3.2. Powder XRD AnalysisThe powder XRD spectra of [email protected] ZIF-L nanoframeworks reveals the crystalline nature of the prepared material. Sharp diffraction peaks at around 15.04°, 16.86°, 21.86°, 24.65°, 27.84°, 29.17°, 30.72°, 32.45°, 35.33°, 36.71°, 40.97°, 43.30° and 46.13° corresponding to (002), (101), (102), (110), (103), (200), (201), (004), (104), (203), (114), (204) and (213) Braggs reflection planes (Figure 2). According to JCPDS No. 01-1136, diffraction peaks at 2θ values and planes represent the two-dimensional flake-like crystal structure. Scherer′s formula has been used to calculate the crystalline size of [email protected] ZIF-L nanoframeworks, which is approximately 48.26 nm. 3.3. FTIR AnalysisThe inclusion of folic acid and methyl gallate active groups in bare Folate ZIF-L and [email protected] ZIF-L was confirmed by FTIR spectroscopy. The FTIR spectra of [email protected] ZIF-L and Folate ZIF-L are shown in Figure 3. With a few additional peaks that show the loading of methyl gallate, both are remarkably comparable. The Zn-N stretching vibrations’ peaks at 396 and 662 cm−1 correlate to the synthesis of ZIF-L between zinc and imid-azole. The C-N stretching vibration of imidazole linkers in Folate ZIF-L and [email protected] ZIF-L nanoframeworks was confirmed by intense peaks at 974 cm−1, 1125 cm−1, and 1154 cm−1. Stretching vibrations of the C=O molecule correspond to the spectral bands at 1554 and 1658 cm−1. The N-H group of the imidazole linker is shown by a tiny peak at about 2910 and 2933 cm−1. The O-H bending vibration of the Folate ZIF-L and [email protected] ZIF-L nanoframeworks is responsible for peaks at about 3385 and 3547 cm−1. 3.6. pH-Responsive Drug Releasing Mechanism of [email protected] ZIF-L NanocompositeStimuli-based drug delivery techniques overcome the drawbacks of classical methods, by efficient drug release on specific cancer sites, hence pH-responsive is a trusted and simple technique for cancer drug delivery application. In zeolitic imidazole frameworks, the organic linker imidazole and zinc iron covalent bonds get cleaved in acidic conditions [5,8,29]. Figure 6 shows the gradual increases in methyl gallate at acidic pH 5 and 6, which reached the stationary phase within the 39 h without changes up to 52 h. However, at the normal physiological pH of 7.4, a very minimum drug release was observed within the transit time. At neutral pH of 7.4, the [email protected] ZIF-L showed a stable and uniform drug release capacity of 13.71%. When the pH of the medium was reduced to acidic conditions 5 and 6, the drug release capacity was gradually raised to 76.32 and 59.81% in the short period of 39 h (Figure 6). The enhanced drug release at lower pH indicates the cleavage of zinc and imidazole bonding from ZIF-L frameworks. Present results revealed that an [email protected] ZIF-L nanoframework is a suitable drug delivery system for pH-responsive anticancer treatments. 3.7. Cytotoxicity of Folate ZIF-L Nanocomposite on Artemia salinaA. salina nauplii lethality assay is one of the reliable alternatives for MTT and other in vivo models. In this present evaluation, synthesized nanoframeworks revealed that the toxicity of [email protected] ZIF-L nanoframeworks is more biocompatible. The cytotoxicity of [email protected] ZIF-L nanoframeworks was quantified from death and survival of treated nauplii (Figure 7). The LC50 was calculated to be 118.43 ± 2.52 µg/mL. The growth inhibition and morphological changes of A. nauplii were identified under the inverted microscope, which shows much less toxicity and no significant changes in morphology and growth up to 125 µg/mL some deaths appeared. Our toxicity study revealed that [email protected] ZIF-L nanoframeworks exhibited non-toxic behavior against A. salina at lower concentrations, which shows mild toxicity in higher doses (125 µg/mL). There is no significant modification in the swimming behavior of treated nauplii at the end of 48 h of testing duration. 3.8. Anti-Biofilm Activity of [email protected] ZIF-L NanoframeworksStaphylococcus aureus is a more dreadful clinical pathogen affecting humans, causing cruel infections and life-threatening diseases. The biofilm forming potential of MRSA provides antibiotic resistance to bacterial cells [30,31,32]. It is associated with unhygienic medical procedures like the use of impure surgical devices and touching people in the crowd. Advancement in the nanomedicine field has diverted the attention of nanotechnology researchers toward the annihilation of bio-film-causing bacteria via nano formulation [33,34]. The anti-biofilm potential of methyl gallate encapsulated Folate ZIF-L was assessed by the measuring color intensity of crystal violet present on the treated cells of MRSA (control—clinical strain—N7, ATC C MRSA 33591). Figure 8 illustrated that [email protected] ZIF-L nanoframeworks prevent and eradicate MRSA 33591 and clinical strain N7 bio-film formation depending on the doses. Figure 9 showed the quantification of biofilm inhibition by the nanoframeworks at different concentrations, which concluded that [email protected] ZIF-L efficiently controls the bacterial growth rate. Methyl gallate encapsulation within Folate ZIF-L enhanced the anti-biofilm efficacy of Folate ZIF-L by the adhesion of MRSA and N7 biofilm surfaces, thereby eradicating bio-film formation. 3.10. Anticancer Potential of [email protected] ZIF-L NanoframeworksLung cancer is a death-dealing form of human disease worldwide. Current therapeutic techniques such as chemotherapy, radiation, and surgery have increased the risk to patient health. Due to these classical treatment strategies, the patient immune system is damaged by the drugs, which leads to side effects. Recently, biomedical researchers have had a great interest in the uses of targeted drug delivery methods in cancer treatments [35,36,37,38]. Today, several synthetic drugs are available in the market for lung cancer cells targeting epidermal growth factor receptor (EGFR). These EGFR inhibits cancer cell growth and delays the spreading of infected cells. In some lung cancer cases, cancer cells developed resistance to epidermal growth factor receptor (EGFR), and these problems promoted a need for new drug development [39,40,41,42].In this study, ZIF-L as a folic acid receptor bonded carrier for methyl gallate molecule delivery depends on the pH-responsive condition. By using the MTT assay on A549 lung cancer cells, the anti-proliferative effectiveness of free methyl gallate, Folate ZIF-L, and [email protected] ZIF-L was determined. At the maximum dose (100 mg/mL), the results showed that Folate ZIF-L had a minimal cytotoxic impact. In this context alone, methyl gallate and [email protected] ZIF-L show reliable inhibition at the same concentration, which increases cytotoxicity with an IC 50 value of 62.32 ± 0.08 mg/mL, respectively (Figure 10). Folate ZIF-L nanocomposite exhibits higher cytotoxicity at the highest dose of 100 mg/mL, when compared to the same dose of methyl gallate encapsulated Folate ZIF-L nanoframeworks, revealing the fact that Folate ZIF-L retains the lethality effect of methyl gallate incorporation. Structural changes in the A549 cells treated with [email protected] ZIF-L nanoframeworks were identified by inverted phase contrast microscopy. Results concluded that (Figure 11B) both methyl gallate and [email protected] ZIF-L treated cells exhibit membrane damages and cell shrinking with condensed chromatin, while the control cells exhibited normal cell morphology, and Folate ZIF-L treated cells show mild changes in cell morphology. The [email protected] ZIF-L treated cells showed effective depletion in cell counting compared to the control group.Nuclear morphological changes of [email protected] ZIF-l treated and control cells were assessed from the Hoechst 33342 staining assay. Control cells show regular cell structure and spherical nuclei. However, Folate ZIF-L and methyl gallate treated cells showed mild alteration in the nuclei region. Figure 11B exposed high blue fluorescence, which indicates the fragmented nuclei. At the same time, [email protected] ZIF-L induces condense nuclei leading the apoptosis. The percentage of normal and abnormal nuclei was evaluated by the quantification method. Figure 11A shows [email protected] ZIF-L and methyl gallate treated cells showed 83.10 ± 1.32% of abnormal and 17.013 ± 1.31% of normal nuclei cells; when compared to control and Folate ZIF-L, the nuclear damage rate is higher in [email protected] ZIF-L treated cells. Overall, the results confirmed that the [email protected] ZIF-L treated cells show a high level of ROS generation, provoking oxidative stress-mediated genotoxicity and mitochondrial dysfunction ultimately leading the apoptosis process.Anticancer efficacy of Chemotherapeutics depends on the generation of reactive oxygen species ROS mediated cell death mechanism. However, a variety of new drugs designing and treatment methods are based on targeting intracellular ROS levels of cancer cells [43,44]. Based on this concept, the generation of intracellular ROS level of lung cancer cells was assessed by DCFDH-DA fluorescence assay. Results revealed that the [email protected] ZIF-L treated cells generate more cells DCF fluorescence intensity compared to Folate ZIF-L and vehicle control (Figure 12B). Spectrofluorimetric results concluded that the [email protected] ZIF-L treated cells had five-fold increases in fluorescence intensity compared with control cells and a threefold increase compared to methyl gallate alone (Figure 12A). The releasing of methyl gallate and folic acid molecules leads cell wall damage and increases the intracellular ROS level.