Folic acid, Methotrexate, N-hydroxysuccinimide (NHS), and N,N’-dicyclohexylcarbodiimide (DCC) were obtained from “Tokyo Chemical Industry” (Tokyo, Japan). Chitosan with a molecular weight of 50 kDa and DDA of 90 was obtained from “Sigma Aldrich” (MO, USA). Both PLGA with a glycolide to lactide ratio of 50:50 and a molecular weight of 40–75 kDa, and poly (vinyl alcohol) (PVA) with a molecular weight of 13–23 kDa (87–89% hydrolyzed) were obtained from “Sigma Aldrich” (MO, USA). RPMI 2650 (Human nasal epithelial cell line; CCL-30^™), Calu-3 (Human bronchial epithelial cell line; HTB-55^™), and A549 (Human alveolar basal epithelial cell line; CCL-185^™) were acquired from “ATCC” (Virginia, USA). Tow cell culture media: Eagle’s minimal essential medium, EMEM, and Kaighn’s Modification of Ham’s F-12 Medium, F-12K were acquired from “ATCC” (Virginia, USA). Penicillin-streptomycin (PS) and heat-inactivated fetal bovine serum (FBS) were obtained from “Biowest” (Nuaillé, France). 0.33 cm² Sterile 96-well plates were purchased from “SPL Life Sciences” (Pocheon-si, South Korea). All other reagents and chemicals used in this work were of analytical grade.

Samples were analyzed for MTX via an in-house validated method, where concentrations of MTX were determined using a Shimadzu HPLC system, Class-VP, (Kyoto, Japan) as previously described by Nagulu et al. with minor modifications (Nagulu et al. 2009). Phosphate buffer (pH=3), methanol, and acetonitrile in a ratio of 70:20:10, v/v were used as the mobile phase. This mixture was filtered and degassed using a vacuum filtration unit with 0.45-µm pore size. C18 column (25 cm × 4.6 mm, 5 µm) was used and 50 µL samples were injected and detected at λ_max of 302 nm. To build up the calibration curve, eight MTX concentrations (0.8–100.0 µg/mL) were prepared using the mobile phase as the solvent. All operations were carried out at room temperature.

FA and CS were conjugated as described by Dhas et al. where 500 mg of FA was dissolved in 10 mL of DMSO that contained 250 µL of trimethylamine (Dhas et al. 2015). Then 470 mg of DCC and 260 mg of NHS were added to the solution. The mixture was stirred at a rate of 400 rpm for 12 hours at room temperature. After that, the insoluble by-product (dicyclohexylurea) was removed by filtration. Then, the filtrate was poured into a mixture of anhydrous diethyl-ether and acetone. This mixture was cooled by a jacket of ice, to precipitate the FA-NHS. Then, the mixture was centrifuged for 15 min at 6000 rpm and washed twice with diethyl-ether at room temperature. Finally, the product was dried using a Nalgene vacuum desiccator (NY, USA) and the pale yellow powder (FA-NHS) was collected.

In order to prepare FA-CS conjugate, 100 mg of FA-NHS powder was dissolved in 10 mL of DMSO, and then 66.8 mg of CS was added to the mixture and stirred at 400 rpm for 4 h and at 60 °C. The mixture was then centrifuged at 6000 rpm for 15 min to collect the formed FA-CS conjugate. FA-CS conjugate was washed three times using deionized water to remove residual DMSO. Finally, the collected FA-CS conjugate was dried using a LyoQuest Telstar freeze drier (Barcelona, Spain). It is worth mentioning that this preparation was carried out in the dark.

IR grade KBr was mixed with samples of FA, CS, FA-NHS, and FA-CS. After grinding each mixture and pressing it into a tablet, the samples were scanned at wavenumber ranges from 400 to 4000 cm^-1 using Fourier-transform infrared spectroscopy (FTIR) (Shimadzu, Kyoto, Japan).

Nuclear magnetic resonance (¹H-NMR) (Bruker Avance Ultra Shield, 300 MHz, USA) was also used, where the samples were dissolved in deuterated water containing 1% v/v deuterated acetic acid in a concentration of 2 mg/mL.

Finally, the Ultima IV X-ray diffractometer (Rigaku, Japan) was used to test FA-CS using cobalt radiation (40 kV and 30 mA). Step scan mode was used with diffraction angles (2θ) from 4° to 60° and an analysis step size of 0.02°.

MTX NPs were prepared using the nanoprecipitation method, where 11.0 mg of MTX, 15.0 mg of FA-CS conjugate, and 100.0 mg of PVA were dissolved in 20 mL of water to prepare the aqueous phase (Madani et al. 2018). In another beaker, 60.0 mg of PLGA was dissolved in 10 mL DCM to prepare the organic phase. Later, the organic phase was added dropwise into the aqueous phase and stirred at 700 rpm. The mixture was then stirred overnight at 500 rpm to evaporate DCM. Then the dispersion was centrifuged at 10,000 rpm for 30 min, and freeze-dried.

Malvern zetasizer (Malvern, UK) was used to assess the mean particle size, the size distribution, as well as the Zeta potential of the formed nanoparticles. Nanoparticles were suspended in deionized water and the analysis was carried out in triplicate, at 25 °C.

The encapsulation efficiency (%EE) and the loading capacity (%LC) of the NPs were determined using the supernatant formed in the centrifugation step in the formation of MXT NPs. This supernatant was collected and analyzed for the free amount of MTX using the previously mentioned HPLC method. The %EE of MTX was calculated according to the following equation:

$\%\mathrm{EE}=\frac{(\text{ Total amount of MTX }-\text{ Free amount of MTX })*100\%}{\text{ Total amount of MTX }}$

On the other hand, %LC was calculated according to the following equation:

$\%\mathrm{LC}=\frac{\left(\text{ Total Encapsulated amount of MTX }\right)*100\%}{\text{ Total weight of NPs }}$

To test the compatibility between MTX and other formulation components, FTIR spectroscopy tests were conducted. The FTIR spectra of FA-CS, PLGA, MTX, the physical mixture (FA-CS, PLGA, and MTX), MTX NPs, and empty NPs were recorded using FTIR spectrometer (Shimadzu, Kyoto, Japan). The analysis was conducted using the KBr disc technique. A small amount of each sample was mixed with KBr, pressed into disks, and scanned from 400–4000 cm^-1.

XRD was conducted to study the physical state of MTX. X-ray patterns of FA-CS, MTX, PLGA, empty NPs, and MTX NPs were recorded using Ultima IV–X-ray diffractometer (Rigaku, Japan) applying the parameters previously.

Finally, samples of lyophilized PLGA NPs were vacuum-coated with gold before being examined with Scanning electron microscopy (SEM). FEI Quanta 450 FEG SEM (FEI) was used to inspect the morphology of the nanoparticles.

The release of MTX from the nanoparticles was studied in vitro using the dialysis bag method (Liu et al. 2017). An amount of freeze-dried nanoparticles equivalent to 500 µg of MTX was accurately weighed and suspended in a phosphate buffer solution (PBS) with a pH of 7.4. The suspension was then transferred into a dialysis bag with a molecular weight cut-off of 12–14 KDa (Spectra por, Rancho Dominguez, CA, USA). The dialysis bag was then dipped into 50 mL falcon tube, and 12 mL of PBS medium was added. Then the tube was horizontally shaken at 100 rpm in a shaking water bath (Daihan Scientific. Co Ltd, Korea), at 37 °C and at 100 rpm. At certain time points, 1.0 mL of the dissolution medium was withdrawn and replaced with another 1.0 mL of blank release medium. The withdrawn samples were filtered by a 45 µm Millipore syringe filter and analyzed to determine the released concentrations of MTX.

Calu-3, A549, and RPMI 2650 cell lines were maintained in culture according to the published protocol by ATCC, ATCC Product Sheet. Both Calu-3 (Anon 2021b [n.d.]), and RPMI 2650 cells (Anon 2021c [n.d.]) were cultured in EMEM culture media, whereas A549 cells (Anon 2021a [n.d.]) were cultured in F-12K culture media. Both media were supplemented with 10% v/v fetal bovine serum (FBS), 0.1 mg/mL of streptomycin and 100 U/mL penicillin. Cells were kept in an incubator at 37 °C, 95% humidified air, and 5% CO₂.

To perform MTT assays, cells were seeded at 37 °C in 96 well-plates (3×10⁵ cells/cm²) and kept at 95% humidified air containing 5% CO₂ in the CO₂ incubator for 24 h. After that, the MTT colorimetric assay by Sigma Aldrich (St. Louis, Missouri, USA) was started.

MTT assay was used to assess the cytotoxicity of the MTX, MTX NPs, and empty NPs. Five concentrations of MTX (18.75, 37.5, 75, 150, and 300 µg/mL) were prepared from 300 µg/mL stock solution using the matching growth media. After that, 200 µL of each concentration was added to cells and incubated in 5% CO_2x and 95% humidified air at 37 °C for 24 h.

Then, 20 µL of MTT reagent (concentration of 5 mg/mL in PBS) was added per well and the well-plates were incubated in 5% CO₂ and 95% humidified air at 37 °C for 4 h. Then, after removing the MTT-containing solution, 200 µL of DMSO were added per well. BioTek Microplate reader (Vermont, USA) was used to measure the absorbance at a wavelength of 570 nm.

The growth media without any cells was used as the negative control, and cells in culture media without any treatment were used as the positive control. It is worth mentioning that blank normalization was done for all readings against the negative control. Cell viability for each sample was calculated as the percentage of the mean viability of positive control.

Statistical analysis was performed using GraphPad software (Prism 5.04), where a student t-test was performed for two groups-comparisons with a p-value less than 0.05 considered statistically significant. Data were presented as mean ± SD.

Fig. 1 shows the FTIR spectra of FA, FA-NHS, CS, and FA-CS. First of all, when FA and FA-NHS are compared, two remarkable peaks appeared in the FA-NHS spectrum at 1700 cm^-1, and at 1600 cm^-1 corresponding to C=O stretching and N-O stretching, respectively, which confirm the conjugation formation (FA-NHS) (Dhas et al. 2015).

Figure 1. 

FTIR spectra of Folic acid (FA), Folic acid-NHS conjugate (FA-NHS), Chitosan (CS), and Folic acid- chitosan conjugate (FA-CS).

On the other hand, when CS and FA-CS FTIR spectra are compared, some differences can be noticed that may confirm the conjugation between FA and CS. For example, the stretching vibrations of broad bands in the FA spectrum related to carboxyl groups that appeared at 3300 and 3500 cm^-1 disappeared from the FA-CS spectrum. Furthermore, new broadband appeared at 3300–3500 cm^-1 related to the formation of the amide bond between the carboxyl groups of FA and the amino groups of CS. Similar results were previously reported and used to confirm the conjugation of CS and FA (Nagulu et al. 2009).

Fig. 2 shows the ¹HNMR spectra of FA, CS, and FA-CS. FA protons showed signals corresponding to H-19, H-22, and H-21 at 4.94, 2.33, and 2.07 ppm, respectively (Salar and Kumar 2016). It can be noticed that the spectrum of CS shows three main peaks at 2.07, 2.24, and 4.79 ppm. On the other hand, the spectrum of FA-CS showed that a new peak at 2.7 ppm is related to the interaction between FA and CS, which proves the formation of a new amide bond between the amine groups in CS and the activated ester in FA (FA-NHS) (Salar and Kumar 2016).

Figure 2. 

NMR spectra of A: folic acid (FA), B: chitosan (CS) and C: folic acid-chitosan conjugate (FA-CS).

Finally, Fig. 3 shows the CS and FA-CS’s XRD patterns. When the two spectrums are compared, it can be noticed that the peak at 2θ = 11° from the CS spectrum disappeared. This may be related to the decrease in the crystallinity of FA-CS in comparison to pure CS. The new strong hydrogen bonds formed between FA and CS during conjugation were related to lower the crystallinity of CS by other researchers (Ji et al. 2012).

Figure 3. 

XRD patterns of chitosan (CS) and folic acid-chitosan conjugate (FA-CS)

Figure 4. 

Size distribution of MTX NPs

Particle size and polydispersity indices of MTX NPs are shown in Fig. 4. The average particle size of the NPs was 384.3 nm and the polydispersity index was 0.27. The Zeta potential of MTX NPs was around +13 mV as shown in Fig. 5. The positive charge may be due to the presence of the positively charged chitosan on the surface of NPs (Dhanaraj et al. 2016).

Figure 5. 

Zeta potential distribution of MTX NPs

The %EE of MTX NPs was 79% and the %LC was 21.2%. Those results show that the method of preparation was very effective in loading MTX in the NPs (Hassan et al. 2017).

FTIR spectra of FA-CS, PLGA, MTX, physical mixture, empty NPs, and MTX NPs are shown in Fig. 6. MTX FTIR spectrum characteristic absorption peaks can be noticed at 1652.9 cm^-1, 3388.9 cm^-1, and 3186.4 cm^-1 due to -OH and -NH₂ bond bending, and aromatic ring stretching, respectively. These results are similar to the results reported by other researchers in the literature (Samra et al. 2013). Additionally, the PLGA FTIR spectrum showed a characteristic absorption peak at 1716 cm^-1 related to C=O stretching (Anon 2021a, b, c, d? [n.d.]). FA-CS spectrum showed a characteristic absorption peak at 1670 cm^-1 that may be related to the formation of an amide bond between the carboxyl group of FA and the amino group of CS (Naghibi Beidokhti et al. 2017). The presence of an absorption peak of C=O of PLGA at 1670 cm^-1 in the MTX NPs spectrum indicates that PLGA was successfully incorporated into the final formulation. The presence of the absorption peak of the amide bond of FA-CS at 1670 cm^-1 in the spectrum of MTX NPs indicates that MTX-PLGA NPs were successfully coated with FA-CS. Finally, MTX NPs FTIR spectrum showed characteristic peaks at 3150 cm^-1 and 1652 cm^-1 due to stretching of the aromatic ring and bending of -OH groups of MTX, which indicates that MTX was compatible with formulation ingredients and successfully incorporated into NPs.

Figure 6. 

FTIR spectra of FA-CS, PLGA, Free MTX, physical mixture, empty NPs, and MTX NPs

X-ray patterns of FA-CS, MTX, PLGA, Empty NPs, and MTX NPs are shown in Fig. 7. FA-CS and PLGA XRD patterns showed no distinct peaks because of the amorphous nature of the polymers (Dhas et al. 2015). Free MTX showed a crystalline pattern as indicated by its characteristic diffraction peaks as shown in Fig. 7 (Nezhad-Mokhtari et al. 2019). MTX distinct peaks are less obvious in the MTX NPs pattern, which may be a result of the dilution effect (Ashwanikumar et al. 2014).

Figure 7. 

XRD patterns of FA-CS, PLGA, Blank NPs, Free MTX, and MTX NPs

Fig. 8 shows the SEM images that were used to explore the morphology of the NPs. It can be seen that both unloaded and loaded NPs are spheres with smooth surfaces.

Figure 8. 

SEM photomicrographs of A. empty NPs, B. MTX NPs

The in vitro release profile of MTX from nanoparticles is shown in Fig. 9. Almost 85% of MTX released after 5 h. The results indicate that loading MTX in PLGA NPs could somehow sustain MTX release from the particles. The release was biphasic in nature with a first phase in which most of the drug was released rapidly (85% in 5 h). This fast initial release may indicate that most of the MTX was accumulated on the surface of the NPs. In the second phase, a slow release of a small portion of the drug (15%) can be noticed. This part could be entrapped inside the NPs, and therefore, required more time to be released.

Figure 9. 

In vitro release profile of MTX nanoparticles

Fig. 10 shows a reduction in cell viability for both MTX and MTX NPs in RPMI 2650, Calu-3, and A549 cell lines. In RPMI 2650, both MTX and MTX NPs reduced the cell viability at all tested concentrations, but the difference between the two treatments was statistically insignificant (p-value>0.05). Furthermore, the effect of MTX and MTX NPs was concentration independent on RPMI 2650 cell viability, as shown in Fig. 10A. In addition, empty NPs showed significantly higher cell viability (p-value<0.05) as compared with the positive control at 18.75 µg/mL and showed significantly lower cell viability compared with the positive control at 300 µg/mL.

Figure 10. 

The effects of different concentrations of methotrexate (MTX), MTX NPs, empty NPs, and positive control on cell viability tested in A. RPMI 2650; B. Calu-3; C. A549 cells. Data are presented as percentages of the mean viability of positive control. Data: Mean ± SD (n=3–5). ^*p-value<0.05, compared to positive control, ^# p-value<0.05, compared to MTX, ^$ p-value<0.05, compared to empty NPs.

As shown in Fig. 10B, MTX and MTX NPs showed a decrease in Calu-3 cell viability. At 37.5 µg/mL, MTX NPs showed significantly (p-value<0.05) lower cell viability in comparison to MTX, which indicates that incorporation of MTX into the coated PLGA NPs could enhance its cytotoxicity effect. Similarly, a significant reduction in A549 cell viability as treated by MTX NPs can be noticed at concentrations of 75 and 150 µg/mL, in comparison to the cells treated with free MTX (p-value<0.05). The effect of both MTX and MTX NPs was concentration-dependent on Calu-3. MTX NPs showed the same trend on A549 cell viability, where the cytotoxicity increases as the MTX concentration increases in each treatment; these results are shown in Fig. 10B, C, respectively.

Empty NPs showed no significant effect on the cell viability (p-value>0.05) of the three cell lines media (RPMI 2650, Calu-3, and A549). This could point out the absence of any significant cytotoxicity in vitro on these tested cell lines (Fig. 10). Consequently, the formulation components can be considered non-toxic and safe on tested cell lines, and any effect reported previously was caused solely by MTX.