Ultrasonic-Assisted Synthesis of Fe-BTC-PEG Metal-Organic Complex: An Effective and Safety Nanocarrier for Anticancer Drug Delivery.

The porous metal-organic complexes are emerging as novel carriers for effective and safe delivery of drugs for cancer treatment, minimizing the side effect of drug overuse during cancer treatment. This study fabricated the Fe-BTC-PEG metal-organic complex from Fe ions, trimesic acid, and poly(ethylene glycol) as precursors using an ultrasonic-assisted method. The morphology and crystallinity of the resultant complex were observed by scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. FTIR spectroscopy was employed to investigate the functional groups on the surface of the Fe-BTC-PEG complex. The result showed that the prepared Fe-BTC-PEG complex was in particle form with low crystallinity and diameter ranging from 100 to 200 nm. The obtained Fe-BTC-PEG complex exhibited a high loading capacity for the 5-fluorouracil (5-FU) anticancer drug with a maximal capacity of 364 mg/g. The releasing behavior of 5-fluorouracil from the 5-FU-loaded Fe-BTC-PEG complex was studied. Notably, the acute oral toxicity of the Fe-BTC-PEG metal-organic complex was also carried out to evaluate the safety of the material in practical application.

Introduction

One of humankind’s most well-known “death penalty” is cancer, which is increasing in the modern world.[1] Several methods could be employed to cure cancer, including surgery, chemotherapy, hormonal therapy, radiation therapy, adjuvant therapy, and immunotherapy; however, their effectiveness is still questionable.[2] Of these cancer treatment techniques, chemotherapy is considered to be the most common.[3] Even though proven to be an effective cancer treatment technique, chemotherapy lacks selectivity toward cells and drug molecules, which could damage the normal cells, causing side effects of significantly weakening other organs.[4] Furthermore, the cancer treatment efficiency of the chemotherapy is also hindered by the low solubility, stability, and bioavailability.[5] Thus, we need to find practical pathways to deliver drug molecules to the targeted tumor and cancer cells. Many strategies have been developed to accurately deliver the anticancer drug to the tumor, such as the direct introduction of the anticancer drug into the tumor, routes of drug delivery, systemic delivery targeted to the tumor, drug delivery targeted to blood vessels of the tumor, special formulation and carriers of anticancer drugs, transmembrane drug delivery to intracellular targets, and biological therapies.[6] Nanoparticles as carriers for drug delivery belong to the unique formulation, and carriers of the anticancer drugs category have been extensively studied and employed for this purpose. The anticancer drugs are encapsulated into nanocarriers and delivered to the targeted tumors before release into the tumor space, resulting in the effective uptake of the drug molecules by tumor cells.[7,8] The major disadvantage of drug delivery using nanocarrier systems is to actively release the drug molecules to the suitable tumors at the right time. Many factors such as heat sensitivity, pH controls, employment of proteases or phospholipases, and controlling the glutathione levels have been considered to design the delivery systems for the anticancer drugs using nanoparticles.[9,10] Controlling these factors enables the release rate of the anticancer drug molecules when they are in the cancer cells while remaining stable in the environment of the normal cells, consequently minimizing the side effects and enhancing the therapeutic efficiency.

The recent emergence of porous metal–organic complexes (MOCs) has attracted significant research interest from scientists. MOCs are porous crystalline materials self-assembled by metal ions or clusters and organic ligands to form frameworks.[11,12] MOCs are known for their extraordinarily high specific surface areas, tunable pore size, and adjustable internal surface properties. These inherent features render MOCs promising in various applications, such as gas adsorption/separation,[13−15] catalysis,[16,17] and drug delivery.[18−20] The MOCs possess several outstanding features, including the mesoporous architecture with mesoporous cages and microporous windows, a giant cell volume, and a high surface area. The unique combination of properties highlighted above makes MOCs an excellent candidate for industrial applications, including drug delivery systems (DDSs).[21] Because of facile synthesis, high loading capacity, biocompatibility, and degradability, MOCs have been extensively employed as carriers for stimuli-sensitive drug delivery.[22−24]

5-Fluorouracil (5-FU) is a chemotherapeutic agent employed to treat several deadly cancers, such as breast, colorectal, and head and neck cancers. It has been demonstrated to be effective in treating various types of cancers as it has a mode of action based on interfering with thymidylate synthesis, which controls the development of cancerous cells.[25] The main challenge of using 5-FU is its short biological half-life, low selectivity, and toxic side effects on the bone, marrow, and gastrointestinal tract.[26,27] To minimize these limitations, drug delivery systems have been considered for the controlled release of 5-FU drugs to targeted tumors.[28,29] Many nanoparticles have been employed for the 5-FU drug delivery, such as mesoporous silica nanoparticles,[30] nanogels,[31] magnetic nanoparticles,[32] metal–organic frameworks (MOFs), or metal–organic complex.[20,33] To the best of our knowledge, the use of the Fe–BTC–PEG complex obtained from the green synthesis method of ultrasonication has not been investigated for the delivery of the 5-FU drug.

In this work, the new Fe–BTC–PEG metal–organic complex is synthesized using a facile approach with the assistance of an ultrasonicator. The detailed characterizations of the prepared metal–organic complex will be thoroughly investigated. The drug-loading capacity and release for the 5-fluorouracil will also be studied. Significantly, the acute oral toxicity and repeated dose 7-day oral toxicity of the 5-FU-loaded Fe–BTC–PEG metal–organic complex are investigated in mice.

Results and Discussion

Characterizations of the Fe–BTC–PEG Complex

The morphologies of the obtained Fe–BTC–PEG complex were observed by scanning electron and transmittance electron microscopies, as shown in Figure . It can be seen from Figure a,b that the Fe–BTC–PEG complex in the shape of the small particles tends to aggregate to form large clusters. The morphology of the Fe–BTC–PEG complex is similar to the Fe–BTC fabricated from the ultrasonication approach; however, the particle size of the Fe–BTC–PEG is significantly smaller than that of Fe–BTC (Figure S1). This indicates that the addition of the PEG modifier could slow down the growing process of the Fe–BTC, reducing the particle size. The TEM images (Figure c) exhibit the round shape of the Fe–BTC–PEG complex with diameters ranging from 100 to 200 nm. The high-resolution TEM image in Figure d indicates that the Fe–BTC–PEG was well prepared and identical in the complex structure. The thin outer layer is ascribed to the coverage of PEG polymer around the Fe–BTC metal–organic particles. This morphology is in good agreement with the Fe–BTC shape of the previous study.[34]

Figure 1

SEM (a, b) and TEM (c, d) images of the prepared Fe–BTC–PEG metal–organic complex.

The crystallinity of the prepared Fe–BTC–PEG metal–organic complex was investigated using a powder X-ray diffraction pattern, as shown in Figure a. The low and relatively broad peaks (or maybe overlapping) observed in the XRD pattern indicate the Fe–BTC–PEG materials’ semiamorphous nature.[34,35] This may be due to the disordered structure (SEM and TEM images) or the effect of the PEG organic coating. Because of the low crystallinity of the Fe–BTC, the XRD characterization for this material was limited, with no reported crystal structural data in the literature. However, the position of the broad peaks in the XRD pattern are well-matched with the ones reported previously.[36,37]Figure b exhibits the FTIR spectrum of the Fe–BTC–PEG complex to investigate the chemical structure and functional groups on the material surface. The broad adsorption from 3300 to 3700 cm–1 is assigned to the O–H bending vibration in adsorbed moisture and the O–H stretching vibrations in the BTC and PEG molecules. The characteristic peaks that appeared at 1632, 1581, 1451, and 1380 cm–1 are ascribed to the asymmetric and symmetric carboxylate groups’ vibrations in BTC molecule (Figure S2), respectively, which demonstrates the coordination between carbonyl group in BTC ligand with the iron sites.[38,39] The C–H bonding of the benzene zings in BTC is evident by the stretching vibrations at 748 cm–1. The vibration bands at around 636 cm–1, which belong to the Fe–O stretching vibrations, also indicate the Fe–BTC’s successful formation. The presence of PEG in the complex is confirmed by the appearance of the absorption bands at 1729 and 1103 cm–2, which are attributed to the C–O–C and C = O stretching vibration of the PEG molecules[25,40] (Figure S3).

Figure 2

XRD pattern (a) and FTIR spectrum (b) of the Fe–BTC–PEG metal–organic complex.

The materials’ surface area and functional groups are among the decisive factors to evaluate the loading capacity for drug delivery. The BET surface area plot of the Fe–BTC–PEG metal–organic complex was determined using and nitrogen adsorption–desorption plot, as shown in Figure . The results show that the Fe–BTC–PEG metal–organic complex has a surface area of 21.4718 m2/g. This relatively low surface area is probably due to the coverage of the PEG molecule inside and outside of the Fe–BTC micropores. Even with low surface area, the drug adsorption capacity by Fe–BTC–PEG metal–organic complex might be significantly enhanced by functional groups induced by the PEG modification, which will be demonstrated by the drug-loading study in the following sections. Furthermore, the Fe–BTC–PEG metal–organic complex’s pore volume and pore diameters are 0.078 cm3/g and 24.86 nm, respectively, making it suitable for the 5-FU drug loading.[41]

Figure 3

Nitrogen adsorption–desorption plot of the Fe–BTC–PEG metal–organic complex.

5-FU Loading and Releasing by the Fe–BTC–PEG Complex

Many drugs have been effectively utilized for cancer treatment. Among them, 5-fluorouracil (5-FU) could be employed for treating many types of cancers such as skin, stomach, breast, anal, and colorectal. As mentioned above, the Fe–BTC–PEG metal–organic complex is suitable for functional groups, particles size, pore diameter, and pore volume to deliver 5-FU to the targeted cell with minimum side effects. In this work, the prepared complex was loaded with 5-FU via immersing approach. The presence of 5-FU in the complex structure after encapsulation was confirmed by FTIR spectroscopy, as shown in Figure . The FTIR spectrum of the 5-FU-loaded Fe–BTC–PEG reveals all characteristics peaks of the Fe–BTC–PEG complex discussed above. Additionally, the appearance of the vibration band at around 750 cm–1 is attributed to the C–H stretching group in the CF=CH plane of the 5-FU; this characteristic peak is also observed in the FTIR spectrum of the 5-FU as shown in Figure S4. The surface morphology of the Fe–BTC–PEG metal–organic complex was also observed, showing a negligible change compared to the morphology of the sample before drug loading (Figure S5). The BET surface area, pore volume, and pore diameter of the Fe–BTC–PEG metal–organic complex after 5-FU loading were determined to be 17.4 m2/g, 0.034 cm3/g, and 8.27 nm, respectively, which decreased in comparison with those of the initial Fe–BTC–PEG sample, further demonstrating the success of the 5-FU loading on the sample (Figure S6).

Figure 4

FTIR spectra of the Fe–BTC–PEG before (black line) and after (red line) loading with 5-fluorouracil.

The drug-loading capacity is one of the critical properties to evaluate the applicability of the materials for drug delivery. To determine the maximal 5-FU-loading capacity of the Fe–BTC–PEG complex, the complex was immersed into the 5-FU solution for 14 days to reach an equilibrium adsorption state. The result showed that the prepared complex has a high loading capacity for 5-FU with a maximal capacity of 364 mg/g material, accounting for 36.4% weight of the carrier material, which was higher than the drug-loading capacity of the zinc-based metal–organic framework.[42]

It is noteworthy that the slow release behavior was one of the crucial properties when using advanced materials for drug delivery. Figure illustrates the releasing profile of the 5-FU-loaded Fe–BTC–PEG metal–organic complex. Two evident releasing stages could be clearly observed during the release process of 5-FU from the loaded complex. The first stage is the release of the 5-FU in free form, which forms no or a weak bond with the complex. This stage is expected to occur very quickly (Figure a). The second stage is a slow-releasing process of the 5-FU drug because of intense interaction with the functional groups on the surface or in the micropores of the BTC–PEG metal–organic complex. For the free-standing 5-FU drugs, more than 90% of the 5-FU could be released in vitro media after 30 min.[43] When loaded with the Fe–BTC–PEG metal–organic complex, only 35% of 5-FU is released after 2 h of dissolution in the first releasing stage (Figure a). In the second stage, the releasing rate is significantly reduced, with only a few percentages of 5-FU released after each immersion (Figure b). The slow release of 5-FU is ascribed to solid host–guest bonding between 5-FU and the Fe–BTC–PEG metal–organic complex. The complete release of 5-FU from the loaded complex in the PBS solution was only evident after 14 days of the experiment, which is considered to be highly stable in the body-simulated media. The drug-releasing time of the Fe–BTC–PEG/5-FU system is much slower than that of the Fe–BTC–PEG/5-FU system, which is advantageous in cancer treatment.[25] This slow 5-FU release minimizes the side effect of the cancer drug to the healthy cells and improve the treatment effectiveness of the drug.

Figure 5

Releasing profile of 5-fluorouracil from the 5-FU-loaded Fe–BTC–PEG metal–organic complex: (a) first releasing stage and (b) second releasing stage.

Acute Oral Toxicity

Evaluating the toxic properties of the Fe–BTC–PEG metal–organic complex is crucial when this material is employed as a carrier for drug delivery in the body. Figure shows the optical images of the Albino BALB/c mice administered Fe–BTC–PEG orally in different doses of 2, 4, 6, 8, and 10 g material/kg mice using a stomach tube. Results reveal that the Fe–BTC–PEG complex does not cause any toxicity in mice even at the highest dose of 10 g/kg bodyweight. All mice treated with Fe–BTC–PEG survived and did not show any toxic symptoms or abnormal behavior at the tested doses over the 7 days of observation. The testing mice treated with Fe–BTC–PEG showed little change in bodyweight, food intake, or water consumption compared to the control group. Acute treatment with Fe–BTC–PEG showed that the healthy white mice (six males and six females) moved and ate usually and responded well to light and sound.

Figure 6

Optical images of Albino BALB/c mice treated with various doses of Fe–BTC–PEG for 7 days.

The change in the bodyweight was also investigated to assess the oral toxicity of the prepared metal–organic complex toward the mice after the oral uptake of materials for 7 days. The result is shown in Figures and 7. It is evident from the figure that the mice normally developed with the bodyweight of tested mice almost the same as that of the control sample when the treatment dose was 2–6 g/kg. After 7 days of treatment with a 2–6 g/kg dose, the bodyweight of mice is approximately 24 g. When the Fe–BTC–PEG increases to 8 g/kg, the bodyweight of the mice slightly decreases compared to that of the controlled sample after 7 days of the experiment. The bodyweight of the mice treated with an 8 g/kg dose is around 22.4 g after 7 days of treatment. Especially at the treatment dose of 10 g/kg, the bodyweight of mice decreases compared to those of the mice before the administration of the Fe–BTC–PEG complex with the bodyweight of only 21.9 g after 7 days of treatment. However, the mice treated with this dose were still healthy with no sign of abnormal behaviors. No change was observed in the food and water consumption of the mice at all groups, including the controlled group and treated groups with various doses of the prepared complex throughout the testing period. The results indicate that the Fe–BTC–PEG metal–organic complex can be a safe carrier for drug delivery for cancer treatment in humans.

Figure 7

Change in the bodyweight of mice treated with Fe–BTC–PEG with various doses for 7 days.

The effectiveness of the 5-FU-loaded Fe–BTC–PEG metal–organic complex compared to the free-standing 5-FU for the inhibition of the human gastric cancer cells in various treatment times was evaluated. For the free-standing 5-FU drug, the cells inhibiting percentage is determined to be around 74% after 24 h of incubation time and tends to decrease along with the increase of the incubation time. When Fe–BTC–PEG/5-FU was employed to treat the human gastric cancer cells, the inhibiting percentage increased along with the incubation time and retained the inhibiting effect of higher than 80% even after 120 h of incubation time (Figure ). This long-lasting effectiveness of the Fe–BTC–PEG/5-FU system is ascribed to the slow release of the 5-FU drug in the Fe–BTC–PEG metal–organic complex, which effectively provides the 5-FU drug for killing cancer cells.

Figure 8

Inhibiting percentages of human gastric cancer cells using the 5-FU-loaded Fe–BTC–PEG metal–organic complex and free-standing 5-FU drugs at various treatment times at the concentration of 10 μg/mL 5-FU.

Conclusions

In summary, the Fe–BTC–PEG metal–organic complex has been successfully synthesized using a facile ultrasonic-assisted approach. The resultant Fe–BTC–PEG complex has diameters ranging from 100 to 200 nm with functional groups on the surface, which is suitable to be utilized as an effective carrier for drug delivery. The Fe–BTC–PEG complex exhibits a high loading capacity for the 5-fluorouracil drug with the maximal capacity of 364 mg/g complex, accounting for 36.4% weight of the carrier material. Significantly, the 5-FU-loaded Fe–BTC–PEG revealed a prolonged release of 5-FU in the in vivo media. The acute oral toxicity also showed no death or signs of toxicity in mice administrated with the Fe–BTC–PEG complex at doses ranging from 2 to 10 g/kg. No change was also observed in the food and water consumption of the mice during the testing period, which indicates that the Fe–BTC–PEG complex could be safely used in the human body. With high drug-loading capacity and safety of use, the Fe–BTC–PEG metal–organic complex is considered a promising carrier for drug delivery for the effective and safe treatment of various cancers.

Experimental Section

Ferric nitrate hydrate (Fe(NO3)3·9H2O, 99.95%), trimesic acid (C6H3(COOH)3, H3BTC, 95%), poly(ethylene glycol) bis(carboxymethyl) ether (PEG diacid), phosphate-buffered saline (PBS) tablet form (1 tablet/200 mL: pH = 7.2–7.6), N,N-dimethylformamide (DMF, 99%), and N,N-dimethylpropionamide ((CH3)2NC(O)H, 98%) were purchased from Sigma-Aldrich. 5-Fluorouracil (5-FU) was obtained from the Vietnam Academy of Science and Technology (Vietnam). All chemicals were used without any further purification.

Synthesis of the Fe–BTC–PEG Metal–Organic Complex

Fe–BTC–PEG was synthesized using a facile method of ultrasonication. First, ferric nitrate hydrate (1 g) was dissolved in 50 mL of distilled water (solution A). One gram of H3BTC and 5 mL of PEG were dissolved in 50 mL of water (solution B). Then, solution A was mixed into solution B. The mixed solution reacted under ultrasonic conditions for 20 min at room temperature. The precipitate was collected by centrifugal separation and washed at least three times with ethanol. The powder was dried at 70 °C under a vacuum. The powder was stored in the vacuum condition and used for further characterizations.

Characterizations

Transmittance electron microscopy (TEM) and scanning electron microscopy (SEM) were performed on Hitachi S-4600 to observe the Fe–BTC–PEG metal–organic complex’s morphology and size distribution. The functional groups of the complex before and after loading with 5-FU were studied by Fourier transform infrared spectroscopy (FTIR, TENSOR II, Bruker). The crystallinity of the samples was investigated by the XRD pattern obtained on the A X’Pert PRO PANalytical instrument with a radiation source of 0.154 nm Cu Kα. The BET TriStar II Plus instrument was used to obtain an N2 adsorption isotherm to measure the surface area of the Fe–BTC–PEG metal–organic complex.

Drug-Loading and Release Batch Experiment

The 5-FU solution with a concentration of 10 g/L was prepared by diluting the 5-FU powder in distilled water. The Fe–BTC–PEG metal–organic complex was then immersed into the 5-FU solution for 10 days before taking out, drying, and determining the loading capacity. The 5-FU drug delivery experiment was carried out as follows: seven samples of the 5-FU-loaded Fe–BTC–PEG metal–organic complex immersed in the body-simulated media (seven vials containing 5 mL of PBS solution) at the temperature of 37 °C. After each interval, one vial was taken out and the vacuum filter removed the material. The 5-FU release was calculated by measuring the absorbance of the remaining filtrate at the wavelength of 265 nm. All experiments were repeated three times to obtain the average value with standard derivation.

Oral Toxicity Study

Animals

Albino BALB/c mice (8 weeks old, weighing 19–22 g) were obtained from the Institute of Biotechnology, Vietnam Academy of Science and Technology (Hanoi, Vietnam). All mice were housed in a temperature-controlled room with a 12 h light and dark cycle. All experiments were performed following the ethical guidelines (Vietnamese ethical laws and European Communities Council Directives of November 24, 1986 (86/609/EEC) for the care and use of laboratory animals).

Acute Oral Toxicity Study

The acute oral toxicity of the Fe–BTC–PEG metal–organic complex was tested following the OECD Guidelines 425.[44] Mice were administered Fe–BTC–PEG orally using a stomach tube. The animals received the complex in serial doses of 2000; 4000; 6000; 8000; and 10 000 mg/kg bodyweight after 12 h diet. They were observed for 3 days to monitor survival and toxic symptoms.

Repeated Dose 7-Day Oral Toxicity Study

A repeated dose 28-day oral toxicity test was determined for mice according to the guidelines of the OECD Test Guideline 407 and Taiwan Food and Drug Administration (TFDA) (2014).[44] Forty-two mice were randomly divided into seven groups with doses of 0, 2, 4, 6, 8, and 10 g material/kg bodyweight, with six mice in each group. The Fe–BTC–PEG suspension was orally administered to mice once a day for seven consecutive days. The controlled mice received water only. The general appearance of all mice was observed daily. The animals were weighed and food consumption was monitored first, fourth, and seventh day of the experiments.

In Vitro Toxicity Study of Fe–BTC–PEG/5-FU

The in vitro toxicity testing of the Fe–BTC–PEG/5-FU was carried out following the Monks’ protocol.[45] The Fe–BTC–PEG/5-FU system and the free-standing 5-FU drug were diluted at the concentration of 10 μg/mL 5-FU. The diluted reagents were added into 96 wells and incubated at various testing times of 24, 48, 72, 96, and 120 h. After a certain time, the cells were fixed using trichloracetic acid and coloring by sulforhodamine B dye in 30 min at 37 °C, then washed with acetic acid, and dried at room temperature. The optical density of dyed cells in the wells was read on the ELISA Plate reader at the wavelength of 515–540 nm. The data were used to calculate the cell-inhibiting percentages.