One-pot construction of gemcitabine loaded zeolitic imidazole framework for the treatment of lung cancer and its apoptosis induction

Abstract The zeolitic imidazole framework (ZIF) is a novel metal-organic framework with distinctive properties, including crystalline form, controllable pore size, wide surface area and biocompatibility. ZIF-L is a good candidate for biological applications due to its outstanding thermal and chemical stabilities. The current study aimed to develop gemcitabine (GEM) encapsulated ZIF-L in a single pot and assess its anticancer efficacy against lung cancer. The GEM@ZIF-L nanoparticles were studied using microscopic and spectroscopic measurements. The outcomes of cell counting kit-8 (CCK-8) demonstrated that two lung cancer cells were significantly cytotoxic to the IC50 values GEM@ZIF-L in A549 and H1299 cells were 9.12 ± 1.28 µg/mL, 5.47 ± 2.90 µg/mL, respectively. A dose-dependent decrease of lung cancer cells (A549 and H1299) was found using GEM@ZIF-L. Additionally, remarkably induced apoptosis was observed in GEM@ZIF-L, validated by fluorescence staining techniques (including acridine orange and ethidium bromide) (AO-EB and nuclear DAPI staining). The mode of cell death was examined by flow cytometry (dual staining Annexin V-FITC/PI) methods. Further, the ELISA analysis confirmed that GEM@ZIF-L induced apoptosis through caspase activation. The multifunctional GEM@ZIF-L nanoparticles may be suitable for biological applications, as the current work demonstrates.


Introduction
Lung cancer is the second most frequent cancer in both men and women and is also one of the deadliest [1].Traditional treatments for lung cancer, including surgery, radiation and chemotherapy, have far from adequate long-term survival rates [2].The systemic side effects of anticancer medicines are undesired.If anticancer medications can be delivered locally to the tumour site while reducing systemic exposure, improved drug delivery might play a significant role in the battle against cancer [3][4][5].Inhalation of chemotherapeutic solutions is the subject of several research [6].However, pulmonary administration has been observed to have comparable systemic adverse effects and dose-limiting toxicity as intravenous treatment [7].
Difluorodeoxycytidine (gemcitabine, termed GEM) is a pyrimidine antimetabolite chemically like deoxycytidine [8].GEMs mode of action has been thoroughly studied and understood.Deoxycytidine kinase may convert GEM to dFdCMP, dFdCDP and dFdCTP or deoxycytidine deaminase can convert it to difluorodeoxyuridine [9].The latter enters DNA and causes a break in the strand.Incorporating dFdCTP into DNA is less efficient than cytosine arabinoside (ara-C), so DNA exonuclease is more challenging to remove [10][11][12].This likely adds to more intracellular accumulation of dFdCTP than ara-C, which may, in part, account for its distinct spectrum of preclinical and clinical action [13].The enzyme ribonucleotide reductase, which generates the deoxynucleotides necessary for DNA synthesis, is also inhibited by GEM.Several human tumour xenografts and a wide range of mouse solid tumours and leukaemias respond to GEM treatment [14].
Nanoparticles (NPs) have gained popularity as potential drug carriers in cancer therapy during the past two decades due to their enhanced permeability and retention (EPR) effect, which causes them to accumulate in cancer tissues [15][16][17].Furthermore, NPs can passively and actively target specific types of cells or tissues, modify and overcome multidrug resistance in vitro, and have a wide range of potential applications [18].However, the buildup of nanosized carriers in the liver following intravenous (i.v.) administration significantly decreases the tumour site's dosage [19].Nanomaterials have emerged in the last several decades as a promising platform in biomedicine, potentially significantly advancing the treatment and diagnostics of various illnesses because of their unique properties [20].Metal-organic frameworks (MOFs) are one type of porous material that has been intensively studied for their potential in biomedical applications, specifically in generating innovative drug formulations with enhanced biological performance compared to standard medications [21].High porosity, tunable surface properties, excellent thermal and mechanical stability and exceptional chemical stability due to high resistance to alkaline water and organic solvents are just some of the desirable properties of zeolitic imidazolate frameworks (ZIFs), a subunit of metal-organic frameworks (MOFs) [22].For these reasons, ZIFs have shown great promise for use in the gas collection, separations, chemical sensors, drug delivery and catalysis fields.From a structural peak, ZIFs are built by coordination between imidazole (Im) anions and M 2+ cations, with Im as a linker to form connecting bridges between the M(Im)4 tetrahedral units of metal centres [23].Most ZIFs are made via solvothermal techniques, either in an organic solvent or water.The functionality of ZIFs may be manipulated by modifying the linkers between the components or by enclosing guest species (such as nanoparticles (NPs)) inside the ZIFs [24].In addition, ZIFs' pore size may be easily adjusted, which allows for controllable molecular mass/ diffusion transfer and the loading of substantial payloads.ZIFs have found several new uses, including catalysis research and pharmaceutical delivery [25].To create pH-responsive drug delivery systems, ZIFs are of great interest due to their stability under physiological settings and their pH-dependent degradability under acidic conditions (DDSs).As a result of the somewhat acidic nature of the TME, ZIFs and ZIF-8, in particular, have been studied extensively as a nanocarrier for cancer ablation, both in vitro and in vivo [26].Furthermore, much study has been devoted to developing innovative multifunctional ZIF-based composites for cancer therapy, antimicrobial applications, bioimaging and theranostics [27][28][29].ZIF-8, as one of the main subgroups of ZIF nanomaterials, has numerous applications thanks to the ease with which Zn 2+ and 2-methylimidazole (2-MeIm) can be polymerized around a wide range of objects, such as drugs, NPs and bio-macromolecules, to confer on them multifunctionality while maintaining the structural crystallinity and porosity of the ZIF shell [30].Therefore, the current investigation has emphasized the efficient one-step construction of GEM-encapsulated zeolitic imidazole frameworks for lung cancer treatment.

Fabrication of GEM@ZIF-L nanoparticles
ZIF-L was synthesised after a prior study, and minor adjustments were made [31][32][33].This technique used a 1:3 combination of Zn(NO 3 ) 2 and 2-methyl imidazole.Separately, 45 mL of double-distilled water was used to dissolve Zn(NO 3 ) 2 .6H 2 O (0.1 g) and 2-methyl imidazole (0.3 g).After swirling Zn(NO 3 ) 2 (0.1 g) in 45 mL of water for 15 min at RT (room temperature), we slowly added 45 mL of 2-methyl imidazole (0.35 g/50 mL) while continuing to stir.The production of zeolitic imidazole frameworks was indicated by the appearance of a white sponge-like colloid after 30 min of churning.The final step was adding a 2 mg/mL GEM solution in a volume of 10 mL while stirring the mixture with a magnet.The agitation continued for an additional hour to get the desired yellow tint.After being centrifuged to separate it, the residue was rinsed three times with double-distilled water and dried at 60 °C for 6 h (Figure 1).
The yield of the synthesis of GEM@ZIF-L nanoparticles was 160 mg, with a pale-yellowish colour.Following the previous method [33], a ZIF-L nanocarrier devoid of the drug was constructed.The absorbance at 280 nm in a UV-Vis spectrophotometer was used to determine the quantity of GEM loaded in ZIF-L nanoparticles.The drug loading efficiency of GEM@ZIF-L was assessed by hydrochloric acid (HCL) and ethanol decomposition methods.GEM quantification was achieved with the use of a GEM calibration curve.

Characterization studies
UV − Vis absorption spectra were noted by a UV-3101PC spectrometer (Shimadzu, Tokyo, Japan).Fourier transform infrared spectroscopy (FTIR) was obtained with a Nicolet 67 spectrometer (Thermo Nicolet).The crystallography of GEM@ZIF-L NPs was analyzed using a mini-X-ray diffractometer with Cu Kα radiation (λ = 1.5418Å, Japan).The morphology and structure of the as-fabricated GEM@ZIF-L NPs were examined using field emission transmission electron microscopes (TEM, Tecnai G2 20, Thermo) and field-emission scanning electron microscopy (SEM, ZEISS Sigma 300).Dynamic light scattering (DLS, Zetasizer Nano S, UK) was employed to establish the distribution of the hydrodynamic size of GEM@ZIF-L NPs.Fluorescence microscopy OLYMPUS X71 was used for fluorescence imaging.

Cell culture and CCK-8 cell viability assay
Human lung cancer A549 and H1299 cells and noncancerous mouse embryonic fibroblast NIH3T3 cells were purchased from China Center for Type Culture Collection (CCTCC) and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin.The cells were incubated under standard culture conditions (20% O 2 , 5% CO 2 , 37 °C).
The A549 and H1299 cells were treated with ZIF-L, GEM and GEM@ZIF-L before the CCK-8 assay to study the effect on cell viability.Cells (5 × 10 3 cells in 100 μL of RPMI-1640 medium each well) were planted in a 96-well plate.After incubation for 24 h, the culture medium was withdrawn, and the new medium containing ZIF-L, GEM and GEM@ZIF-L loaded nanoparticles was added (5 mg/mL of NPs in 100 μL of medium per well).After 48 h, CCK-8 was added (10 μL per well).After 4 h, the absorbance of the solution was assessed at 450 nm by a microplate reader (Bio-Rad 550, USA).The % growth inhibition has been determined using the following formula [34][35][36][37].

Cell viability
Absorbance of eacwell Absorbance of control w % /

Morphological changes in the cells (AO-EB and DAPI staining)
Targeting tumour cells and activating their programmed cell death is the keen purpose of potent anticancer drugs.Dual AO-EB staining analysis was achieved to point out the fundamental mechanism of cytotoxicity and antiproliferative properties of the fabricated nanoparticles.A549 and H1299 cells (1 × 10 5 cells of RPMI-1640 medium each well) were planted in a 48-well plate.After incubation for 24 h, the culture medium was removed and 2 mL of fresh medium containing ZIF-L, GEM and GEM@ZIF-L was added (IC 50 concentration).After 24 h, the cells were collected and stained with acridine orange and ethidium bromide (AO-EB) for 15 min at 37 °C.Finally, they were rinsed with PBS to remove the extra dye and examined using a fluorescence microscope.Then, the morphological changes were tested using the AO-EB Kit according to the manufacturer's protocol [38][39][40][41].
DAPI nuclear staining method was performed to examine nuclear morphologic characteristics.A549 and H1299 cells (1 × 10 5 cells of 1640 medium per well) were planted in a 48-well plate.After incubation for 24 h, the medium was removed and 2 mL of fresh medium containing ZIF-L, GEM and GEM@ZIF-L was added (IC 50 concentration).After 24 h, the cells were collected and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at 37 °C.Finally, they were rinsed with PBS to remove the extra dye and examined using a fluorescence microscope [42][43][44].Then, the nuclear changes were tested using the DAPI Kit according to the manufacturer's protocol.

Cell apoptosis assay
Conversely, precise quantification of apoptosis is essential to understand better the function and effectiveness of potential anticancer drug candidates.Apoptosis can be assessed quantitatively via the double staining using annexin V-FITC and PI (propidium iodide).A549 and H1299 cells (2 × 10 5 cells in 2 mL of 1640 medium per well) were seeded in a 6-well plate.After incubation for 24 h, the medium was removed, and 2 mL of fresh medium containing ZIF-L, GEM and GEM@ZIF-L was added (IC 50 concentration).After 24 h, the cells were collected, stained by an Annexin V-FITC/PI Staining Assay Kit [45], and analyzed by flow cytometry (Dakewe EXFLOW-206, Shenzhen, China).

Caspase activity assay
A chromogenic assay using caspase-3, caspase-8 and -9 colorimeter activity assay kits determined the caspase activity assay.A549 and H1299 cells (2 × 10 5 cells per well) were seeded in a 6-well plate.After incubation for 24 h, the medium was removed, and 2 mL of fresh medium containing ZIF-L, GEM and GEM@ZIF-L was added (IC 50 concentration), the cells lysed with cell lysis buffer (50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 100 mM EDTA) followed by centrifugation at 10,000 rpm for 1 min.About 50 mL of supernatant was incubated with the specific substrate (at 37 °C) for 2 h in a water bath.The absorbance of the cleaved substrate was measured at 405 nm using a microtiter plate reader (Bio-Rad 550, USA).

Statistical analysis
All data were presented as mean ± standard deviation (SD).The experimental results were analyzed by one-way analysis of variance (ANOVA) by using GraphPad Prism software.p-value < .05,.01 and .001were considered statistically significant difference and marked with *, ** and ***, respectively.

Characterization studies
ZIF-L and GEM@ZIF-L nanoframeworks are crystalline when analyzed by powder X-ray diffraction (PXRD) (Figure 2).Diffraction peaks at 15.73°, 16.58°, 21.48°, 21.356°, 27.44°, 30.46° and 35.62° were shown in ZIF-L, and they correspond to the (002), (101), ( 102), (103), ( 201) and (104) Bragg's reflection planes.In line with JCPDS: 01-1136, ZIF-L was discovered to have a specific hexagonal crystal structure, as evidenced by a distinctive reflection at 2 values and planes.Consistent with the findings of Yao et al. the XRD pattern of ZIF-L was also consistent [46].Diffraction peaks were shown in the PXRD pattern of GEM@ZIF-L that were not displayed in the pattern of ZIF-L alone.The XRD spectrum of GEM matched the report of Zhang et al. [47], with the addition of diffraction peaks at 2θ values of 16.22°, 19.00°, 23.48° and 40.82°, corresponding to the (101), (111), ( 112) and (404) planes, respectively, demonstrates that GEM is uniformly encapsulated inside ZIF-L, providing evidence of the emergence of a nanoparticle.The PXRD spectra of the nanoparticles closely matched that of JCPDS: , showing that the nanoparticles had a rhombohedral crystal structure.It was determined that the crystalline diameters of ZIF-L and the nanoparticles were around 44.18 and 45.18 nm, respectively.GEM encapsulation within ZIF-L appeared to upset the crystallinity of the ZIF-L nano framework, as evidenced by the structural instability of the resulting nanoparticles.
Comparing the FTIR spectra of ZIF-L and GEM@ZIF-L, we found they were quite comparable (Figure 3).The large area of the IR spectra between 3400 and 3100 cm −1 indicated that the O-H bond was being stretched.C-H stretching of the imidazole ring and the methyl group in 2-methyl imidazole were responsible for IR spectral bands at 3136 cm −1 and 2927 cm −1 .There was a stretch in the C-N bond, which was reflected in the 995 cm −1 band.The prominent bands exhibited the C-O and C = C bond stretching at 1116 and 1659 cm −1 .During the formation of the ZIF-L nano-frameworks, the stretching of Zn-N bonds between the zinc atoms of ZIF-L and the nitrogen atoms of the 2-methyl imidazole linker was shown as prominent IR band at 428 cm −1 , 755 cm −1 .Slight changes in the FTIR spectrum of GEM@ZIF-L might be due to the encapsulation of GEM within ZIF-L frameworks.
A colour change was first noticed in GEM@ZIF-L from white to a pale yellow in water as a solvent, later confirmed by UV-Visible spectroscopy (Figure 4).There was an absorption band at approximately 280 nm for GEM and approximately 224 nm for pure ZIF-L.The absorption band demonstrated uniform encapsulation of GEM inside ZIF-L   frameworks at approximately 224 nm for GEM@ZIF-L.One-pot synthesis often does not allow observation of the individual spectra of the enclosing molecules.
The drug loading efficiency of GEM@ZIF-L was assessed by hydrochloric acid (HCL) and ethanol decomposition methods.The drug loading capacity (DLC) of CA@ZIF-L was calculated to be 17.24% [48].
Thermogravimetric analysis was performed on both ZIF-L and GEM@ZIF-L to determine thermal stability.Thermogravimetric analysis showed that ZIF-L nanoparticles retained their bulkiness after being heated to 500 °C.Thermogravimetric analysis (TGA) of the uncoated ZIF-L revealed two zones of weight loss with a little change in overall mass at temperatures of 100-500 °C and 500-800 °C, respectively.At 500 °C, the loss of 5.56% of ZIF-mass L's revealed that water molecules had been removed.At temperatures between 500 °C and 800 °C, the deformation of ZIF-L frameworks was visible in the second, extended plateau zone, where weight loss averaged 30.13% (Figure 5).According to TGA, the GEM@ZIF-L nanoparticles had three distinct zones of weight loss between 100 °C and 220 °C, 220 °C and 400 °C and 400 °C and 800 °C (Figure 5).Removal of water molecules from GEM@ZIF-L frameworks was initially shown by a 6.25% weight reduction in the 100-220 °C temperature range.At temperatures between 220 °C and 400 °C, GEM in GEM@ZIF-L was decomposed, as evidenced by a second weight loss of 30.55%.At temperatures between 500 °C and 800 °C, the ZIF-L frameworks completely deformed, resulting in a further 19.00%mass decrease.
SEM and TEM analyzed the GEM@ZIF-L nanoparticles to determine their size and shape as prepared.The GEM@ZIF-L crystals were homogeneous in size and shape when viewed using a scanning electron microscope (Figure 6A), suggesting a two-dimensional leaf-like crystal morphology.Two-dimensional leaf-like zeolitic imidazole frameworks with cushion-shaped voids were shown in the crystal morphology of GEM@ZIF-L during the room-temperature synthesis, which was found to be in excellent agreement with the earlier result by He et al. [49].Nanoscale flakes with an average particle diameter of approximately 120 nm were visible in the TEM image of GEM@ZIF-L (Figure 6B).Anticancer properties improved in the nanoscale 2D constructed GEM@ZIF-L.Compared to other zeolitic imidazole frameworks, such as ZIF-95 and ZIF-100, ZIF-L has a larger surface area and greater porosity, making it a promising option for use in biomedicine.
Their size profoundly affects nanoparticle absorption by cells and subsequent movement through biological membranes.A previous study found that the average particle size of the GEM@ZIF-L nanoparticles was 220 nm, suggesting the nanoparticles may be taken up efficiently by cells (Figure 6C).The GEM@ZIF-L nanocomposite was measured to have a negative zeta potential of −6.7 mV.In solution, the GEM@ZIF-L nanoparticles were spread evenly, and the particles did not aggregate due to electrostatic repulsion.

In vitro cytotoxicity of the fabricated NPs
The evaluation of the anticancer efficacy of the ZIF-L, GEM and GEM@ZIF-L has been initiated with the cell counting lit-8 (CCK-8) assay.All the Nanoparticles were assessed against A549 and H1299 (lung carcinoma) and NIH3T3 (noncancerous mouse embryonic fibroblast) cells by the CCK-8 assay at different concentrations for 48 h.The results reveal that ZIF-L did not show cell proliferation even at a high concentration of 500 μM.GEM@ ZIF-L exhibit significantly higher cytotoxic activity than the ZIF-L, which could be attributed to GEM encapsulation to the ZIF.In A549 cells, the cytotoxicity (IC 50 values) of the ZIF-L, GEM, GEM@ZIF-L and standard drug cisplatin was found to be 47.68 ± 3.25 µg/mL, 16.32 ± 2.21 µg/mL, 5.78 ± 1.49 µg/mL and 18.11 ± 3.01 µg/mL, respectively (Figure 7A).In H1299 cells, the cytotoxicity (IC 50 values) of the ZIF-L, GEM, GEM@ZIF-L and standard drug cisplatin was found to be 43.09± 6.54 µg/mL, 14.19 ± 3.58 µg/mL, 5.47 ± 2.90 µg/mL and 21.50 ± 5.14 µg/mL, respectively (Figure 7B).The results above reveal that the newly fabricated nanoparticles have lower inhibitory dose than the cisplatin.In addition, the samples of ZIF-L, GEM, GEM@ZIF-L and standard drug cisplatin were treated with noncancerous NIH3T3 cells, showing high cytocompatibility (Figure 7C).The results highlight the significance of GEM@ZIF-L as it promisingly kills cancer cells without affecting noncancerous cells.

Morphological changes by AO-EB assay
Therapeutic anticancer drugs may aim to kill cancer cells by stimulating their apoptotic process.Alterations in cell morphology and discovering apoptotic cell death can be shown using dual labelling using nucleic acid binding dyes like acridine orange (AO) and ethidium bromide (EB), which rely on fluorescence emission to identify the phenomenon.This reveals the mechanism by which synthetic complexes inhibit cancer cell proliferation and kill them.Changes in cell structure, such as cytoplasmic shrinkage, nuclear condensation, plasma membrane blebbing, DNA breakage and translocation of phosphatidylserine to the extracellular side, are diagnostic of cells that have been induced to undergo apoptosis [50][51][52].Both live and dead cells are stained with acridine orange during the fluorescence staining procedure.Ethidium bromide, conversely, stains cells with compromised membranes.Through a fluorescence microscope, living cells appear green.Although they stain red, necrotic cells have a nucleus structure not dissimilar to that of healthy ones.Cells undergoing the morphological alterations linked with apoptosis appear as reddish-orange areas.Our experiment's control cells are alive, have a normal, well-ordered structure, green shine and consistently emit green fluorescence.The A549 and H1299 cells stained with AO-EB were exposed to the ZIF-L, GEM and GEM@ ZIF-L.By disrupting the membrane of cancer cells, the novel chemicals promoted apoptosis, with EB penetrating the cells to obscure AO fluorescence and generate a greenish-orange stain.ZIF-L, GEM and GEM@ZIF-L, as found in Figure 8A, trigger cell death.GEM@ZIF-L shows a high level of apoptosis compared to the free ZIF-L and GEM.The quantified ratio of the morphological changes during cell death is shown in Figure 8B.

DAPI staining assays
The AO-EB experiment demonstrated that apoptotic cell death was indeed triggered.The DAPI staining experiment was performed on A549 and H1299 cells with all the ZIF-L, GEM and GEM@ZIF-L to validate the apoptotic mechanistic route behind the anticancer action.Following 24 h of treatment with the IC 50 of all ZIF-L, GEM and GEM@ZIF-L, the cells' activity was assessed by observing for any changes in the nucleus's morphology.The fluorescence of the control cells is relatively low under a fluorescence microscope.Cells treated with the named complexes glow blue in specific locations, revealing morphology indicative of apoptotic cell death, such as chromatin condensation and nucleus fragmentation.Nuclear damage and apoptosis induction were displayed for all synthetic complexes under identical circumstances.Apoptosis was triggered more by GEM@ZIF-L than by ZIF-L and GEM (Figure 9A).The quantified ratio of the nuclear changes during cell death is shown in Figure 9B.

Quantification of cell apoptosis by flow cytometry
ZIF-L, GEM and GEM@ZIF-L were shown to induce apoptosis in A549 and H1299 cancer cells in prior staining studies of in this work.Therefore, flow cytometry with the annexin V-FITC/propidium iodide double-staining approach employing ZIF-L, GEM and GEM@ZIF-L has been used to assess the quantification of apoptosis (Figure 10A).When a cell undergoes apoptosis, phosphatidylserine (PS), a phospholipid involved in cell cycle signalling, is moved from the interior of the membrane to the outside [53][54][55].As a  result, the interaction of fluorescently tagged annexin V, which flow cytometry detects, reveals that PS has been displaced, a morphological feature of apoptosis.ZIF-L, GEM and GEM@ZIF-L at their IC 50 concentration with A549 and H1299 cells induce various apoptotic cell death.The result proves the high level of apoptotic cells with GEM@ZIF-L compared to ZIF-L and GEM.These outcomes of the staining model and flow cytometry investigation reveal that the GEM@ZIF-L nanoparticles effectively induce apoptosis in A549 and H1299 lung cancer cells.The quantified ratio of the apoptosis is shown in Figure 10B.

Assessment of caspase-3/8/9 activation
The apoptotic cell death was confirmed by caspase activation by ELISA analysis.In eukaryotes, caspases (protease enzymes) play a significant role in amplification, transduction and induction of intracellular apoptotic signals.Based on the amino acid composition, caspases are separated into three subfamilies: mediator, executioner and activator.Relevantly, the executioner and initiator caspases perceive within the apoptotic signalling cascade.Caspase-8 and -9 are the initiators of the extrinsic and intrinsic cell death pathways, respectively.Both mechanisms converge in the activation of the executioner caspase-3.Hence, the IC 50 concentration of ZIF-L, GEM and GEM@ZIF-L at 24 h were treated on A549 and H1299 cells with caspases-3, -8 and -9.The outcomes confirmed that GEM@ZIF-L efficiently upregulated the expression caspase-8 among other caspases-3 and -9 for triggering cancer cell apoptosis.The results also showed a GEM@ZIF-L increase in caspase activity than control cells (Figure 11).

Conclusion
In the current work, the encapsulation of the GEM within developed ZIF-L was found.Compared to conventional chemical synthesis techniques, the novel room-temperature fabrication of GEM@ZIF-L offers significant advantages in speed, reliability and environmental friendliness.GEM@ZIF-L showed a dose-dependent reduction of lung cancer cells  (A549 and H1299).In addition, GEM@ZIF-L exhibited excellently induced apoptosis, and it was confirmed by various fluorescence staining methods (such as acridine orange and ethidium bromide (AO-EB and nuclear DAPI staining).The present work highlights the possibility of employing the multifunctional GEM@ZIF-L nanoparticles as a suitable material for biomedical applications.Interestingly, GEM@ZIF-L is more potent in A549 and H1299 cells.AO-EB and DAPI fluorescent staining confirm that the GEM@ZIF-L triggers apoptosis in A549 and H1299 cells.Further, the quantification of apoptosis was determined with flow cytometry analyses.In addition, the apoptotic mode of cell death was confirmed by caspase-8 enzyme activation.The cancer target specificity and apoptosis induction behaviours of the present GEM@ZIF-L open the prospect of promoting efficient GEM@ZIF-L anticancer drug candidates.

Figure 1 .
Figure 1.Graphical representation of fabrication process of Zinc nitrate hexahydrate (Zn(no 3 ) 2 .6H 2 o) and 2-methyl imidazole to obtain Zif-l and encapsulation of anticancer drug gemcitabine (GEm) termed as GEm@Zif-l for this work.

Figure 7 .
Figure 7. (a, B) cell viability effects of Zif-l, GEm and GEm@Zif-l on a549 and H1299 cells after 48 h. the figure shows that Zif-l, GEm@Zif-l and GEm caused cell cytotoxicity dose-dependently.(c) cell viability effects of Zif-l, GEm and GEm@Zif-l on niH3t3 (noncancerous) cells after 48 h.Bars represent the mean ± standard deviation of individual experiments performed in triplicate.Graphpad prism 8.0 was used to evaluate statistical significance.p-value < .05,.01 and .001were considered statistically significant difference and marked with *, ** and ***, respectively.

Figure 8 .
Figure 8.(a) microscopic images of a549 and H1299 cells with the ao-EB staining after treatment with ic 50 concentration of Zif-l, GEm and GEm@Zif-l.(B) Quantification analysis of Zif-l, GEm and GEm@Zif-l.scale bar 100 µm.Bars represent the mean ± standard deviation of individual experiments performed in triplicate.Graphpad prism 8.0 was used to evaluate statistical significance.p-value < .001were considered statistically significant difference and marked with ***, respectively.

Figure 9 .
Figure 9. (a) microscopic images of a549 and H1299 cells with the Dapi staining after treatment with ic 50 concentration of Zif-l, GEm and GEm@Zif-l.(B) Quantification analysis of Zif-l, GEm and GEm@Zif-l.scale bar 100 µm.the arrow reveals that nuclear damages the cells.Bars represent the mean ± standard deviation of individual experiments performed in triplicate.Graphpad prism 8.0 was used to evaluate statistical significance.p-value < .001were considered statistically significant difference and marked with ***, respectively.

Figure 10 .
Figure 10.(a) flow cytometry analysis of a549 and H1299 cells with the Dapi staining after treatment with ic 50 concentration of Zif-l, GEm and GEm@Zif-l.(B) Quantification analysis of Zif-l, GEm and GEm@Zif-l.Bars represent the mean ± standard deviation of individual experiments performed in triplicate.Graphpad prism 8.0 was used to evaluate statistical significance.p-value < .001were considered statistically significant difference and marked with ***, respectively.

Figure 11 .
Figure 11.Effect of Zif-l, GEm and GEm@Zif-l on caspase-3, -8, -9 in a549 and H1299 cells by the Elisa assay for 24 h.Bars represent the mean ± standard deviation of individual experiments performed in triplicate.p-value < .05 and .01 were considered statistically significant difference and marked with * and **, respectively.