Facile engineering of chitosan-coated aminopterin loaded zeolitic imidazolate framework: promising drug delivery system for breast cancer

Abstract In this study, we have designed an anticancer drug delivery framework by a one-pot technique using a zeolitic imidazolate framework (ZIF) as the carrier. The chitosan-coated Aminopterin (AMT) loaded with zeolitic imidazolate framework (ZIF-90) CS@AMT@ZIF-90 (CAZ-90) for breast cancer cells. The particle’s outer layer changed Chitosan (CS), a pH-sensitive biomaterial, to increase the composite CAZ-90’s reactivity to pH during drug release. CAZ-90 displayed target-selective and pH-responsive by releasing a significant ratio of AMT under an acidic milieu and a considerably reduced amount of AMT in a normal milieu. Additionally, CAZ-90 was found to have low toxicity to normal Human umbilical vein endothelial cells (HUVECs) cells while inhibiting breast cancer 4T1, MDA-MB-231, and MCF-7 cells in vitro. Further, cell death was investigated by two different staining methods (AO-EB and DAPI nuclear staining). RNase-PI staining by flow cytometry techniques investigated the cell cycle arrest in breast (4T1, MDA-MB-231, and MCF-7) cancer cells. CAZ-90 showed high AMT drug loading, cancer-targeted release, and excellent biocompatibility in the related tests, making it a promising option for an anticancer drug delivery system.


Introduction
The World Health Organization (WHO) presently ranks cancer as the second leading cause of death.The WHO estimates that in poor and middle-income countries, cancer is the leading cause of death [1].However, breast cancer is the second most prevalent cancer overall and the leading cause of cancer death in women worldwide, after lung cancer.It typically begins in the breast, mammary, or lobular glands [2].Breast cancer tumors are typically non-malignant and thus amenable to surgical removal if caught early.The therapy is more difficult when the tumor is malignant because cancer can travel through the body's bloodstream or lymphatic system [3].Surgery, followed by drugs, radiation therapy, or both, might be a possibility for patients, depending on the severity of their tumor.There are three different classification systems for breast cancer [4].Grade 1 tumors develop slowly and rarely spread to other body areas, hence the name.Tumors of grade 2 are intermediate between grades 1 and 3, showing some degree of differentiation from normal cells [5].The third-grade malignancy is characterized by rapid cell growth and total differentiation from the normal cells from which it originated.Even though conventional treatments have become the standard of care over the past few decades, their negative adverse effects and low success rate are not to be overlooked [6][7][8].Cannabidiol (CBD) treatment, photodynamic therapy (PDT), or a combination of the two, is a less intrusive yet successful method of treating cancer [9].
Aminopterin, or 4-aminopterin acid, is a well-known folate blocker.Several folate-mediated processes, including nucleotide synthesis, may be blocked by AMT and its active compounds, which could reduce DNA synthesis [10].AMT is an extremely popular chemotherapy drug in clinical settings [11].Treatment of acute lymphocytic leukemia, choriocarcinoma, breast carcinoma, head and neck cancer, oat cell carcinoma, mycosis fungoides, and osteogenic sarcoma can all benefit from this approach, either on their own or in conjunction with other therapies [12].Its therapeutic application is constrained by its toxic side effects, including bone marrow inhibition, anemia, thrombocytopenia, and gastric and liver problems.Several active research initiatives are currently aimed at developing compounds with enhanced tumor-cell selectivity, decreased toxicity, enhanced delivery characteristics, and enhanced lipid solubility and membrane permeability [11,13,14].
Metal-organic frameworks (MOFs) are a new class of molecule crystalline materials with interesting properties and a wide range of potential applications [15].Due to their nanoscale dimensions, MOFs find applications in various fields of nanomedicine, including but not limited to biosensing, molecular imaging, and drug delivery [16].Biocompatibility, pore size tunability, chemical stability, and ease of endosomal release are desirable multifunctional properties that have piqued interest in searching for new medicinal approaches [17].MOFs are ripe for additional functionalization with many organic compounds and inorganic building elements [18][19][20].In addition, novel multifunctional composites/hybrids for cancer treatments like photodynamic therapy (PDT), photothermal therapy (PTT), chemotherapy, immunotherapy, combination therapy, and so on are being developed through the merging of MOFs and functional materials [21][22][23].Multifunctional MOFs with the ability to deliver tailored and multimodal medicine show tremendous promise for enhancing the efficacy of cancer treatments [16].
Because zeolitic imidazolate frameworks (ZIFs) have large frameworks, and minimal toxicity, they have been the subject of extensive research into their possible uses as drug carriers among metal-organic frameworks (MOFs) [24].ZIF-90 is a different composition than the well-studied ZIF-8 [25].The mechanism of Schiff base aldehyde bonds with the amino groups to fabricate the ZIF-90 and effectively load drug molecules [26].ZIF-90's coordination bond is disrupted upon reaching a mildly acidic cancerous site, causing the skeleton to collapse and releasing the drug via a Schiff base bond breakdown and hydrolysis process [27].ZIF-90's favorable properties as drug delivery for penetrating tumors are summarised above.Emerging as inorganic-organic composite coordination polymers, metal-organic frameworks (MOFs) have been widely investigated for their potential in medicine [28].Zeolitic imidazolate frameworks (ZIFs) are a subfamily of metal-organic frameworks (MOFs) composed of transition metal ions (M) coordinated to imidazole or imidazole derivatives (Im) in the form of molecules written as M-Im-M.ZIFs have a zeolite-like shape due to their structural similarities to aluminosilicate zeolites, with metal ions mimicking the function of imidazolate anions and silicon replacing the function of oxygen in zeolites [29].Therefore, ZIFs express the key qualities of both MOFs and zeolites, such as high pore sizes and large surface areas recognition to the exposed sides of the linkers, that give ZIFs maximum load ability; ZIFs can be new functionalities by presenting drug ingredients or adapting ligands; and chemical resistance and superior thermal stability in alkaline water or organic solvents [28].Furthermore, ZIFs' framework endows them with additional intriguing characteristics.These are the breakdowns: Some ZIFs (such as ZIF-90) can decompose in response to the subcellular adenosine triphosphate due to much greater coordination among adenosine triphosphate and Zn 2+ , allowing ZIFs to achieve the exact release of molecular structures and drugs in tumor site; additionally, for the common ZIF, ZIF-90, the modest strength of covalent bond facilitates ZIFs to remain stable under the physiological environment, but shows slow biodegradability [30][31][32].
Due to its acid solubility, chitosan undergoes protonation of its amino groups, transforming it into a cationic polymer capable of interacting with multiple molecules [33].It is widely accepted that chitosan is the only marine protein that contains cations [34].Chitosan's antimicrobial properties stem from the contact of the positively charged molecule with the negatively charged membranes of microbes [35].One of the chitosan's biological applications is the ability to selectively chelate metals like iron, copper, cadmium, and magnesium.The compound's stability allows it to take many shapes, including films, nanofibers, hydrogels, and pastes [36].Controlled drug release, increased drug stability, decreased adverse drug responses, and increased bioavailability have all been proposed as benefits of using chitosan and its derivatives as carriers for drug delivery in treating illnesses like cancer [37].More than enough experiments have shown that chitosan works as a drug delivery system for gastric cancer [38].
Based on these outcomes, we attempt to construct the AMT-loaded@ZIF-90 (AZ) and CS@AMT@ZIF-90 (CAZ-90) were effectively fabricated and thoroughly described in this study using the biocompatible composites ZIF-90, the modified composites CS, and the anticancer drug AMT.About 250 mg/g of substance was within the AMT's loading capability.Meanwhile, in vitro tumor milieu and healthy tissue models confirmed CAZ-90's low pH reaction drug release behavior.The novel drug-loaded particles' biocompatibility and targeting properties were tested via MTT assays using 4T1, MDA-MB-231, MCF-7 (breast cancer cells), and HUVECs (non-cancerous cells).This investigation demonstrates one type of efficient drug carrier for Aminopterin and suggests an approach to change that could improve the efficiency of anticancer drug delivery.

Synthesis of ZIF-90
10 mL methanol solution was used to mix with trimethylamine (0.2 mL) added to disperse the Zn(NO 3 ) 2 .6H 2 O (114 mg) and imidazole-2-carboxaldehyde (ligand termed as 2-ICA, 75 mg).After refluxing, the combination was agitated at ambient temperature for 1-h and stirred at 70 °C for 25 min.To obtain purified ZIF-90, the yellowish precipitates were gathered and rinsed with the methanolic solution.The composites were air-dried in a normal environment.Zn-based output estimates put it at 98%.

Fabrication of AMT@ZIF-90 (AZ) and CS@AMT@ZIF-90 (CAZ-90)
To acquire the AZ and CAZ-90 composites, we used a one-pot technique to dissolve 2-ICA (75 mg) in MeOH (10 mL) with triethylamine (TEA, 0.2 mL).In the meantime, AMT (45 mg) was dissolved in 10 mL of aqueous containing a suitable quantity of NaOH, and the Zn(NO 3 ) 2 .6H 2 O (108 mg) was combined.All the liquids were combined and warmed slowly at 35 °C.After 2-h of stirring at ambient temperature, the solution crystallized into golden AZ.
Furthermore, the prepared 1% CS solution was added during the room-temperature agitation procedure for making AZ (1.5 g of CS was mixed with 5% acetic acid solution in 150 mL of water and stirred at room temperature for 24-h); after 12-h of stirring, a solution formed.Yellow CAZ-90 was produced after the crystal-clear products were rinsed three times with methanol and water, then desiccated at 30 °C.

Assessment of drug release and loading
At 37 °C, the AMT release from CS@AMT@ZIF-90 was calculated in phosphate-buffered saline (PBS) solution at pH 5.5 and 7.4.Buffer solution from PBS was used to immerse samples containing 10 mg of AMT (40 mL).The drug content was determined by high-performance liquid chromatography (HPLC) by periodically removing a sample of the solution (0.5 mL) and replacing it with a new PBS buffer solution [39].

Cell cytotoxicity and anticancer activity assay
The 4T1, MDA-MB-231, and MCF-7 (breast cancer cells) and HUVECs (non-cancerous cells) were purchased from the Chinese Academy of Sciences.All the cells were maintained in 90% RPMI-1640 Medium containing 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin.Cells were maintained at 37 °C in a humidified atmosphere of 5% CO 2 .
4T1, MDA-MB-231 and MCF-7 cells density of (1 × 10 3 cells/well) in 96 well plates.Cell monolayers were treated with ZIF-90, AMT, and CS@AMT@ZIF-90 for 24-h.At the end of exposure, MTT solution (5 mg/mL in PBS) was added to each well and incubated for 2-h.The formation of formazan crystals formed proved using inverted microscopy.DMSO (100 µL/well) was added to dissipate the formazan crystals for 10 min after the absorbance was read at 575 nm against the ELIZA microplate reader [40][41][42][43].The inhibitory concentration IC 50 values were calculated using GraphPad Prism software.

Cell cycle assay by PI staining in flow cytometry
Evaluation of cell cycle development was observed by using flow cytometry.The cells density of (3 × 10 5 cells/well) in 6 well plates.4T1, MDA-MB-231 and MCF-7 cells were treated with IC 50 concentration of ZIF-90, AMT, and CS@AMT@ZIF-90 for 24-h.After 15 min at 4 °C, cells were trypsinized, collected, and preserved in 1 mL of 75% cold ethanol in flow tubes.After incubation, cell pellets were resuspended in 500 µL of propidium iodine (10 µg/mL) containing 300 µg/mL RNase and centrifuged at 1500 rpm for 5 min (Promega, USA).Then cells were incubated on cold for 30 min and filtered with 53 μm nylon gauze.Cell cycle distribution was examined using a Beckmann flow cytometer [52].The samples were collected and analyzed with cell Flow JO software.

Fabrication of CS@AMT@ZIF-90 and its characterization
The conventional approaches to fabricating ZIF-90 incorporate solvent diffusion and solvothermal methods, with PVP/alcohol/water or DMF as the major solvents for these reactions.Although excellent crystallinity results can be obtained using these techniques, the processes involved are laborious, and the output is low (around 40%) [29].In this study, TEA could deprotonate imidazole-based ligands into ZIFs, preventing the development of metal hydroxides that are not required.The synthesis presented here extends the above strategy for improving synthetic processes.In particular, the reaction duration has been reduced (more than 1-h), and the starting components can be dissolved in water and used in a room-temperature reaction.Despite the milder and more handy reaction circumstances, the yield can still reach 98%.This study improves the speed with which ZIF-90 can be prepared due to a revised synthesis procedure.
ZIF-90, AZ, and CAZ-90 shape and SEM images are displayed in Figure 1, respectively.The synthesized ZIF-90 has a uniform cubic crystalline morphology; its particles are evenly dispersed and grey-white.After AMT loading and CS change, AZ and CAZ-90 particles become golden instead of white like ZIF-90.There has been a load of AMT into the containers because of the noticeable hue shifts.Furthermore, it is clear from the SEM images that the AZ and CAZ-90 particles were well formed, albeit with a noticeable change in the particles' general structure.FT-IR was utilized to verify CAZ-90's composites.Figure 2 displays the FT-IR bands of fabricated composites.The absorption bands of ZIF-90 (C = O and C-H) at 1685 cm −1 and 2842 cm −1 indicate the presence of 2-ICA aldehyde groups, while the stretching bands of the 2-ICA become visible at 1150-1450 cm −1 following coupled with the Zn 2+ .CAZ-90 reveals that AMT is covalent bonding into ZIF-90s via the formation between the amino and aldehyde group of ZIF-90s.A solid Figure 1.scanning electron microscopic (sEm) images of Zif-90, amt@Zif-90, and cs@amt@Zif-90 (caZ-90).(scale bar = 1.00 μm).
stretching absorption vibration developed at 1650 cm −1 (C = N).The effective coating of chitosan on ZIF-90s are further illustrated by the presence of distinctive bands of the chitosan at 1085 cm −1 and 1215 cm −1 in the spectral of the CAZ-90, which validate the formation of CAZ-90.The PXRD analysis showed that the as-synthesized ZIF-90 was structurally and chemically pure.The phases of ZIF-90 were achieved, as evidenced by the excellent agreement between the computed and as-synthesized X-ray diffraction patterns (Figure 3B).However, the CAZ-90 structure is maintained when CS is modified with CAZ-90, as the diffraction pattern of CAZ-90 is nearly identical to that of ZIF-90.In addition, the PXRD of AZ products synthesized with varying concentrations of AMT was evaluated (Figure 3B).PXRD patterns reveal a stable AZ framework in the substance at 20, 30, and 40 mg dosages.A shift in the composites PXRD and the absence of AZ were observed at 50 mg of AMT.One potential explanation is that any promising drugs attempt to fabricate the CAZ-90 composites simultaneously.Consequently, 40 mg is the highest dosing increment for this response system.The zeta potential was measured in a water solution to establish the surface characteristics of the various samples.Figure 4 shows that the zeta potential (δ) ZIF-90s were −4.52 mV before being loaded with AMT.After loading the AMT, the zeta potential marginally dropped to −19.2 mV.In addition, the zeta potential shifted dramatically from −19.2 mV (AZ) to +2.98 mV (CAZ-90) after CS coating for AZ.The positively charged particle surfaces aided cellular absorption because cell walls are negatively charged.

Thermogravimetry (TGA) and gas adsorption assessment
As demonstrated in Figure 5A, TGA is also used to investigate the thermal stability of AZ, ZIF-90, and CAZ-90.Due to the elimination of foreign molecules in the pore, the ZIF-90 experiences a dramatic decrease in mass from RT to 75 °C.In comparison, AZ and CAZ-90 exhibit only a slight decrease in weight over that range, suggesting significantly fewer solvent molecules bound in the pores.This is likely due to the AMT loading.Additionally, there is a 10% weight difference between CAZ-90 and AZ from 70 °C to 240 °C because of the change in CS.Furthermore, after 300 °C, the major decomposition phases of ZIF-90, AZ, and CAZ-90 are similar, indicating that AMT loading and CS modification have a little discernible effect on ZIF-90's thermal stability.
N 2 sorption tests were carried out to determine the porosity of the synthesized ZIF-90, AZ, and CAZ-90 after they had been triggered by drying in pressure at 50 °C.The type I isotherm for N 2 absorption at 77 K in Figure 5B demonstrates that ZIF-90 has a persistent porosity.Because of micro holes, isothermal temperatures rose sharply even at moderate pressures.In contrast, the isotherm of ZIF-90 exhibited a hysteresis loop at p/p0 = 0.3, possibly because of the small reduction in microparticle size brought about by the presence of 2-ICA.The highest adsorption capability of a ZIF-90 was around 400 cm 3 /g.The surface area determined using the Brunauer-Emmett-Teller (BET) technique for ZIF-90 and AZ showed a significant drop from 1226.1 to 419.5 m 2 /g.These findings show that AMT molecules filled all the empty spaces in the ZIF-90 structure.Chitosan coating confirmed that the CAZ-90 pores were prevented by polymeric coating because of the lack of a hysteresis ring and the reduced N 2 adsorption.
framework contributed to the comparatively large AMT load.ZIF-90 pH stability was demonstrated by submerging samples in PBS solutions of varying pH for 48-h, after which the samples' X-ray powder diffraction (PXRD) profiles were observed (Figure 3B).ZIF-90's structure was more robust at high pH than at low pH.pH values of 5.5-6.5, typical of the area around a tumor, rendered the ZIF-90 scaffold unsustainable.This indicated that the pH significantly influenced the stability of ZIF-90s.Therefore, ZIF-90s was an attractive choice of drug carrier because of its possibility for tumor-targeted delivery due to the frameworks in the surroundings of the tumor's region.
The drug (AMT) release assessment was performed in healthy and cancer tissue models (pH 5.5 and 7.4).Indicators of prolonged-release behavior were visible in AMT-loaded AZ and CAZ-90.AZ drug release graphs exhibited minimal pH reactivity (Figure 6).As shown in Figure 6, AMT was gradually released from the CAZ-90 particles, and under the acidic environment, it was almost fully released in about 100-h.(pH 5.5).Around 60% of connected AMT was released in the PBS solution at pH 7.4 within 25-h.Drug release was more pH-sensitive in CAZ-90 because of the alterations to CS.These data demonstrated that CAZ-90's pH reactivity during drug release could be enhanced via CS surface modification, which was associated with CS's intrinsic characteristics.The CS extended into a polymeric matrix at acidic pH, exposing AZ to an acidic environment that eventually degraded the AZ structure and released AMT.On the other hand, a constriction process was observed in the CS layer at pH 7.4, resulting in a restricted AMT release.Due to CS's growth in a low pH milieu, AMT molecules were released when CAZ-90 invaded cancer cells.By blocking the usual synthesis of cancer cell genetic composites, the unleashed AMT was able to slow the growth of malignant cells and achieve its goal of stopping cancer from spreading.

In vitro proliferation assay
Understanding the toxicity of drug delivery is essential [53].The cytotoxic action of ZIF-90 and CAZ-90 was initially tested using the MTT method against the Human umbilical vein endothelial cells (HUVECs).Cell survival was greater than 80% and 75% for ZIF-90 and CAZ-90, respectively, at 20 µg/mL (Figure 7), indicating that these proteins exhibited very low cytotoxicity.As displayed in Figure 7, various concentrations of three different breast cancer cells (4T1, MDA-MB-231, and MCF-7) were assigned to three different administration groups (AMT, ZIF-90, and CAZ-90) further to assess the cytotoxicity of CAZ-90.After 48-h of incubation, the cells treatment with AMT and CAZ-90 at 20 µg/mL had been destroyed.Both free AMT and AMT-loaded CAZ-90 particles displayed superior inhibitory effects and therapeutic effects on cancer cell proliferation, more apparent with the rise in concentration.The drug-loaded CAZ-90 group also exhibited comparable levels of inhibition to the free AMT drug group across all concentrations and accomplished a greater inhibitory impact on cancer cells.CAZ-90, a compound particle, was found to have a selectively toxic impact on cancer cells when tested in vitro against normal and malignant tissue samples.Based on these findings, CAZ-90 could be used as a highly effective anticancer delivery method because it is biocompatible and has minimal cytotoxicity.
Anticancer complexes commonly cause cancer cells to undergo apoptosis and necrosis, resulting in structural alterations.Ethidium bromide and acridine orange (AO/EB) distinguish viable, apoptotic, and necrotic cells [54].Live cells exhibit bright green fluorescence along with normal cytoplasm and nuclei, whereas early apoptotic cells exhibit green fluorescence along with nuclear shrinkage and chromatin condensation.Necrotic cells fluoresce red without chromatin fragmentation, while late apoptotic cells fluoresce red with nucleus atrophy and chromatin condensation (Figure 8).Analysis results using AO/EB indicate that the apoptotic mode of cell death is stimulated by IC 50 concentration of ZIF-90, AMT, and CS@AMT@ZIF-90 in 4T1, MDA-MB-231, and MCF-7 breast cancer cells (Figure 8).Fluorescent microscopy examination of DAPI-stained cells has been used to assess the distinctive morphological changes in the cells by therapy with ZIF-90, AMT, and CS@ AMT@ZIF-90.As a result, the nucleus and cytoplasm of the dying cell condense, the plasma membrane blebs, and the cell disassemble into apoptotic bodies, which are membrane-enclosed objects holding organelles and fragments of the nucleus that have survived [55].In the next step, expert phagocytes and adjacent cells quickly identify, consume, and destroy these apoptotic bodies.Figure 9 shows some of the most prominent morphological alterations in ZIF-90, AMT, and CS@AMT@ZIF-90, including cytoplasmic blebbing, nuclear swelling, cytoplasmic vacuolation, bi-and multinucleation, chromatin fragmentation, and a late apoptotic sign of dot-like chromatin condensation.In contrast to treated cancer cells with an extremely intense blue emission, untreated control cells, and normal cells have less intensely stained nuclei and a more uniform color.Cells exhibiting necrotic and apoptotic morphologies caused by treatment with the IC 50 concentration of ZIF-90, AMT, and CS@AMT@ZIF-90 in 4T1, MDA-MB-231, and MCF-7 breast cancer cells were by staining with AO-EB and DAPI, gathered from hand cell counting (Figure 9).
The cell division and replication process are known as the cell cycle.There are four stages to this process: the G1 phase, the S phase (synthesis), the interphase (G2), and the M phase (mitosis) [56][57][58].An unchecked growth of cells, leading to cancer, occurs whenever there is a change in the basic genetic regulation of cell division.Cell cycle disruption in tumor cells would be a promising target for anticancer drug development.Flow cytometry, which measures the amount of DNA in cells quantitatively, has been   used to study the various stages of the cell cycle in ZIF-90, AMT, and CS@AMT@ZIF-90 in 4T1, MDA-MB-231, and MCF-7 breast cancer cells.Results from an analysis of cell cycle development after 24-h of incubation with cells treated at the IC 50 dose of ZIF-90, AMT, and CS@AMT@ZIF-90 are shown in Figure 10.The results reveal that CS@AMT@ ZIF-90 effectively arrests all breast cancer cells.This is clear evidence to use the animal model and future pre-clinical applications.

Conclusions
In conclusion, we developed a unique one-pot technique for constructing CAZ-90's antitumor drug delivery system.The anticancer drug model AMT was chosen for particle loading, with drug loading efficiencies reaching 250 mg/g.ZIF-90's synthesis process has been fine-tuned to reduce harsh reaction conditions and speed up the reaction time.This results in a final yield of up to 98% and a more effective way of preparing composite products based on MOF.Substrate reactivity to changes in pH was effectively enhanced by surface alteration of drug-loaded particles using CS.The compound particle CAZ-90 also shows a tailored inhibitory impact on cancer cells while being relatively non-toxic to normal human umbilical vein endothelial cells (HUVECs) cells.In conclusion, CAZ-90 is a promising anti-cancer drug delivery system because of its excellent drug-loading ability, regulated drug delivery, and minimal toxicity.