The manufacture of CZIF-8 nanocomposites was based on the basic principle that curcumin is soluble at different pH values31.
It was more potent than doxorubicin in HepG-2 cells and comparable in MDA-MB-231 cells.
Likewise, CZIF-8/HA caused more evidence on G1 arrest and a slight decrease in S phase cells compared with those of the untreated cells.
In this regard, CZIF-8/HA formulation induced a more pronounced apoptotic response in MDA-MB-231 cells compared to HepG-2 cells.
Similarly, in MDA-MB-231 cells, caspase-3 protein level was elevated after treatment with both nanocomposites compared to control cells.
The manufacture of CZIF-8 nanocomposites was based on the basic principle that curcumin is soluble at different pH values31. The following steps were taken in order to improve the encapsulating of curcumin in nanoparticles forms. Firstly, the pH was elevated to an alkaline level, which dissolved the curcumin; next, the pH was dropped to an acidic level, which precipitated the curcumin. Consequently, curcumin was directly dissolved in 2-methelyimidazole aqueous solution as was carried out in this study at pH around 1132. Zinc ions were then added to ZIF-8 to create CZIF-8 and then the resulted orange nanocomposites were coated with hyaluronic acid (HA) in order to increase hydrophilicity of curcumin and its dispersity in aqueous medium. A molar ratio of 1:100 (zinc to 2-methylimidazole) was employed in the production of CZIF-8. The substantial excess of ligand is crucial for shifting the equilibrium towards the development of the zeolitic framework while inhibiting the emergence of impurity phases like Zn (OH) 2 33.The basic environment was established utilizing 2-methylimidazole itself which function as both the organic linker and the base. While OH ions are present and the rapid coordination between Zn²⁺ ions and imidazole nitrogen is significantly stronger than that of Zn(OH) 2 under these specific molar ratio between imidazole and zinc ions (1:100).Thus, ZIF-8 framework was formed over Zn(OH)₂ precipitation, as widely reported for ZIF-8 synthesis34.
The capacity of the encapsulated curcumin was determined to be 3.32% for CZIF-8/HA, which is slightly lower than the value for CZIF-8 is 4.36%, similar to results obtained by Li et al.19 and Yu et al.17.The elevated DLC% and EE% values for CZIF-8 suggest minimal curcumin loss during the surface modification with HA, attributable to the inherent structural properties of ZIF-8, such as its high porosity, extensive specific surface area, and well-defined pore architecture, which facilitate effective drug incorporation35. Previous studies have demonstrated that curcumin is predominantly contained within the ZIF-8 framework during its production. The diminutive pore size of ZIF-8 (~ 3.4 Å)36, which is considerably less than the molecular dimensions of curcumin, prevents the drug from diffusing into existing pores, resulting in its physical entrapment during the in situ crystallization process. Alongside size constriction, secondary noncovalent interactions especially π–π stacking between the aromatic rings of curcumin and the imidazole linkers of ZIF-8 enhance the stabilization of the encapsulated drug within the framework37.
In contrast, the decreased DLC% and EE% values noted for CZIF-8/HA align with findings on surface-modified or coated MOFs systems. The deposition of a hyaluronic layer on ZIF-8 particles may obstruct pore entrances or diminish the available pore volume, consequently restricting curcumin diffusion into the internal framework after loading19. Furthermore, the supplementary mass from the HA coating elevates the overall weight of the nanocomposite without a corresponding rise in curcumin content, leading to a diminished estimated DLC%. Comparable decreases in drug loading metrics after the polymeric or inorganic surface coating of metal-organic frameworks MOFs have been extensively documented12,38.
The resulted nanocomposites were characterized by Zeta potential, SEM, TEM, and PXRD. Zeta potential results suggested that, increasing negative surface charge in the CZIF-8/HA system is attributed to the presence of HA, which introduces additional negatively charged functional groups on the nanocomposites surface compared to CZIF-8 alone. Colloidal literature firmly establishes that absolute zeta potential levels beyond approximately 25–30 mV (either positive or negative) signify robust electrostatic stability against aggregation39. This enhances its stability in biological fluids, making it a more reliable and effective carrier for the delivery of curcumin.
TEM results confirms the Irregular semi-spherical morphology and nanoscale particle size (100–200 nm) of CZIF-8 align with the findings of Zheng et al.37, as ZIF-8 generally forms polyhedral or near-spherical nanoparticles contingent upon synthesis conditions. Furthermore, the encapsulation of hydrophobic drugs, such as curcumin, within the ZIF-8 framework has been demonstrated to maintain the overall morphology while augmenting particle size due to curcumin interactions within the pores and on the surface. Increasing aggregation and the slight alternation in particle morphology and average size observed in CZIF-8/HA nanocomposites as the addition of HA seems to affect the overall particle structure, ionic interactions and intrinsic surface chemistry. The presence of HA may be the cause of clustering. Because of its hydrophilic and bioactive properties, HA may be potentially have improved antiparticle interactions, which could explain the observed particle agglomeration, This is consistent with the interpretations provided by Li et al.19.As a result, improvement in stability, biocompatibility, and possible targeting qualities are expected. These results show the morphological changes brought about by the addition of hyaluronic acid and validate the effective creation.
SEM analysis indicates that the rough and porous morphology of CZIF-8 aligns with previously documented ZIF-8-based drug delivery systems, wherein the encapsulation of curcumin results in partial surface coverage and particle aggregation40.In contrast, HA incorporation modifies surface architecture by producing denser, less porous particles. This morphological transformation suggests successful HA integration into the ZIF-8 matrix, likely due to HA’s tendency to form amorphous or layered structures that adhere to and fill the pores of ZIF-8 particles41,42.
The observed PXRD peaks for CZIF-8 align with the broader, attenuated peaks observed in the crystal structure of ZIF-8 documented by Tiwari et al.24.The retention of peak locations upon curcumin encapsulation does not modify the long-range order of the ZIF-8 peaks (2θ = 7–20°), corroborating prior studies that indicate the structural stability of ZIF-8 upon the inclusion of small-molecule drugs. Post-HA modification, the PXRD pattern preserves all distinctive ZIF-8 reflections without any peak displacement; nonetheless, the noted decrease in peak intensity and broadening aligns with surface coating effects and partial amorphization caused by HA deposition, rather than structural failure. A comparable reduction in diffraction intensity without phase change has been documented for ZIF-8 composites covered with HA43.
Using MTT assay, curcumin nanocomposites were tested for their cytotoxic effect against HepG-2and MDA-MB-231 cancerous cells and MRC-5 normal cells. The cytotoxic effect of CZIF-8/HA is much greater than CZIF-8 in both cancerous cell lines; especially in the case of HepG-2.This results suggested that, the incorporation of HA into the ZIF-8 framework enhances anticancer effect of the HA-containing composite and this may be attributed to HA bioactivity, which could be facilitate cellular internalization and support the sustained release of curcumin. Our findings are nearly in agreement with those of Li et al.19, who demonstrated that the incorporation of HA enhanced the cytotoxicity of curcumin-loaded ZIF-8 nanocomposites across a wider concentration range (12.5–100 µg/ml). These inconsistencies highlight the possibility that even slight differences in nanocomposites preparation methods can result in significant difference in biological activity. In this study incorporation of hyaluronic acid was the case.
CZIF-8/HA nanocomposites showed significantly much lower IC₅₀ values (5.63 µg/mL and 9.15 µg/mL) respectively for HepG-2 and MDA-MB-231. It was more potent than doxorubicin in HepG-2 cells and comparable in MDA-MB-231 cells. Additionally, ZIF-8/HA loaded curcumin showed relatively lower toxicity to normal MRC-5 cells than the uncoated CZIF-8, indicating higher selectivity and a better therapeutic index. In our in vitro viability assay doxorubicin showed higher TI, but known in vivo toxicities. This is what we will examine in vivo on HepG-2 model.
Therapeutic index (TI) values show that CZIF-8 demonstrated relatively low TI values compared with HA coated nanocomposites, indicating limited selectivity especially for the breast cancer model. In contrast, functionalization with HA significantly enhanced the therapeutic index in both cell lines. This improvement can be ascribed to functionalization with HA, which is known to be selectively bind to CD-44 receptors overexpressed on the surface of various tumor cells32,42, this may be facilate receptor-mediated endocytosis and improving intracellular drug accumulation. Doxorubicin displayed the highest TI values among the tested treatments. These results highlight doxorubicin’s potent cytotoxicity and high cancer selectivity, particularly against MDA-MB-231 cells. So, further, in vivo study is essential to fully assess the biocompatibility and therapeutic index of these formulations.
Unchecked proliferation and continuous advancement are hallmarks of the cancer cell cycle. Any alteration in the control of the cell cycle phase causes cancer cells to proliferate quickly. Therefore, stopping the unchecked cell cycle at different points is essential for treating cancer44. In the current study, the cell cycle was studied in vitro on HepG-2 and MDA-MB-231 cells-treated with CZIF-8 and CZIF-8/HA and after it’s loading in an equivalent dose using the flow cytometric assay. The cell cycle assay results showed that, after treatment the HepG-2 cells with CZIF-8, a notable increase in G1 phase cells was observed with reduction in S phase cells and G2/M suggesting induction of G1 phase arrest. However, treatment with CZIF-8/HA also increased the G1 population but to a lesser extent than CZIF-8. On the other hand, a lower proportion of cells in the S phase indicate G1/S phase arrest and inhibition of DNA during synthetic phase. In case of MDA-MB-231 untreated cell, a high percentage of cells in S phase. After treatment with CZIF-8, a reduction of G1 phase cells was observed indicating G1/S-phase arrest. Likewise, CZIF-8/HA caused more evidence on G1 arrest and a slight decrease in S phase cells compared with those of the untreated cells. All together indicates that the HA coated nanocarriers was more effectively halted the tumor cell cycle before DNA synthesis. Our findings suggest that both nanocomposites interfered with the regular distribution of the cell cycle in HepG-2 and MDA-MB-231 cells because they may interact with intracellular nucleic acid and proteins, which could contribute to the observed effect; however, direct binding was not assessed in this study. Cell death results from both interaction with cellular proteins and the activation of certain apoptotic enzymes, such as caspase-345,46. However the effects varied depending on the phase. These diversities could be linked to the two cancer cell lines’ distinct intracellular trafficking strategies and the levels of HA receptor expression.
In order to delineate the mode of cell death which can occur through two major mechanisms apoptosis (programmed cell death) or necrosis (uncontrolled cell death), annexin V-FITC assay was used to demonstrate the cytotoxic effect of the nanocomposites on the tested cells. Our finding confirms that, treatment of HepG-2 and MDA-MB-231 cells with CZIF-8 and CZIF-8/HA led to a significant increase in apoptotic cells than necrotic ones. In this regard, CZIF-8/HA formulation induced a more pronounced apoptotic response in MDA-MB-231 cells compared to HepG-2 cells. The negligible apoptosis in the untreated cells clarify the ability of both nanocomposites to induce cell death via apoptosis mainly through early apoptosis not through necrosis.
Caspase- 3 is a crucial effector and most prevalent in family of caspases, widely concede for their triggered proteolytic functions in enforcing apoptosis in cells reacting to particular internal or extrinsic triggers of this type of cell death47. Apoptosis resistance is one of the most popular causes of carcinogenesis. Therefore, the majority chemotherapeutic agents mediate induction of apoptosis in cancer cells via different mechanisms. Thus, the dominant idea in many cytotoxic chemotherapeutic treatments for cancer has been the activation of caspases to cause apoptosis36,37, . In this study we use the ELISA technique to track the expression of the apoptosis-regulated caspase-3 protein. Our findings showed a significant increase of caspase-3 protein in both cell lines following treatment with curcumin-loaded nanocomposites, indicating the induction of apoptosis. In HepG-2 cells, caspase-3 baseline protein level of the untreated cells increases significantly with CZIF-8/HA and reached a maximum of with CZIF-8. This substantial elevation indicates a strong pro-apoptotic response, particularly with the ZIF-8 formulation lacking HA. These results suggest a more rapid or potent release of curcumin. Similarly, in MDA-MB-231 cells, caspase-3 protein level was elevated after treatment with both nanocomposites compared to control cells. This evaluation is low if compared to HepG-2 cells. However, CZIF-8 enhances apoptotic signaling, particularly in HepG-2 cells, where the response is more pronounced. HepG-2 cells’ greater apoptotic response in contrast to MDA-MB-231 might be the result of variations in innate sensitivity to curcumin-mediated apoptosis, internalization of nanocomposites, or cellular uptake efficiency. Interestingly, CZIF-8/HA preserved similar efficacy in MDA-MB-231 but marginally decreased caspase-3 activation in HepG-2, possibly as a result of HA-mediated targeting or possibly by inducing apoptosis through activation of another apoptosis-regulation genes48. Overall, our earlier findings indicate that the curcumin-loaded nanoformulations’ potential act as therapeutic agents for hepatocellular and breast carcinomas possibly via apoptosis induction and cell cycle arrest. A key limitation of the present study is the lack of in vitro drug release data and control experiments comparing curcumin delivery with and without ZIF-8 and ZIF-8/HA, which will be addressed in future work to more accurately correlate curcumin release with the observed cytotoxic effects and clarify the nanocomposites’ role.
