Materials

Ethanol (absolute degree) and hydrochloric acid (HCl) are common reagents supply. MES buffer was purchased from ACROS Organics (New Jersey, NY, USA) and poloxamer 407 (Kolliphor® P407) from BASF (Geismar, LA, USA). An FcRn-targeted peptide with the following amino acid sequence, CQRFVTGHFGGLYPANG, was requested from GenScript Biotech (Leiden, Netherlands). Zein, insulin, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS), O-methyl-O′-succinylpolyethylene glycol 5′000 (mPEG-COOH), gelatin from porcine skin, endothelial cell growth supplement (ECGS), dexamethasone (Dex), M-199 medium, and sulfuric acid (H2SO4) were all purchased from Sigma-Aldrich (St. Louis, USA). Paraformaldehyde (PFA) was purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Heparin sodium salt from porcine intestinal mucosa was purchased from Alfa-Aesar (Haverhill, USA). Matrigel® basement membrane matrix was purchased from Corning (New York, USA). Insulin-transferrin-selenium (ITS), Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, and Hanks’ Balanced Salt solution (HBSS) were purchased from Gibco (Waltham, USA). Fetal bovine serum (FBS) was purchased from Biochrom AG (Berlin, Germany). Human FcRn antibody (MAB8639) was obtained from Bio-Techne (Minneapolis, USA), goat anti-mouse Alexa 594 (A-11020) from Invitrogen (California, USA), and anti-insulin + proinsulin mouse monoclonal capture antibody (ab8304) and HRP anti-insulin + proinsulin mouse monoclonal antibody (ab28063) from Abcam (Cambridge, UK).

Nanoparticles preparation methodPreparation of zein nanoparticles

A desolvation procedure previously described [18] was applied to prepare ZNPs with minor modifications (Fig. 1(1)). Briefly, 5 mL of a hydroalcoholic solution (61% (v/v) ethanol in water) was used to dissolve 50 mg of zein under continuous magnetic stirring, while 5 mg of insulin were dissolved in 0.5 mL of freshly prepared 10 mM HCl aqueous solution. Both solutions were mixed under continuous magnetic stirring for 30 min, and then the desolvation step of zein was induced by the addition of an equivalent volume of 5 mL of acidified Milli-Q® ultrapure water at pH 5.3, under continuous magnetic stirring at 400 rpm. The evaporation of ethanol occurred under the fume hood for 4 h. Afterward, the suspension of ZNPs was collected and centrifuged at 2000 g for 30 min at room temperature (RT) through a filter device with a molecular weight cutoff of 100 kDa (Amicon Ultra filter, Ultracel membrane with 100,000 MWCO, Millipore Corporation, Bedford, USA). Empty ZNPs were prepared using the same protocol, but without insulin.

Fig. 1

Methodology of preparation of (1) insulin-loaded ZNPs (i-ZNPs) using desolvation method; (2) FcRn-targeted i-ZNPs (F-i-ZNPs) using carbodiimide chemistry; (3) covalent PEGylated F-i-ZNPs (mPEG-F-i-ZNPs) using carbodiimide chemistry; and (4) physically adsorbed PEO-PPO-PEO (P 407) polymer F-i-ZNPs (P407-F-i-ZNPs). The figure was made using Adobe Illustrator and ChemDraw

Selection of FcRn-targeted ligand

The FcRn-targeted ligand selected to functionalize the ZNPs was based on the SYN746 peptide framework (QRFCTGHFGGLYPCNGP), developed in Mezo et al.’s work [23]. This cyclic peptide was further converted to a linear peptide (QRFVTGHFGGLYPANG) by replacing both cysteine residues with valine and alanine, at 4 and 14 positions, respectively, as previously described by Sockolosky et al. and Datta-Mannan et al. [24, 25]. A cysteine residue was added to the N-terminal of the linear FcRn-targeted peptide framework (CQRFVTGHFGGLYPANG) to facilitate its conjugation to the candidate nanocarrier by different possible chemistry reactions, namely maleimide-thiol and EDC/NHS chemistries.

Conjugation of FcRn-targeted ligand to zein nanoparticles

ZNPs were conjugated to the FcRn-targeted peptide mentioned in the previous section, the “Selection of FcRn-targeted ligand” section, using carbodiimide chemistry, in which the carboxyl (–COOH) groups of ZNPs were covalently linked to the primary amines (–NH2) groups of the peptide (Fig. 1(2)). Firstly, 1 mL of ZNPs (4 mg/mL) was diluted in acidic buffer (MES buffer, pH 5.3) and then reacted with EDC/NHS for 1 h to form ZNPs with amide bonds. The formed by-products were removed by filtration with Amicon tubes (100 kDa) at 2000 g for 15 min at RT. The formed ZNPs with amide bonds were added to the FcRn-targeted peptide (CQRFVTGHFGGLYPANG), previously dissolved in basic buffer (PBS, pH 8.5), under continuous stirring at RT for 1 h. The unconjugated FcRn-targeted peptide was removed with Amicon tubes (100 kDa) at 2000 g for 15 min at RT, and the functionalized ZNPs (F-ZNPs) were collected to a final volume of 1 mL and 4 mg/mL concentration.

Preparation of PEGylated functionalized zein nanoparticles

The production of PEGylated F-i-ZNPs was conducted using two methodologies: covalent binding of ZNPs with mPEG and physical mixture of ZNPs with poloxamer 407 (PEO-PPO-PEO (P 407)). Regarding the first methodology, mPEG-COOH:EDC:NHS in the molar ratio of 1:10:25 were dissolved in MES buffer (pH 5.35) to activate the -COOH group on mPEG-COOH by stirring for 3 h at RT (Fig. 1(3)). Then, the mixture was added dropwise to F-i-ZNPs for 1 h at RT to obtain covalently PEGylated targeted ZNPs (mPEG-F-i-ZNPs), whereas the physical adsorbance of P407 on F-ZNPs (P407-F-i-ZNPs) was obtained by adding dropwise 0.1 mL of F-i-ZNPs (20 mg/mL) into 0.9 mL of 0.1% (w/v) P407 and incubating overnight at 4 °C (Fig. 1(4)). In both formulations, samples were further filtered with Amicon tubes (100 kDa) at 2000 rpm for 15 min at RT to remove the free polymers. The formulations were stored at 4 °C until further use.

Freeze-drying of zein nanoparticles

The ZNPs of concentration 1 mg/mL and final volume 2 mL were poured into semi-stoppered glass vials with slotted rubber closures. ZNPs were initially frozen at −80 °C for 24 h followed by lyophilization using a Modulyo 4 K freeze-dryer (Edwards, Crawley, UK) at 0.09 mbar for 72 h. The temperature of the condenser surface was maintained at −60 °C ± 5 °C. After lyophilization, ZNPs were stored in a 4 °C chamber until future use.

Physical–chemical characterization of zein nanoparticlesHydrodynamic diameter, polydispersity index, and zeta potential

The freshly prepared ZNPs formulations were characterized regarding their hydrodynamic diameter and polydispersity index (PdI) through dynamic light scattering (DLS) and zeta potential (ZP) by laser Doppler electrophoresis (LDE), using the Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For that, samples were previously diluted in a ratio of 1:100 in 10 mM NaCl adjusted to pH 5 with HCl 0.5 M. All measurements were performed in triplicate at 25 °C. Data was collected and analyzed using Zetasizer software (version 7.12).

Stability of unloaded zein nanoparticles

The stability of the liquid suspensions of unloaded ZNPs stored in dark at 4 °C was evaluated until day 51 of production. The ZNPs were dispersed in 10 mM NaCl adjusted to pH 5 with 0.5 M HCl and characterized in terms of hydrodynamic diameter, PdI, and ZP, using Zetasizer equipment.

Morphological evaluation

The morphology of ZNPs, mPEG-F-ZNPs, and P407-F-ZNPs was confirmed by transmission electron microscopy (TEM). TEM images were obtained by placing 10 µL of 0.033 mg/mL NPs suspension of concentration on Formvar/carbon film-coated mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA) and left standing for 2 min. Then, the excess liquid was removed with filter paper. The visualization was carried out on a JEOL JEM 1400 TEM at 120 kV (Tokyo, Japan). Images were digitally recorded using a CCD digital camera Orious 1100 W (Tokyo, Japan), at the HEMS/i3S of the University of Porto.

The surface morphology of freeze-dried unloaded ZNPs was further studied by high-resolution scanning electron microscopy (SEM) with X-Ray Microanalysis and CryoSEM experimental facilities: JEOL JSM 6301F/Oxford INCA Energy 350/Gatan Alto 2500. ZNPs were mounted onto metal stubs with carbon tape and sputter-coated with a thin layer of gold/palladium using the SPI Module Sputter Coater equipment (Structure Probe, Inc., West Chester, PA, USA).

Attenuated total reflectance–Fourier transform infrared spectroscopy

In order to confirm the covalent bonds between FcRn-targeted peptide and ZNPs as well as mPEG-COOH and F-ZNPs, the Fourier transform infrared spectroscopy (FTIR) spectra of the raw materials and the NPs were obtained using a Fourier transform spectrophotometer IR Affinity-1S (Shimadzu, Kyoto, Japan) coupled to a Specac Golden Gate Attenuated Total Reflectance (ATR). For that purpose, NPs suspensions were placed over the diamond, and the reflectance spectra were obtained by scanning from 400 to 4000 cm−1 at 4 cm−1 of the resolution, and 32 scans per spectrum. The PerkinElmer Spectrum IR software was used to analyze the spectra.

Insulin association efficiency and drug loading

The association efficiency (AE) and drug loading (DL) of insulin in freshly prepared ZNPs were evaluated by an indirect method using ultracentrifugation. For that purpose, ZNPs were centrifuged at 30,000 g for 30 min at 4 °C, and the supernatants containing insulin were collected. The amount of loaded insulin into ZNPs was calculated by the difference between the initial amount of insulin used to produce the ZNPs and the remaining drug collected in the supernatant. An insulin calibration curve was prepared in a range of concentrations from 0.5 to 75 µg/mL, using the supernatant obtained from unloaded ZNPs as a solvent and as a blank, to remove possible interference from zein. Standards and samples were evaluated in triplicates by micro-BCA assay. The AE and DL were calculated by the following equations:

$$\mathrm\;\mathrm\;\mathrm\;\left(\%\right)=\frac\;\mathrm\;\mathrm\;\mathrm\!-\!\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\mathrm\;\mathrm\;\mathrm}\times100$$

(1)

$$\mathrm\;\mathrm\left(\%\right)=\frac\;\mathrm\;\mathrm\;\mathrm\!-\!\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm}\times100$$

(2)

FcRn-targeted peptide conjugation efficiency

The conjugation efficiency (CE) of the FcRn-targeted peptide was determined by indirect method. Briefly, after unloaded ZNPs were functionalized with the FcRn-targeted peptide by carbodiimide chemistry, as described in the “Conjugation of FcRn-targeted ligand to zein nanoparticles” section, samples were transferred to Amicon tubes (100 kDa), and the unconjugated peptide was removed by centrifuging at 2000 g for 15 min at RT. Filtrates were collected, and the mass of conjugated peptide on ZNPs was determined by the difference between the initial mass of peptide and the mass of peptide detected in the filtrate. The amount of peptide non-conjugated was quantified using micro-BCA assay, with relevant controls, unconjugated ZNPs. Similarly to the insulin AE experiment, an FcRn peptide calibration curve was prepared in a range of concentrations from 0.5 to 50 µg/mL, using the filtrates obtained from unconjugated ZNPs as a solvent and as a blank, to remove possible interference from zein. Six independent experiments were performed (n = 6). The CE was determined by the following formula:

$$\mathrm\left(\%\right)=\frac\;\mathrm\;\mathrm\;\mathrm-\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\mathrm\;\mathrm\;\mathrm}\times100$$

(3)

In vitro release study

The release profile of insulin in i-ZNPs, F-i-ZNPs, mPEG-F-i-ZNPs, and P407-F-i-ZNPs was determined by incubating 2 mL of NPs (corresponding to an insulin concentration of 53 µg/mL) in phosphate buffer (20 mM) at pH 7.4. Aliquots of 400 µL were withdrawn from each formulation at specific time points (0.25, 0.5, 1, 2, 5, 8, and 24 h), and the same volume was replaced with warmed PBS. Samples were centrifuged at 13,000 g for 10 min at RT, and the supernatants were collected and quantified by micro-BCA assay to evaluate the amount of insulin released from each formulation throughout the experiment [26]. An insulin calibration curve was prepared in PBS, and samples were evaluated in triplicates with relevant controls (unloaded ZNPs). The experiment was performed at 37 °C, 120 rpm, and in sink conditions [27].

Cell cultureCell culture maintenance

Human lung carcinoma epithelial cell lines (A549, NCI-H441, and Calu-3 cells), human colorectal adenocarcinoma cell line (Caco-2 cells), and human cervical adenocarcinoma cell line (HeLa cells) were purchased from the American Type Culture Collection (ATCC, Manassas, USA). NCI-H441 cell line (passage number between 89 and 103) was cultured in T75 cm2 cell culture flasks with RPMI1640 culture medium supplemented with 10% (v/v) FBS, penicillin (100 IU/mL), streptomycin (100 mg/mL), 1% (v/v) non-essential amino acids (NEAA) 100 × concentrate (Gibco, Paisley, UK), and 1% (v/v) sodium pyruvate. Caco-2, HeLa, A549, and Calu-3 cell lines (passage number between 25 and 35, 20 and 35, 18 and 32, 83 and 93, respectively) were cultivated in T75 cm2 cell culture flasks with DMEM culture medium supplemented with 10% (v/v) FBS, penicillin (100 IU/mL), streptomycin (100 mg/mL), 1% (v/v) NEAA 100 × concentrate, and 1% (v/v) sodium pyruvate. HPMEC-ST1.6R cells were provided by professor C. James Kirkpatrick (Institute of Pathology, Johannes Gutenberg University of Mainz, Germany). The T75 cm2 cell culture flasks were coated with gelatin solution (0.2% (w/v)) for 20–30 min at 37 °C, and then HPMEC-ST1.6R cells (passage number between 34 and 48) were seeded. Cells were cultured with M-199 medium supplemented with 20% (v/v) FBS, penicillin (100 IU/mL), streptomycin (100 mg/mL), 1% (v/v) NEAA 100 × concentrate, 25 µg/mL of ECGS, and 25 µg/mL of heparin sodium salt. All cell lines were maintained in an incubator at 37 °C and 5% CO2 in a water-saturated atmosphere, and the medium was changed every 2–3 days. Once cells reached confluence (70–80%), they were washed with PBS and treated with trypsin–EDTA (for 5 min at 37 °C). After the inactivation of trypsin–EDTA by the addition of a complete medium, cells were centrifuged and seeded into new cell culture flasks.

Evaluation of FcRn expression by flow cytometry

FcRn expression on the membrane surface of NCI-H441, A549, and Calu-3 cells was evaluated through flow cytometry. The Caco-2 cell line was used as a positive control and the HeLa cell line as a negative control. Cells were grown inside an incubator with the humidified atmosphere at 37 °C and 5% CO2, and when reached 70–80% of confluence, cells were detached with Versene (Gibco Laboratories, Grand Island, NY), to maintain the integrity of cell surface molecules. Then, cells were counted and resuspended at 1 × 105 cells/mL in FACS buffer (PBS supplemented with 10% FBS and 0.1% sodium azide), and 100 µL of cell suspension was added to each well of a round-bottom 96-well plate. The plate was centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was discarded, and the cells were resuspended in 50 µL of primary antibody (anti-mouse-FcRn (#MAB8639)) diluted in FACS buffer (2:1000 (v/v)), according to the manufacturer, for 2 h. Afterward, cells were washed in 100 µL of ice-cold FACS buffer and resuspended in a fluorescently labeled secondary antibody (goat anti-mouse Alexa 594 (#A-11020)), which was diluted in FACS buffer (2.5:1000 (v/v)) for 60 min at 4 °C. Samples were prepared in triplicates and analyzed using FACS Aria II (BD Bioscience, New Jersey, USA). Data were treated using FlowJo software (TreeStar Inc.) and presented as median fluorescence intensity (MFI).

Cell viability assay

NCI-H441 and HPMEC-ST1.6R cells were seeded in 96-well plates (n = 3) with a density of 4 × 104 and 5 × 103 cells per well, respectively [28, 29]. Cells were left to adhere and grow overnight in a 5% CO2 incubator at 37 °C. On the following day, the medium was discarded, and cells were incubated with different concentrations of ZNPs formulations diluted in the respective complete medium for 24 h at 37 °C and 5% CO2 incubator. A negative control, corresponding to cells incubated with 1% (v/v) Triton X-100 in medium, a positive control, consisting of cells incubated with medium, and a blank, comprising medium without cells, were also prepared. After 24 h of incubation, 10% (v/v) of Resazurin reagent diluted in the medium was added to each well and incubated at 37 °C for 2 h in an incubator with humidified atmosphere and 5% CO2. Resazurin is reduced to resorufin, resulting in a highly fluorescent compound, with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Fluorescence was measured using a microplate reader (Synergy™ Mx HM550, BioTek). Cell viability is expressed based on Eq. 4, in which the background signal refers to the blank.

$$\mathrm\;\mathrm\left(\%\right)=\frac\;\mathrm\;\mathrm\;\mathrm-\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\mathrm\;\mathrm\;\mathrm\;\mathrm-\mathrm\;\mathrm}\times100$$

(4)

Permeability assay

The permeability assay was performed using a lung monoculture model and a lung co-culture model, which were produced as previously described in the literature [22]. THP-1 macrophage-like cells were excluded from both models since the aim was to evaluate the permeability of ZNPs in a healthy model [22]. The monoculture model consists in culturing 1.0 × 105 of NCI-H441 cell line on the apical side of the Transwell® inserts of 12-well insert (cellQART®, Northeim, Germany) of 12 µm diameter, 1.1 cm2 area, and pore size of 1.0 µm. The co-culture model consists of NCI-H441 cell line cultured on the apical side (1.0 × 105/insert), as well as HPMEC-ST1.6R cells seeded on the basolateral side (5.0 × 104/insert) of a 12-well insert (cellQART®, Northeim, Germany) of 12 µm diameter, 1.1 cm2 area, and pore size of 1.0 µm. Before seeding HPMEC-ST1.6R cell line, the cell culture insert was previously coated with 50 µL/cm2 of Matrigel® with a concentration of 4.0 mg/mL. Both models were incubated for 24 h with RPMI 1640 medium (0.5 mL and 1.5 mL of the medium at the apical and basolateral sides, respectively), supplemented with 5% (v/v) FBS, penicillin (100 IU/mL), and streptomycin (100 mg/mL), 1% (v/v) sodium pyruvate, 25 µg/mL ECGS, and 25 µg/mL sodium heparin. After 24 h, the medium was discarded, and a complete fresh medium, further supplemented with 200 nM of Dex and 1% (v/v) ITS, was added to each insert. Cells were kept under liquid–liquid conditions (LLC) for 48 h and then transferred to air–liquid conditions (ALI), in which the medium from the apical side was removed and the medium from the basolateral side was replaced by 0.5 mL of fresh pre-heated complete medium (including Dex and ITS). Cells were kept under ALI until the day of the experiment (day 5).

Transepithelial electrical resistance (TEER) was measured every 2 days and also during the permeability assay to evaluate the integrity of the cell barrier. During the culturing of the models at LLC and throughout the permeability assay, TEER was measured directly by placing the electrode, connected to an EVOM2 Voltohmmeter (both from World Precision Instrument, Sarasota, USA), on the lateral side of the insert. However, during the ALI culture conditions, 0.5 mL and 1 mL of culture medium were added to each well, on the apical and basolateral sides, respectively, for 1 h at 37 °C with a humidified atmosphere of 5% CO2, before TEER measurements. This parameter was calculated based on Eq. 5:

$$\mathrm=\left(}_}-}_}\right)\times\mathrm\;\mathrm$$

(5)

Insulin permeability was investigated as described elsewhere [22, 30]. 0.5 mL of insulin (control), mPEG-i-ZNPs, mPEG-F-i-ZNPs, P407-i-ZNPs, and P407-F-i-ZNPs, contained 50 µg/mL of insulin, diluted in HBSS buffer (pH 7.4) were added to the apical chamber, and the well plates were shaken at 100 rpm at 37 °C. Aliquots of 200 µL were withdrawn from the basolateral chamber at different time points (10, 30, 60, 90, 120, and 180 min) and replaced with the same volume of pre-warmed HBSS buffer (pH 7.4) to maintain a constant medium volume. The amount of insulin permeated across cell monolayers was determined by enzyme-linked immunosorbent assay (ELISA), which will be further described in the next section.

The cumulative permeability of insulin across cell monolayers was calculated from the concentrations measured in the basolateral compartment (Eq. 6), and the apparent permeability coefficients (Papp) were calculated using Eq. 7:

$$\mathrm\;\mathrm\;\mathrm\,\left(\%\right)=\,\left(\frac\;\mathrm\;\mathrm}\;\mathrm}\right)\times100$$

(6)

$$_}=\left(\frac\triangle\right)\times\left(\frac1_0}\right)$$

(7)

where ΔQ/Δt is the steady-state flux (µg/s), C0 is the initial insulin concentration in the apical compartment (µg/mL), and A is the membrane area of the insert (cm2). The Papp values were estimated using C0 values based on the complete insulin release from NPs. The experiments were carried out in triplicate (n = 3).

Enzyme-linked immunosorbent assay

Sandwich ELISA was used to quantitatively analyze permeability assay samples. Briefly, high-binding 96-well plates (Corning Costar, Durham, NC, USA) were coated with 50 µL of anti-insulin antibody (Abcam, ref. ab8304) diluted 1:1000 in PBS and incubated overnight at 4 °C under orbital agitation at 65 rpm. After incubation, wells were washed three times with 200 µL of PBST and blocked with 100 µL of 4% skimmed milk (PanReac AppliChem, Barcelona, Spain) diluted in PBST (0.05% Tween-20 in PBS) for 1 h at RT. Wells were washed again three times with 200 µL of PBST. An insulin calibration curve was prepared in the range of 10 to 2000 pg/mL. Fifty microliters of standards and diluted samples were added in triplicates to wells and incubated for 2 h at RT. Wells were washed as described before, and 50 µL per well of HRP anti-insulin antibody (Abcam, ref. ab28063), diluted 1:2000 in PBS, were added for 1 h at RT, followed by a washing step. Afterward, 100 µL per well of 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific, Rockford, IL, USA) were added and incubated in the dark for 15 min. Then, 100 µL of 2 M of sulfuric acid was added to each well to stop the reaction. Absorbance was measured at 450 nm using a microplate reader (SynergyTM Mx HM550, BioTek).

Statistical analysis

All experiments were performed at least in triplicates (n ≥ 3), and results are reported as mean or mean ± standard deviation (s.d.). Statistical significances were analyzed by one-way analysis of variance (ANOVA) for the quantification of hFcRn expression in human lung cell lines by flow cytometry followed by Dunnett’s multiple comparison test, and for the Papp followed by Sidak’s multiple comparisons test. Two-way ANOVA was used for the cumulative insulin release from ZNPs formulations followed by Dunnett’s multiple comparison test and for the in vitro cumulative permeability analysis followed by Dunnett’s multiple comparison test (GraphPad Prism, GraphPad Software Inc., CA, USA). The levels of significance were set at probabilities of *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.