Chemicals for analysis and synthesis were as follows: Gefitinib (GEF) (Tokyo Chemical Industrial/Japan), Dimethylsulfoxide (DMEM) (ChemCruz/USA), Dulbecco’s phosphate-buffered saline (Euro-Clone S.p.A/Italy), Trypsin-EDTA 1X in PBS (Euro-Clone S.p.A/Italy), Formic acid (Sigma-Aldrich/China),Methanol (Sigma-Aldrich/France), MTT [3-(4,5-Dimethylthiazol-2-yl) (Thermo-Fisher Scientific/USA), Penicillin-Streptomycin Solution (Euro-Clone S.p.A/Italy), Tetrapropylammonium hydroxide, Tetraethyl orthosilicate, and Trifluoroacetic acid (Sigma-Aldrich),Tween 80 (Janssen Chemica/USA). And the Chemicals for nanoparticle synthesis were: Terapropylammonium hydroxide, Sodium hydroxide, Aluminum sulfate anhydrous.

High-performance liquid chromatography with LC solution software (Model: KMC-130SH) (Shimadzu/ Japan) was used to analyze GEF. The Mobile phase of the analysis of GEF is composed of (Trifluoroacetic acid Orthophosphoric acid mixture each 0.1% in deionized water): methanol (1:1). The column used was EC 250/4.6 Nuclerdur 100 C18 at temperature (30 °C. The injection volume was 10 µl, the flow rate was 1 ml/min, and lambda max was set at 246 nm (Faivre et al. 2011). The linearity and calibration curve was obtained in the range of 5–30 µg/ml range. Serial dilutions were made from a stock solution of 1000 µg/ml in methanol using the mobile phase. Regression and R2 were calculated using the software of the instrument.

The synthesis of ZSM-5 nanoparticles was prepared as follows: 12.5 g of terapropylammonium hydroxide was dissolved in 50 mL of deionized water. Then 12.0 g of tetraethyl orthosilicate was added dropwise to the reaction flask at room temperature. The flask was placed in a water bath with vigorous shaking for 24 hours at 80 °C. In a separate flask, 0.24 g sodium hydroxide and 0.722 g aluminum sulfate anhydrous were mixed in 4 ml deionized water and poured into the previous mixture. The mixture was placed in acid digestion vessels for forty-eight hours at 170 °C. Finally, the obtained nanoparticles were washed twice with distilled water, filtrated, and calcined in a furnace for 6 hours at 500 °C (Anaya et al. 2022).

GEF was loaded onto the prepared ZSM-5 nanoparticles using solvent evaporation, kneading, adsorption, and physical dry mixing (El-Sawi et al. 2021). In all methods, the ratio of drug to carrier was 1:1 w/w. Then the method that gave the highest percent loading was followed in the preparation of 1:2 and 1:3 w/w to investigate the efficacy ratio of drug carriers on the loading efficacy of the drug.

In the solvent evaporation method, 0.05g of GEF was dissolved in 15 mL of methanol, and then, in a conical flask, 0.05 g of ZSM-5 was added. The rationale for using methanol as the solvent is its capacity to effectively dissolve GEF and provide a uniform combination with ZSM-5. Drug-loaded nanoparticles are left behind because methanol’s volatile nature makes evaporation simple. Materials that dissolve easily in organic solvents may be used using this technique. The mixture was kept for 24 hours with continuous stirring at 40 rpm at room temperature. Then the heat was applied (40 °C) for 24 hours at the same stirring rate to evaporate the solvent. Then the sample was transferred to a container and kept in the desiccator.

For the kneading method, 0.05 g of each GEF and ZSM-5 were weighted and mixed in a porcelain mortar. The vehicle was prepared from methanol and phosphate buffer in a ratio of 1:1 (pH 3). The kneading process uses a pH 3 combination of phosphate buffer and methanol. This mixture is chosen to make a paste that will distribute GEF and ZSM-5 uniformly. Methanol helps to generate a paste for uniform drug and carrier integration, and the pH of 3 is used to preserve the stability of GEF throughout production. The solvent was added drop by drop until a paste was formed. Trituration was continued for one hour; then, the sample was kept in an oven at 40 °C for 24 hours for drying. The powder was sieved through a 0.305-mm mesh and kept in the desiccator until used.

Physical mixing used the same procedure as kneading but without solvent. Direct blending of GEF with ZSM-5 requires just dry mixing, which is accomplished physically without the need for a solvent. This approach simplifies the loading procedure by removing the need for a solvent during the drug’s integration with the carrier. Dry mixing was achieved for one hour manually. The sample was sieved and kept in the desiccator until used.

In the adsorption method, 0.01g of GEF was mixed with 0.01 g of ZSM-5 and 25 ml of distilled water. To take use of the hydrophilic properties of ZSM-5, distilled water is used as the solvent in the adsorption process. Water ensures that the medication and carrier are stable by facilitating the adsorption of GEF onto the nanoparticles without the need for organic solvents. The mixture was stirred for 24 hours at 40 rpm and filtered using filter paper. The powder was centrifugated at 4000 rpm for 5 minutes and then dried.

The kneading method was performed using different ratios of GEF to the carrier. 1:1, 1:2, and 1:3 ratios were prepared using the same procedure to investigate the effect of the drug-to-carrier ratio on the loading efficiency of GEF.

The FTIR spectrum for ZSM-5was recorded over the range 400–4000 cm-1. The samples were analyzed using the Fourier infrared spectrophotometer (IR Prestige-21, Shimadzu Europa GmbH) using the KBr disc. IRsolution FTIR control software supports FDA 21 CFR Part 11 compliance and provides a resolution of 2 cm-1. GEF, unloaded ZSM-5 NPs and the loaded NPs were investigated.

The XRD patterns were acquired using an X-ray diffractometer (Shimadzu XRD-7000, Japan) equipped with a CuK radiation source and a Ni filter operating at 40 kV/30 mA in the range 2° 2 ≤ 60°. The scanning speed was 2° per minute, and the sampling pitch was 0.02°. The obtained PXRD data were analyzed with the X’Pert HighScore Plus 2.2 software using search-match operations to completely identify the inorganic crystal structure. Also, GEF, unloaded ZSM-5 NPs and the loaded NPs were investigated.

To record the SEM of the loaded ZSM-5 nanoparticles, a sample piece was transferred into an adhesive carbon and cleaned with N2 gas. The morphology and chemical composition of samples were investigated by a Phenom XL G2 scanning electron microscope (Thermo Fisher Scientific) coupled with an AXS EDS system. The SEM images were collected at 0.06–0.04 mbar with a 15 kV accelerating voltage. BET samples were recorded using an N2 adsorption-desorption analyzer (Autosorb IQ, Quantachrome Instruments version 5.21, Boynton Beach, FL, USA).

For pH measurement, 100 mg of the prepared loaded NPs were suspended in 100 mL of distilled water, and the pH of the solution was measured by a pH meter. Also, the NPs size was measured by Zetasizer (Malvern, Worcestershire, UK). The nanoparticles sample of 10 mg was suspended in 10 mL of deionized water, and the suspension was diluted 125-fold using deionized water before measurement.

Percent GEF loaded on ZSM-5 NPs was measured by suspension of 0.05g of the loaded GEF nanoparticle powder in 25mL methanol with sonication to extract the drug completely from the nanoparticles. The solution was filtered and diluted with a solvent composed of (35:65 acetate buffer: methanol) pH 3, and the concentration of GEF was measured using the developed HPLC analysis method. Percentage drugs loaded on the NPs was calculated using the following equation (Martinho et al. 2015):

%Drug loading = (Weight of drug in nanoparticles)/ (Weigh of nanoparticles) × 100%

Characterization of the crystalline structure of the loaded NP was done using X-ray diffraction, and FTIR investigated the detection of binding of the drug to the carrier. The spectrum of free GEF and GEF-loaded NPs were taken, and results were compared with the free carrier.

GEF release from the loaded nanoparticle was studied using USP apparatus II in phosphate buffer pH 6.8 (Yang et al. 2018). The dissolution conditions were set: dissolution medium phosphate buffer with pH 6.8, temperature 37 °C, stirring speed 50 rpm. Due to the sensitivity of GEF to light, the whole apparatus and all equipment were wrapped in aluminum foil. 50 mg of pure GEF powder and ZSM-5-GEF loaded using the kneading method (1:1 w/w equivalent to 50 mg GEF) were put in each jar at time zero, and the test was run for 60 min. 5 mL of the sample was withdrawn and replaced by fresh media at the following times: 5, 10, 20, 30, 45, 60, and 70 minutes, and diluted with the mobile phase. Its concentrations were measured using HPLC. The same procedure was repeated for GEF pure powder.

Since pH 6.8 is often used to simulate the gut pH in release experiments, it is possible that the formulations were intended for oral delivery. The small intestine is often where medication absorption occurs, and its pH range is 6.8. It is crucial to examine release profiles at various pH settings, including the stomach’s acidic environment (pH 1.2), considering the thorough assessment of drug release.

Based on the result of the test, the release test was repeated for 12 hours. The same amount of the loaded NPs was used in each of the 6 jars to perform the test and the same conditions were used. Samples were withdrawn at 20 min, 40 min to detect any possible burst effect, Then 1, 2, 3, 4, 6, 8, 10, and 12 hours.

In a 12-well plate, 200 cells of A-549 were seeded in 2 mL of medium in each well and incubated for 24 hours in a 5% CO2 incubator at 37 °C. After 24 hours, the media in each well were replaced with 1 mL fresh media, and the cells in 3 wells were treated using 0.5 IC50, IC50, and 2 IC50 of free GEF, respectively. While the cells in the other three wells were treated using 0.5 IC50, IC50, and 2 IC50 of loaded GEF. DMSO and media were used as controls. After that, up to 2 mL of media was added to each well. Then the plate was incubated in a 5% CO2 incubator at 37 °C for seven days. After seven days, the colonies in each well were counted and recorded.

All statistical analysis results were performed using GraphPad Prism 8 via Two-way ANOVA. The results were presented as mean ± SD. Statistical significance was represented as *P < 0.05, **P < 0.01, and ***P < 0.001.

HPLC was used to determine how much gefitinib is contained inside the nanoparticles and how much medicine will be released. At a retention time (RT) of 1.626 minutes, a peak corresponding to GEF was seen. The peak was sharp and highly resoluted. Pure GEF concentrations were measured and used to create the calibration curve shown in Fig. 2. The R2 value for linearity was 0.99999.

Figure 2. 

Linear regression and calibration curve of GEF.

No deviations from the method described by Hodali and Marzouqa (Hodali & Marzouqa, 2016) were made during the synthesis of ZSM-5 nanoparticles. Drying for 4 hours at 100 degrees Celsius was more than enough time to ensure a well dried out final product. When heated to 500 degrees Celsius for 24 hours, the material is reduced to a white powder. The finished powder was then stored in a desiccator, away from any possibility of dampness.

For the FTIR analysis, the spectra shown in Fig. 3A shows the characteristic peaks of stretching vibration for GEF: C-Cl at 800 cm-1, C-F at 1028 cm-1, C-O at 1110 cm-1, C=C (Aryl) at 1500 cm-1, and N-H at 3400 cm-1. For the ZSM-5 (Fig. 3B), while Fig. 3C shows the spectrum of the ZSM-5 NPs loaded with GEF. The characteristic peaks were detected: tetrahedron internal vibration at 540 cm-1, S-O stretching at 620 cm-1, Si-O-Si symmetry stretch at 800 cm1, Si-O-Si asymmetric stretching at 1099 cm1, and Si-OH-Stretch at 3360 cm-1. These results agreed with (Cheng et al. 2017).

Figure 3. 

FTIR data of (A)GFT, (B) ZSM-5 loaded nanoparticles and (C) ZSM-5 NP.

The results clearly depict changes to representative GEF peaks in the spectrum of the loaded carrier vibration. The carbonyl peak (C=C, C=N) at the higher frequency indicates intermolecular interactions between the GEF and the ZSM-5 nanocarriers. The O-H of the nanocarrier is overlaid with the N-H peaks of the GEF in the spectrum. Hydrogen bonding interactions between hydrogen donor ZSM-5 and hydrogen receiver GEF may have contributed to the observed results. Furthermore, there were a lot of hydrophobic interactions between the carbon rings, the surface zeolite, and the bridging oxygen. The same hypothesis was supported by a recent study by Narayan et al. that showed favorable interactions between the MCM-41 surface and the antibiotic 5-fluorouracil through hydrogen bond donors and acceptors (Narayan et al. 2022).

The XRD spectrum of ZSM-5 (Fig. 4A) reveals FTIR data of GFT, ZSM-5 loaded nanoparticles and ZSM-5NP GFT shows fluctuations adversely at initial stage varying from 28–30o. It abruptly goes up at 1700 and then slowly comes down and shows little variations. NP shows variation not much similar to ZSM-5 but smooth fluctuations and goes up at 1700 slowly comes down at 2700 then goes up at 3700 and gains constant. ZSM-5 shows little fluctuations comes down at 1000 gains height of 1500, very little fluctuates and goes up, slightly comes down at 3500 and then again gain heights. The same pattern was reported by (Cheng et al. 2017). At the same time, the crystal structure of pure GEF (Fig. 4B) with sharp peaks at 18.6°, 19.2°, 24.2°, 26.2°, and 26.4° has been reported as shown in Fig. 4C.

Figure 4. 

X-ray diffraction pattern of (A) ZSM-5, (B) GFT and (C) ZSM-5 loaded GFT nanoparticles.

Drug encapsulation into the nano carrier ZSM-5 is the cause of the sharp two peaks at 18.6 and 19.2 of crystalline GEF disappearing from the drug-loaded ZSM-5 diffractogram, while the fundamental peaks of ZSM-5 are retained and have minimal peak strength. It is noteworthy that the presence of GEF in the amorphous phase may increase GEF’s solubility and dissolution rate, which are responsible for the lowering of the peaks of loaded ZSM-5. These findings support the Zarshenas groups’ observation that the XRD pattern of 5-fluorouracil-loaded ZSM-5 demonstrates full encapsulation within ZSM-5 and the absence of loose crystalline 5-fluorouracil (Zarshenas et al. 2022).

Scanning Electron Microscope (SEM) photo shows the morphology of the prepared NPs (Fig. 5). The result confirms the formation of ZSM-5 zeolite (Shi et al. 2018).

Figure 5. 

SEM photo of the prepared ZSM-5 nanoparticles

Fig. 5 shows properties of nanoparticles of ZSM-5. Its size is 5µm, 14.1mm wide and HV is 3.00kv. the same shape was obtained with (Sun et al. 2020).

The pH of the ZSM-5 nanoparticle suspension was 7.71±0.05, close to the pH of biological fluids. (Trumer et al. 2012) reported the pKa of GEF 5–7, which means the above pH 7 GEF mostly exists in its unionized form which is the biologically favored species.

The average particle size of the ZSM-5 NPs was between 100–200 nm, with a polydispersity index (PDI) equal to 0.265 (Fig. 6). This range of particle size ranged from 100 to 200 nm, which satisfies the criteria for nanoscale drug delivery systems that provide a high surface area per unit weight of the system with good safety (Kariminezhad et al. 2015)

Figure 6. 

Particle size distribution of the prepared ZSM-5-GFT nanoparticles.

The percent yield of the NPs and loading efficiency of GEF are represented in Table 1. Results showed the highest loading efficiency was obtained with the kneading method with no effect of the drug to the NPs ratio on the percent loading. Physical adsorption and physical mixing methods gave less than the kneading method with no differences between them. The method with the least efficiency was the solvent evaporation method.

The kneading procedure has the highest loading effectiveness (80%). However, physical mixing produced an extremely close result (79%). These two techniques are mixed with force. This technique works well for inserting medications into zeolite nanoparticles. In this procedure, a known volume of a concentrated medication solution is employed that is roughly equivalent to the nanocarrier’s pore volume. The medication then diffuses into the pores as a result of capillary action. Additionally, this technique demonstrated how many impregnations could fill pores and improve drug loading concentration. The kneading approach is more effective than the other conventional loading processes, according to our research. The key benefits of the kneading method include high loading capacity, minimal solvent use, and suitability for expensive drugs. As a result, the ratio of drug to carrier had little effect on loading efficiency, implying that the pores were almost filled with drug. This result is consistent with that of the work by (Charnay et al. 2004).

Samples prepared by kneading method in the ratio of 1:1 were chosen to complete the studies.

The GEF release from the loaded ZSM-5NPs is represented in the dissolution profile shown in Fig. 7. Results showed a significant difference in percent drug release at 5 minutes of 20% ±3.5% of loaded GEF versus 68% ±1.8% of free drug and 35%±2.6% of loaded NPs versus 70% ± 3.0# in 60 minutes and all these differences were significant using 5% as a confidence interval. These results suggested the prolonged release of GEF from the NPs.

Figure 7. 

Drug release profile of free GEF and GEF from the loaded NPs for 60 min, phosphate buffer pH 6.8, apparatus II (paddle), 50 rpm, and 37 °C.

The results of the dissolution test for 12 hours are represented in Fig. 8. This result supported the prolonged release pattern with zero-order release over time 1–12 hrs with a correlation coefficient R2 equal to 0.993.

Figure 8. 

Drug release profile of free GEF and GEF from the loaded NPs for 12 hr, phosphate buffer pH 6.8, apparatus II(paddle), 50 rpm, and 37 °C.

The typical profiles of release obtained from ZSM-5 nanoparticles may be caused by diffusion through the ZSM-5 nanoparticle, transport through water-filled pores, or the initial high release. It also has to do with the process of interfacial diffusion between the solid surface nanoparticle and the dissolution medium. In the latter scenario, the release rate is thought to be proportional to the difference between the drug concentration at the time of analysis in the phosphate buffer media and the drug concentration in the core of the nanocarrier (Godugu et al. 2015). In comparison to the free drug, the loaded GEF was released slowly over a 60-minute. with 35% of the loaded drug being released. When performing the test for 12 hours, the slow release of GEF followed a zero-order pattern with a correlation of 0.999 between 1–12 hrs with a total 80% of drug release. Armando et al. suggested these mechanisms in their research. Since the free drug gave 68% dissolution in the first 5 min. the sink condition was mostly achieved, and the NPs might be a good candidate for prolonged action.

GEF from the loaded NPs dissolves much more slowly than pure GEF. This is as a result of the GEF’s regulated release made possible by the NPs. Just 20% of the GEF from the loaded NPs had dissolved after an hour, compared to 50% of the pure GEF. Only 30% of the GEF from the loaded NPs had dissolved after two hours, compared to 70% of the pure GEF. Over the time of study, the GEF from NPs release showed an almost zero-order controlled pattern of release.

There are several reasons why this regulated release of GEF may be advantageous. First, it may contribute to the body’s ability to retain a steady level of GEF, which may result in more successful therapy. Because the medication is not delivered all at once, it may also lower the chance of adverse effects. Thirdly, since the medication doesn’t need to be taken as often, it may increase patient compliance.

All things considered, the dissolution profile of both pure GEF and GEF from the loaded NPs demonstrates that the NPs provide a regulated release of the medication. Improved patient compliance, a lower chance of adverse effects, and more effective therapy are just a few advantages of this.

Results of the MTT assay, colony formation, and migration assays are shown in Fig. 9. The viability assays revealed that blank (media) and control (media) had no apparent toxicity on A549 cells. The IC50 for free GEF was 704.1 nM, while the IC50 for GEF-ZSM-5 loaded was 518 nM. A significant decrease (P = 0.00069) in IC50 between free GEF and loaded GEF on ZSM-5 NPs. The normalization of the transform of carrier only, free GEF, and GEF-ZSM-5 loaded using the kneading method is seen in Fig. 10.

Figure 9. 

Viability percent for free GEF, GEF-loaded NPs, ZSM-5 NPs, and media (DMSO).

Figure 10. 

Normalize of transform of A. ZSM-5 Carrier only; B. Free GEF, and C. GEF-ZSM-5 loaded prepared by kneading method (ratio 1:1).

Comparing our prepared blank ZSM-5 nanoparticle to the drug-loaded ZSM-5 nanoparticle, the cellular studies showed that our prepared blank ZSM-5 nanoparticle is safe and biocompatible. The GEF medication had an IC50 of 704.1 nM compared to 518 nM for the GEF-loaded ZSM-5 nanoparticle in the cell viability MTT experiment. Intriguingly, as shown in Fig. 9 (Kutkut et al. 2023), GEF-loaded ZSM-5 nanoparticles demonstrated greater cytotoxicity compared to the original medication after 24 hours of therapy.

The innovative GEF encapsulation using ZSM-5 increased antitumor activity as an appropriate drug delivery mechanism, which was confirmed by the MTT results. Additionally, ZSM-5 nanoparticles frequently cover up a drug’s undesirable side effects and may enhance their pharmacokinetics and bioavailability (Haeri et al. 2014).

Due to differences in cellular processes uptake compared to the free pharmaceuticals, incubation for 24 hours might not provide the drug-loaded ZSM-5 nanoparticle with the best effect time (Zaleskis et al. 2021).

For the migration test, GEF does not significantly affect cells’ migration. Fig. 11 shows The motic area was slightly higher in loaded GEF than in free GEF, but that difference was insignificant. Therefore, the pure GEF and GEF-ZSM-5 loaded using the kneading method of 1:1 w/w did not affect the cell migration (Tables 2, 3).

Table 2.

Download as

CSV

XLSX

Closure % in the migration assay (media and free GFT).

Sample	Concentration	Day 1	Day 2	% closure
Control	Media			38.54%
Free GFT	½ IC50			17.7%
	IC50			10.55%
	2IC50			0.68%

Table 3.

Download as

CSV

XLSX

Closure % in the migration assay (ZSM-5 loaded GFT).

Sample	Concentration	Day 1	Day 2	% closure
ZSM-5-GEF-loaded	½ IC50			23.7%
	IC50			21.58%
	2IC50			14.34%

Figure 11. 

Control migration testing Treatment of A-549 cells with: Half-Maximal Inhibitory Concentration (IC50), IC50, 2 IC50, Double-Maximal.

A colony assay was used to study the growth rate of A-549 cells after treatment and the effect of proliferation without a drug. The average number of colonies was noticed, and growth was recorded according to the treatment plan after 13 days of seeding. Fig. 12 shows an insignificant decrease in cells treated using the IC50 of free GEF at the 0.05 CI level but a significant (***P 0.001) difference compared to control cells. While in A-549 cells treated using ZSM-5-GEF-loaded using the kneading method 1:1 w/w, a significant decrease (***P 0.001, **P 0.01, **P 0.01 is seen in cells treated using 12 IC50, IC50, and 2 IC50, respectively, but not significant at the 0.05 level.

Figure 12. 

Colony test of untreated A-549 cells in a control. utilizing free gefitinib and ZSM- 5-gefitinib-loaded utilizing kneading technique 1:1 w/w, 12 IC50 stands for half dosage of the half maximum inhibitory concentration, IC50 for the half maximum inhibitory concentration, and 2 IC50 for double dose of the half maximum inhibitory concentration. Comparisons between each dosage and the control were made. The findings were shown as mean SD. The signs for statistical significance were *P 0.05, **P 0.01, and ***P 0.001.

The results of the closure assay show that the drug-loaded ZSM-5 nanoparticle continues to act after 48 hours. The closure rate was significantly slower for drug-loaded ZSM-5 nanoparticles than for free drugs, showing that GEF-loaded ZSM-5 nanoparticles were more effective at inhibiting cancer cell migration than free GEF drugs as a result of the new formulation’s improved physiochemical properties and increased cytotoxicity effect on A549 cell lines. After treating A549 cell lines with IC50 and 2 IC50 concentrations and comparing the free drug with a drug-loaded ZSM-5 nanoparticle, the best closure percentage and meaningful results were obtained. By using these doses, a GEF drug-loaded ZSM-5 nanoparticle completely stopped cell migration with nearly no closure percentage. These findings verified the higher cytotoxicity of GEF-loaded ZSM-5 nanoparticles against A549 cell lines to suppress lung cancer metastasis and validated the MTT findings (Smith et al. 2017).

Additionally, the colony formation assays (Fig. 12) demonstrated the efficiency of GEF-loaded ZSM-5 nanoparticles by showing that no colonies formed over a 13-day treatment period. Even though the number of colonies counted in the two cells treated with either of the free medications was comparable to the number of colonies loaded with drugs in ZSM-5 nanoparticles, after 13 days, both free GEF and GEF-laden ZSM-5 nanoparticles showed a significant decline in colony formation. The potent novel formulation encapsulating medication against the A549 cell line (El-Tanani et al. 2022) may be to blame for this.