp SW 22, Julabo, Italy). At scheduled time intervals, the samples were centrifuged at 7000 rcf for 20 min to isolate hNPs. The release medium (1 mL) was withdrawn and reconstituted with the same amount of fresh PBS. The withdrawn medium was analyzed for PNA* content by spectrofluorimetric analysis as described above. Experiments were carried out in triplicate, and the results are expressed as the percentage of released PNA* ± SD. To better resemble in vivo conditions, the release of PNA* from hNPs was followed in vitro also by membrane dialysis from either phosphate buffer at pH 7.2 (PBS; 120 mM NaCl, 2.7 mM KCl, 10 mM of phosphate salts) or artificial CF mucus (AM) to simulated interstitial lung fluid (SILF) according to the procedure previously described by De Stefano et al.30. Briefly, SILF was prepared by dissolving in water 0.095 g magnesium chloride, 6.019 g of sodium chloride, 0.298 g of potassium chloride, 0.126 g of sodium phosphate dibasic, 0.063 g of sodium sulfate, 0.368 g calcium chloride dihydrate, 0.574 g of sodium acetate, 2.604 g of sodium bicarbonate, 0.097 g of sodium citrate dihydrate. The AM was prepared by adding 250 μL of sterile egg yolk emulsion, 250 mg of mucin, 0.295 mg diethylenetriamine pentaacetic acid (DTPA), 250 mg NaCl, 110 mg KCl, 1 mL of RPMI to 50 mL of water, and the dispersion was stirred until a homogenous mixture was obtained. The water dispersion of PNA*-hNPs (0.1 mL), corresponding to an amount of PNA* of 1 nmol, was added to 0.3 mL of donor medium (AM) and placed in a dialysis membrane bag (MWCO: 5000 Da, Spectra/Por®). The dialysis membrane leads to the diffusion of the free PNA* and not the diffusion of the hNPs. The dialysis bag was placed into 3 mL of external medium and kept at 37 °C. At scheduled time intervals, 1 mL of external medium was withdrawn and analyzed for PNA* content by spectrofluorimetric analysis as described above. The medium was replaced by the same amount of fresh SILF. At the end of release kinetics, the amount of residual PNA* in the dialysis bag was assessed upon hNP degradation in NaOH 0.5 N, as described for PNA actual loading. The diffusion of free PNA* from AM to SILF and from PBS to SILF was evaluated in the same conditions as a control. Experiments were run in triplicate for each time point, and results are reported as the amount of PNA* diffused (%) in the time.
RP-HPLC analysis of PNA* in vitro release from hNPs.
The in vitro release assay of PNA* dissolved in AM to SILF was assessed by reverse-phase (RP) HPLC following the absorbance of PNA bases at 260 nm. At scheduled time point, 500 µL of the stock solution of PNA*-hNPs from AM to SILF was withdrawn to perform the HPLC analysis, monitoring the UV signal of the PNA bases at 260 nm. The chromatogram of free PNA* in water was also analyzed in the same experimental conditions. The analyses were performed with a linear gradient of CH3CN containing 0.1% (v/v) TFA in H2O from 0 to 100% in 45 min, flow 1.2 mL/min at RT. Calculation of peak areas determined in pixel2 was performed using ImageJ software v1.52a.
hNP transport assay.
H.N.E.C.s were treated with 1 mg/mL of empty Rhod-hNPs. The basal medium was recovered at different time points, 6, 24, and 72 h, and transferred to a clean 24-multiwell plate. The fluorescence intensity was measured by loading the 24-multiwell plate into a multi-plate reader (Enspire, PerkinElmer). The excitation and emission wavelengths were set at 540 nm and 630 nm, respectively.
Nasal epithelial cells brushing, culture, and hNPs treatments.
Human nasal epithelial cells (HNEC) were sampled by nasal brushing of both nostrils as reported by Di Lullo, A. M. et al23. Briefly, the nasal brushing was performed after nasal washings with a physiological saline solution to remove the mucus. Then, a soft sterile interdental brush (Paro Isola long, ProfiMed) with 2.5–3 mm bristles was used to scrap along the middle portion of the inferior turbinate, under direct vision, using a frontal light and without anesthesia. The brushes were transported to the cell culture lab in RPMI 1640 medium (supplemented with 1 mg/mL of primocin antibiotics – Invitrogen). The use of nasal epithelial cells from the human subjects was approved by the Ethical Committee of the University of Naples Federico II (protocol number 197/2015), in agreement with the Declaration of Helsinki. Written informed consent to collect and study the cells for scientific research purposes was obtained from the patients or, in the case of minors, from parents. Once in the lab, the cells were centrifuged at 350 g for 10 min, resuspended in PneumaCult–EX Medium (STEMCELL), and seeded in a TPP T10 TC Flask/Tube 0.22 μm filter cap (Techno Plastic Products, Switzerland). Cell culture medium was changed every day. Then, about 33000 cells were seeded on porous filters (0.33 cm2, Transwell, Corning) in PneumaCult–EX Medium until confluence. The PneumaCultTM–EX Medium was replaced by PneumaCult-ALI Maintenance Medium for air-liquid interface (ALI) cultures. Treatments with hNPs (0.5 mg/mL), loaded or not with PNA*, were performed after 21 days of ALI culture when the pseudostratified epithelium was perfectly formed. Transfection of PNA* was performed using Attractene reagent (Qiagen).
Fluorescent Microscopy images acquisition.
Cells were fixed in 10% neutral buffered formalin for 15 minutes, then rinsed in PBS and permeabilized with 0.1% triton x-100 in PBS for 10 minutes. After rinsing, the cells were blocked in 1% BSA solution for 30 min. then rinsed in PBS and then stained with phalloidin and Hoechst. The fluorescence images were acquired using a Leica SP5 confocal microscope, while the images of Fig. 3 were acquired using a THUNDER imager 3D cell cultures system (Leica).