5-FU@DHA-UIO-66-NH₂ potentiates chemotherapy sensitivity of breast cancer cells through a microRNA let-7a-dependent mechanism

Introduction

Breast cancer (BC) represents the most common cancer and the leading cause of cancer-specific death among women worldwide (1). 5-fluorouracil (5-FU), first introduced as a synthesized anticancer agent several decades ago, continues to be widely used for the treatment of a variety of common malignancies, including BC (2). However, like other conventional agents used in chemotherapy, 5-FU shows various disadvantages such as poor bioavailability and great non-productive drug distribution as a result of the lack of specificity for tumors (3). Due to the non-selectively, 5-FU has been reported to cause adverse side effects, serious drug resistance, and limited concentration at the target location (4). Herein, the construction of novel drug delivery system for 5-FU is of great significance in regard of improving its performance in BC.

Metal-organic frameworks (MOFs), also known as porous coordination polymers, are highly crystalline materials composed of organic linkers and metal clusters (5). As a new class of materials characterized by high specific surface areas and defined structures, MOFs are recognized as promising precursors for preparing drug delivery systems (6). Among MOFs, UIO-66-NH₂ is a zirconium-based type that has been highlighted for its low cytotoxicity and relatively high biocompatibility (7). More importantly, the amine on UIO-66-NH₂ contributes to the feasibility of post-modifying UIO-66-NH₂ to construct multifunctional MOFs, which may overcome the limitations of functional simplicity and be applied for drug delivery (8).

Docosahexaenoic acid (DHA) refers to a highly unsaturated fatty acid and is indispensable for human body, and the critical role of DHA has been recognized in maintaining normal physiological activity of nerve cells (9,10). Intriguingly, a previous study has indicated that DHA can up-regulate the expression of let-7a, thereby increasing the sensitivity of BC cells to 5-FU (11). Meanwhile, accumulating evidence has highlighted that 5-FU and DHA can upregulate the expression of let-7a in BC cells and BC cell-derived exosomes, respectively, and inhibit the occurrence and development of BC (12-14). Besides, the low-pH microenvironment in cancer tissues has revealed the potential of novel pH-sensitive drug delivery systems, for which DHA serves as a promising modifier (15). Taken together, in the present study, we synthesized DHA-modified UIO-66-NH₂ to evaluate its effectiveness as a carrier of 5-FU in BC treatment and hypothesized that 5-FU-incorporated DHA-UIO-66-NH₂ (5-FU@DHA-UIO-66-NH₂) reduce the resistance of BC cells to 5-FU by up-regulating the level of let-7a. Although there is literature reporting the effects of magnetic DHA on tumor cells (16), in this study, MOFs were used as drug carriers different from the Fe₃O₄ liposome nanomaterials as drug delivery media used in the literature. Due to the ultrahigh surface area and porosity of MOFs, the 5-FU drug loading capacity can be greatly increased; in addition, MOFs were modified with DHA and loaded with 5-FU drugs, which can play a synergistic effect in the treatment of BC. We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi.org/10.21037/atm-21-5978).

Methods

Ethical statement

Animal experiments were approved by the Animal Ethics Committee of Harbin Medical University Cancer Hospital (approval number: 2019-185) and performed according to the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health. Extensive efforts were made to ensure minimal suffering of the animals used in the study.

Preparation of 5-FU-incorporated DHA-UIO-66-NH₂ (5-FU@DHA-UIO-66-NH₂)

For the synthesis of UIO-66-NH₂ framework, 0.125 g ZrCl4 and 0.123 g BDC-NH2 were added to the mixed solution of 1 mL HCl and 15 mL N, N-dimethylformamide (DMF), followed by 4-h heating and stirring at 80°C. Then, for the preparation of DHA-UIO-66-NH₂, DHA and UIO-66-NH₂ were dissolved in 20 mL of DMF at different ratios (0, 1/8, 1/4, and 1/2), followed by stirring at room temperature for one day. Prepared DHA-UIO-66-NH₂ was washed with DMF to remove uncombined DHA. Subsequently, for the construction of 5-FU@DHA-UIO-66, 20 mg of 5-FU (dissolved in 20 mL PBS) was added with 5 mg DHA@UIO-66-NH₂ and stirred for 24 h in the dark.

Characterization of 5-FU@DHA-UIO-66

The morphology of 5-FU@DHA-UIO-66 was observed under a scanning electron microscope (SEM, Zeiss Field Emission SEM Supra55VP, Oberkochen, Germany). Fourier transform infrared (FT-IR) spectroscopy was performed using a Spectrum100 FT-IR spectrometer (4000–400 cm⁻¹). Powder X-ray diffraction (PXRD) data were collected with a DX-2700B diffractometer (Dandong Haoyuan Instrument, Dandong, Liaoning, China) in the range of 5–50° (2θ) at a scan rate of 5°/min. Further, thermogravimetric analysis (TGA) was conducted under nitrogen utilizing a TGA/SDTA 851e analyzer (Mettler-Toledo, Highstown, NJ, USA) with the temperature rising from ambient temperature to 800 °C at a heating rate of 10 °C/min.

5-FU@DHA-UIO-66 was also subjected to ultraviolet (UV)-VIS spectroscopy utilizing a UV-3010 spectrophotometer (Hitachi, Tokyo, Japan). The solid-state 13C MAS NMR spectrum was recorded utilizing a contact time of 3 milliseconds on a Bruker AM-400 (400 MHz) spectrometer equipped with a 5.0 mm chemical probe (Bruker Daltonik GmbH, Bremen, Germany). The nitrogen adsorption-desorption isotherm was measured with a Autosorb AS-6B/IQ2 apparatus (Quantachrome Instruments, Boynton Beach, FL, USA) under nitrogen (77 K). Moreover, the concentration of 5-FU was measured utilizing UV-VIS spectroscopy.

Loading and release of 5-FU

The loading amount of 5-FU in DHA-UIO-66-NH₂ was calculated based on the determination of the concentration of 5-FU in the solution and supernatant at different time points by UV-3010 spectrophotometer (Hitachi). The release rate of 5-FU in PBS (pH =7.4 or 5.5) was measured by dialysis. Briefly, 200 µg 5-FU@DHA-UIO-66-NH₂ was dispersed in 20 mL of PBS and subjected to dialysis (37±1 °C, 110 rpm). At different time points during the dialysis, 1 mL of the sample was taken from the supernatant for UV detection of 5-FU content, and 1 mL of fresh PBS was simultaneously added to the original system.

Cellular uptake

The uptake of constructed MOFs by MCF-7 human BC cells was observed with confocal laser scanning microscope (CLSM). MCF-7 cells (5×10⁴ per well) were first seeded on 6-well plates and incubated for 12 h, and then incubated with 10 µg/mL FITC-DHA-UIO-66-NH₂ or FITC-UIO-66-NH₂ for 0.5, 1, 3 or 6 h, followed by PBS washing. After that, the cells were fixed with 2.5% formaldehyde at 37 °C for 10 min, treated with DAPI (20 µg/mL in PBS) for 10 min to stain the nucleus, and observed with CLSM (Leica TCS SP8, Leica, Buffalo, NY, USA).

3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay

The biocompatibility and cytotoxicity of 5-FU@UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH2 in MCF-7 cells were examined utilizing MTT assay. The cells were cultured in plates (100,000 strains per well) for 24 h (37 °C, 5% CO₂) and transferred to new plates (100 µL medium/well). Subsequently, the cells were incubated for 48 h with 0, 0.195, 0.39, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL⁻¹ 5-FU drugs: 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂.^.After 48 h, the cells were incubated with 20 µL of MTT solution (5.0 mg/mL). After the supernatant removed and 150 µL of DMSO added, the optical density (OD) of the substance in each well at 570 nm was determined, and the data were characterized by the mean value ± standard deviation (SD).

Cell viability detection

MCF-7 cells cultured in 96-well plates (5×10⁴ cells/well) were incubated with 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ of different concentrations (0.195, 0.39, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL) for 24 h. Then, the inhibiting effects of the drug on MCF-7 cell viability were detected by trypan blue staining of viable cells.

Flow cytometry

Cell apoptosis was detected following the protocols of Annexin V-fluorescein isothiocyanate (Annexin V-FITC)/propidium iodide (PI) counterstaining kit (BD Biosciences, San Diego, CA, USA). Briefly, MCF-7 cells (upon cell confluence reaching 70%) were incubated with 10 µg/mL 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ for 48 h, digested with EDTA-free trypsin, and stained by Annexin V-FITC and PI, followed by detection using a flow cytometer (BD FACSVerse, BD Biosciences, Bedford, MA, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

Live/dead cell staining

MCF-7 cells were cultured overnight in 8-well plates (3×10⁴ cells/well) and then incubated for 24 h with 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ at a concentration of 10 µg/mL. Following that, the cells were stained with Calcein (final concentration of 2 µM) and PI (50 µg/mL) for 10 min and then subjected to CLSM observation.

Cell transfection

MCF-7 cells and MCF7 sublines resistant to cisplatin (MCF-7/DDP) were incubated in high-sugar DMEM medium (DMEM: FBS =9:1) supplemented with 1% antibodies against streptomycin and penicillin. For transfection, plasmids expressing let-7a mimic or let-7a inhibitor, or corresponding negative control (NC) were commercially constructed (RIBOBio, Guangzhou, Guangdong, China). The cells in the logarithmic growth phase were digested, centrifuged, mixed with 1 mL of prepared culture medium, and then counted. The cells were seeded into 24-well plates (2.5×10⁵ cells/well) and cell transfection was conducted when the cell confluence reached 70%. Entraster-R4000 dilution was prepared by mixing 1 µL of Entraster-R4000 with 24 µL of serum-free medium to reach a final volume of 25 µL, allowed to stand at room temperature for 5 min, and added with the aforementioned plasmids to a final volume of 50 µL for transfection.

Total RNA extraction from cells

The Eppendorf tube and other materials used for RNA extraction were cooled before the assay, and total RNA was extracted from the cells following the protocols of Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and purity of the extracted total RNA was determined utilizing a microplate reader.

miRNA reverse transcription

The reagents and samples involved were placed on ice, and miRNA reverse transcription was performed on ice. The reaction system of reverse transcription is shown in Table S1. After centrifugation (42 °C, 60 min) and degeneration under 95 °C for 3 min, miRNA reverse transcription was terminated at 4 °C. Prepared cDNA was immediately subjected to polymerase chain reaction (PCR) or labeled and stored at −20 °C for subsequent experiments.

mRNA reverse transcription

mRNA reverse transcription was also performed on ice, and the reaction system is listed in Tables S2,S3. The first reaction was terminated at 4 °C following a 5-min centrifugation at 65 °C. For the second reaction, centrifugation was performed at 42 °C for 60 min and the reaction was terminated at 4 °C.

miRNA quantification

The primers used for PCR was synthesized by RIBOBio (Guangzhou, Guangdong, China), shown in Table S4. SYBR Green was selected for fluorescent staining. The assay was carried out on ice without direct lighting, and the reaction system is shown in Table S5. Prepared reaction system was determined based on the protocols of the PCR kit, with U6 serving as the housekeeping gene. Then, 2^−∆∆CT method was utilized to calculate the relative expression level of the target gene.

Cell Counting Kit-8 (CCK-8) assay

Cells in the logarithmic growth phase were digested, centrifuged, and seeded onto 96-well plates (3,000 cells/well, 100 µL medium/well) for 24-h incubation (37 °C, 5% CO₂), followed by liposome transfection of plasmids expressing let-7a mimic or inhibitor or corresponding NC. Three repeated wells were set for each group. After 24 hours of transfection, cells of each group were treated with 5-FU of different concentrations (3, 6, 12, 24, 48 µM) for 24 h. After that, CCK- 8 assay was performed to assess the inhibiting effects of 5-FU on cell viability. A microplate reader was employed to measure OD at 450 nm, and the half-maximal inhibitory concentration (IC₅₀) of 5-FU was calculated.

Establishment of a nude mouse model of BC

A BC mouse model was established to observe the anti-tumor effects of 5-FU-DHA-UIO-66-NH₂in vivo. Nude mice with xenografted tumors were then classified into four groups, a Control group and other three groups subjected to treatment of 5-FU or 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂. Briefly, when the tumor volume reached 300 mm³, 100 mL of normal saline (for the Control group), 5-FU, 5-FU@UIO-66-NH₂, or 5-FU@DHA-UIO-66-NH₂ solution were injected into mice through the tail vein. The volume of xenografted tumors was measured with a caliper every three days. The administered dose of 5-FU was 5 mg/kg (17).

Biosafety assessment of 5-FU@DHA-UIO-66-NH2

To evaluate the biosafety of 5-FU@DHA-UIO-66-NH₂, 100 µL of PBS or 5-FU@DHA-UIO-66-NH₂ (5 mg/kg 5-FU) was injected through tail vein into BALB/c nude mice, which were sacrificed 7 days later to collect major organs (heart, liver, spleen, lung and kidney). Collected organs were fixed in 4% paraformaldehyde, dehydrated with ethanol of different concentrations (50%, 70%, 90%, and, 100%), permeabilized with xylene, and embedded in paraffin. Then, the sections were subjected HE staining, followed by observation with a microscope (BX53, Olympus, Tokyo, Japan). Besides, blood cell counting and analysis of serum biochemical parameters were performed on the 1st and 7th day after the intravenous injection of 5-FU@DHA-UIO-66-NH₂.

Statistical analysis

Data in this study were processed using SPSS v.21.0 software (IBM, Armonk, NY, USA). Measurement data were summarized as mean ± SD. Unpaired t-test was applied to compare data of two groups. One-way analysis of variance (ANOVA) with Tukey’s post-hoc test was performed to compare data of multiple groups. Tumor volume and weight at various time points were compared by repeated measures ANOVA with Bonferroni’s post-hoc test. Survival rate at different time points was analyzed by two-way ANOVA. Moreover, P<0.05 indicated statistically significant difference.

Results

Successful construction and characterization of UIO-66-NH₂, DHA-UIO-66-NH₂, and 5-FU@DHA-UIO-66-NH₂

We synthesized UIO-66-NH₂ through hot solvent diffusion method and then DHA-UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂. Then, SEM characterization revealed that the particle size of UIO-66-NH₂ was about 100 nm when the molar ratio of zirconium metal salt to ligand was 0.125:0.123 (Figure 1A). FT-IR spectroscopy of the prepared UIO-66-NH₂ indicated an obvious red shift of carboxyl functional group, identifying that the metal ion had been successfully combined with the ligand (Figure 1B).

Figure 1 Construction and characterization of UIO-66-NH₂, DHA-UIO-66-NH₂, and 5-FU@DHA-UIO-66-NH₂. (A) SEM images of UIO-66-NH₂ (×10,000); (B) infrared spectrum of UIO-66-NH₂ and H₂BDC ligand; (C) NMR spectrum of UIO-66-NH₂; (D) infrared spectrum of DHA, UIO-66-NH₂ and DHA-UIO-66-NH₂ infrared spectrum; (E) adsorption and desorption curves of UIO-66-NH₂ and DHA-UIO-66-NH₂ under nitrogen (77 k); (F) PXRD pattern of DHA-UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂. Each experiment was repeated three times. * indicates the characteristic peak of DHA. DHA, docosahexaenoic acid; 5-FU, 5-fluorouracil; SEM, scanning electron microscope; NMR, nuclear magnetic resonance; PXRD, powder X-ray diffraction.

Subsequently, DHA was modified to the surface of UIO-66-NH₂, and the carboxyl group at the end of DHA allow it to easily bind to UIO-66-NH₂ that has many metal clusters and aminated ligands on the surface can easily bind to the metal clusters and amino ligands on the surface of the nano-MOF. Constructed DHA-UIO-66-NH₂ was then characterized by FT-IR, PXRD, solid-state NMR, and gas adsorption. The characteristic peak of DHA appeared on the NMR spectrum of DHA-UIO-66-NH₂, showing successfully combination of DHA and UIO-66-NH₂ (Figure 1C). FT-IR spectrum DHA-UIO-66-NH₂ also displayed the absorption peak of DHA (Figure 1D), and the nitrogen gas absorption (BET) of DHA-modified UIO-66-NH₂ was lower than that of newly synthesized UIO-66-NH₂ (Figure 1E). Moreover, PXRD pattern of 5-FU@DHA-UIO-66-NH₂ and UIO-66-NH₂ showed no obvious differences, indicating that the framework remained intact (Figure 1F). Collectively, these results demonstrated the successful construction of UIO-66-NH₂, DHA-UIO-66-NH₂, and 5-FU@DHA-UIO-66-NH₂.

DHA-UIO-66-NH₂ framework effectively loads 5-FU

Following the construction of 5-FU@DHA-UIO-66-NH₂, we explored the loading of 5-FU by DHA-UIO-66-NH₂. Results of TGA uncovered decreased thermal stability of DHA-UIO-66-NH₂ as compared with 5-FU@DHA-UIO-66-NH₂ when the temperature reached 480 °C (Figure 2A). Then, the pore size of the MOFs was found to be mainly distributed in the range of micropores (0.5–1.5 nm) and slightly in the range of mesopores (1.5–3 nm), and the pore size of DHA-modified UIO-66-NH₂ was smaller than that of UIO-66-NH₂ (Figure 2B). Further, loading capability of 5-FU was tested in PBS, and the loading amount of DHA-UIO-NH₂ for 5-FU was 30.31% (Figure 2C). Moreover, 5-FU@DHA-UIO-66-NH₂ existed stably for 24 hours in the solution of pH from 4 to 7, showing good water stability (Figure 2D).

Figure 2 Thermal stability as well as 5-FU loading and release of DHA-UIO-66-NH₂. (A) Thermogravimetric curve of DHA-UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂; (B) pore size distribution of UIO-66-NH₂ and DHA-UIO-66-NH₂; (C) 5-FU loading rate on DHA-UIO-66-NH₂; (D) water stability of 5-FU@DHA-UIO-66-NH₂; (E) material stability detected by PXRD test after three rounds of drug release; (F) the drug release rate of 5-FU in solution of pH =5.5 or 7.4. Each experiment was repeated three times. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid; PXRD, powder X-ray diffraction.

Furthermore, the 5-FU-carrying DHA-UIO-66-NH₂ framework remained stable even after three rounds of drug release (Figure 2E). Considering the nearly neutral pH value in normal human cells and the acidic condition in tumor cells, we detected drug release under the conditions of pH 5.5 and pH 7.4. Approximately 37% of 5-FU was released from DHA-UIO-66-NH₂ under pH 7.4 and 62–75% was released under pH 5.5 (Figure 2F). In other words, the release of drugs was augmented by the weak acid microenvironment of the tumor. Taken together, our data suggested that DHA-UIO-66-NH₂ effectively loaded 5-FU and that 5-FU release may be promoted in tumors.

DHA-UIO-66-NH₂ and UIO-66-NH₂ particles can be effectively endocytosed by BC cells

Subsequently, we incubated MCF-7 cells with FITC-labeled DHA-UIO-66-NH₂ or UIO-66-NH₂ to observe the uptake of the two MOFs by MCF-7 cells. In the first 0.5 h, the cells presented with no obvious green fluorescence, indicating that only a few DHA-UIO-66-NH₂ or UIO-66-NH₂ were endocytosed. With the incubation time extending to 3 and 6 h, stronger FITC green fluorescence was observed in the cytoplasm and nucleus, suggesting that more MOFs penetrated the membrane and entered the cells (Figure 3A,3B). Hence, DHA-UIO-66-NH₂ and UIO-66-NH₂ particles were effectively endocytosed by MCF-7 BC cells.

Figure 3 The uptake of DHA-UIO-66-NH₂ and UIO-66-NH2 by MCF-7 cells. (A) Representative images of CLSM of MCF-7 cells incubated with FITC-DHA-UIO-66-NH₂ at 37 °C for 0.5, 1, 3, and 6 h. Green fluorescence, staining of FITC-DHA-UIO-66-NH₂; blue fluorescence, staining of nuclei; (B) representative images of CLSM of MCF-7 cells incubated with FITC-UIO-66-NH₂ at 37 °C for 0.5, 1, 3, and 6 h. Green fluorescence, staining of FITC-UIO-66-NH₂; blue fluorescence, staining of nuclei. Each cellular experiment was repeated three times. DHA, docosahexaenoic acid; CLSM, confocal laser scanning microscope.

5-FU@DHA-UIO-66-NH₂ represses the proliferation of BC cells and augments the apoptosis

Following cellular uptake, we then explored the cytotoxicity of these 5-FU-carrying MOFs. After treatment with UIO-66-NH₂, 5-FU@UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂ of different concentrations separately, the cell viability of MCF-7 cells was measured by MTT. According to the results, the concentration of 5-FU@UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂ was linearly related to the cell viability of MCF-7 cells, and 5-FU@DHA-UIO-66-NH₂ showed greater cytotoxicity relative to 5-FU@UIO-66-NH₂ (Figure 4A,4B). This may be explained from two aspects: DHA may increase the ability of the MOF to penetrate through the cell membrane; the drug can be released more effectively by DHA-modified MOF into MCF-7 cells.

Figure 4 The effects of 5-FU@DHA-UIO-66-NH₂ on the viability and apoptosis of MCF-7 cells. (A) The effects of UIO-66-NH₂, 5-FU@UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂ on MCF-7 cell viability; (B) the effects of UIO-66-NH₂, 5-FU@UIO-66-NH₂ and 5-FU@DHA-UIO-66-NH₂ on the number of MCF-7 cells; (C) representative images of optical microscopy detection of MCF-7 cells treated with UIO-66-NH₂, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂; (D) flow cytometry to detect the apoptosis of MCF-7 cells treated with UIO-66-NH₂, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂; (E) quantitative analysis of CLSM of MCF-7 cancer cells after incubation with UIO-66-NH₂, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂. ^#, P<0.05 vs. UIO-66-NH₂ group. Each cellular experiment was repeated three times. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid.

Then, the morphology and viability of the cells treated with 5-FU@DHA-UIO-66-NH₂ were evaluated by optical microscopy. As shown in Figure 4C, cell viability of MCF-7 cells was reduced in response to 24-h treatment with 3.125 µg/mL of 5-FU@DHA-UIO-66-NH₂. Co-incubation with 5-FU or 5-FU@UIO-66-NH₂ alone also led to decreased MCF-7 cell viability, but not as obvious as that of 5-FU@DHA-UIO-66-NH₂.

In addition, we employed flow cytometry to assess the effects of 5-FU@DHA-UIO-66-NH₂ on MCF-7 cell apoptosis. After 12-h co-incubation, the cells presented with the highest apoptotic rate in response to 5-FU@DHA-UIO-66-NH₂ as compared with 5-FU or 5-FU@UIO-66-NH₂ (Figure 4D). Further, stronger red fluorescence was observed after 5-FU@DHA-UIO-66-NH₂ treatment versus 5-FU@UIO-66-NH₂, indicating that most MCF-7 cells had been killed by 5-FU@DHA-UIO-66-NH₂; in contrast, cells of the control group and cells treated with UIO-66-NH₂ showed strong green fluorescence, showing that most of the cells were still alive (Figure 4E). In summary, 5-FU@DHA-UIO-66-NH₂ exhibited stronger inhibiting effects on BC cell viability as well as stimulative effects on BC cell apoptosis.

5-FU@DHA-UIO-66-NH₂ up-regulates let-7a to reduce the resistance of BC cells to 5-FU

Further to explore the downstream mechanisms, we determined the expression of let-7a in MCF-7 cells with different treatments. The level of let-7a in MCF-7 cells in response to 5-FU@DHA-UIO-66-NH₂ (1.000±0.131) was observed to be higher than that in 5-FU@UIO-66-NH₂-treated cells (0.398±0.045), and the latter was higher than that in UIO-66-NH₂-treated cells (0.226±0.031) (Figure 5A,5B).

Figure 5 5-FU@DHA-UIO-66-NH₂ up-regulates let-7a to reduce the resistance of MCF-7 cells to 5-FU. (A) 5-FU@DHA-UIO-66-NH₂-induced up-regulation of let-7a in MCF-7 cells as compared with 5-FU@UIO-66-NH₂; (B) 5-FU@DHA-UIO-66-NH₂-induced up-regulation of let-7a in MCF-7 cells as compared with UIO-66-NH₂; (C) the survival rate of 5-FU-treated MCF-7/DDP cells in the presence of let-7a overexpression; (D) the survival rate of 5-FU-treated MCF-7/DDP cells in the presence of let-7a inhibition; (E) IC₅₀ of 5-FU-treated MCF-7/DDP cells in response to let-7a inhibition/overexpression. *, P<0.05 between two groups. Each cellular experiment was repeated three times. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid.

To investigate the role of let-7a in BC cell resistance, we performed functional assays and unraveled that let-7a overexpression led to reduced survival rate of 5-FU-treated MCF-7/DDP cells and let-7a inhibition led to the opposite (Figure 5C,5D). Moreover, our data indicated that the IC₅₀ of 5-FU against MCF-7/DDP cells was decreased in the presence of let-7a mimic (10.82±0.53) relative to mimic NC (23.55±0.92), and increased in response to let-7a inhibitor (339.3±2.60) to inhibitor NC (13.15±0.86) (Figure 5E). Collectively, these results demonstrated that 5-FU@DHA-UIO-66-NH₂ up-regulated the level of let-7a to reduce the resistance of BC cells to 5-FU.

5-FU@DHA-UIO-66-NH₂ attenuates tumorigenesis and increases chemosensitivity in BC cells in vivo

After the aforementioned in vitro assay, we then evaluated the anti-tumor effects of 5-FU@DHA-UIO-66-NH₂in vivo. Compared with BC mice of the control group (treated with saline), those treated with 5-FU, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ exhibited repressed tumorigenesis, as reflected by reduced volume of xenografted tumors within 25 days (Figure 6A). Besides, the body weight of the BC mice in all groups showed similar rate of rise throughout the 25 days following drug administration (Figure 6B). Of note, the inhibiting effects on tumorigenesis of 5-FU@UIO-66-NH₂ was stronger than that of 5-FU alone (corresponding to diminished weight of xenografted tumors), while 5-FU@DHA-UIO-66-NH₂ exerted the strongest tumor-inhibiting effects (Figure 6C,6D). Taken together, our data revealed that 5-FU@DHA-UIO-66-NH₂ effectively suppressed the tumorigenic ability of BC cells in nude mice and enhanced chemosensitivity in BC.

Figure 6 The effects of 5-FU@DHA-UIO-66-NH₂ on the tumorigenesis and chemosensitivity of MCF-7 cells in nude mice. (A) Tumor volume within 25 days from the intravenous injection of 5-FU, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ in mice (*, P<0.05); (B) body weight of mice within 25 days from the intravenous injection of 5-FU, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂; (C) tumor weight 25 days after the intravenous injection of 5-FU, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ in mice; (D) photos of xenografted tumor of mice on the 25th day after treatment with 5-FU, 5-FU@UIO-66-NH₂ or 5-FU@DHA-UIO-66-NH₂ (*, P<0.05). n=10. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid.

5-FU@DHA-UIO-66-NH2 is a safe drug-carrier for chemotherapy

Subsequently, we investigated the biosafety of 5-FU@DHA-UIO-66-NH₂ through the detection of physiological toxicity in vivo. Results of HE staining showed no obvious damage to the five major organs of BC mice treated by 5-FU@DHA-UIO-66-NH₂ (Figure 7). Then, through routine blood analysis we found that the blood parameters of the mice were in the normal range on the 1st and 7th day from 5-FU@DHA-UIO-66-NH₂ administration (Table 1). Furthermore, 5-FU@DHA-UIO-66-NH₂ treatment only slightly affected the level of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) indicators, thus causing no damage to the kidney and liver (Table 1). In summary, 5-FU@DHA-UIO-66-NH₂ could be metabolized by BC mice and executed minimized toxic side effects on normal tissues. In this sense, 5-FU@DHA-UIO-66-NH₂ may serve as a safe drug carrier for BC treatment.

Figure 7 Observation on the damage of five major organs in nude mice after 5-FU@DHA-UIO-66-NH₂ treatment. Biosafety of 5-FU@DHA-UIO-66-NH₂ was evaluated by detecting through HE staining the damage on the major organs (heart, liver, spleen, lung, kidney) of mice that have been injected intravenously with 5-FU@DHA-UIO-66-NH₂ or PBS (n=10) seven days before. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid; PBS, phosphate buffered saline.

Discussion

Drug delivery systems with magnetization have attracted considerable interest for their capability of promoting the accumulation of drug at the target site (18). Especially for cancer treatment, drug loading and release based on nano-carrier with high biocompatibility and low cytotoxicity is a promising approach to improve the efficacy of conventional chemotherapy, which has long been limited by drug resistance, side effects, and low effectiveness (19). In the present study, we observed the satisfying performance of DHA-modified UIO-66-NH₂, a Zr-based MOF, when it was utilized as a carrier for 5-FU in BC treatment. We further illuminated that 5-FU@DHA-UIO-66-NH₂ can reduce the resistance of BC cells to 5-FU via regulation of let-7.

Initially, we successfully constructed UIO-66-NH₂ and DHA-UIO-66-NH₂ and characterized them with FT-IR, PXRD, solid-state NMR, and gas adsorption. As a member of MOFs, UIO-66-NH₂ is a crystalline material composed of inorganic zirconium ions connected by organic NH₂-H₂BDC nodes (20). Of note, UIO-66-NH₂ has been highlighted for properties such as large specific surface area and metal ions with various binding sites (8). Microorganism@UIO-66-NH₂ has been reported to be a prospective barcoding candidate for the detection of colorectal cancer-related miRNAs due to the synergy between microorganisms and UIO-66-NH₂ (21) and Fe₃O₄@UIO-66-NH₂ has been employed to develop a drug delivery system possessing sustained drug release, ideal biocompatibility, and effective therapeutic effects (7). Following previous efforts, in this study we modified UIO-66-NH₂ with DHA to deliver 5-FU, and our data unraveled that 5-FU@DHA-UIO-66-NH₂ existed stably for 24 hours in the solution of pH from 4 to 7 and possessed good water stability. Further, we substantiated that DHA-UIO-66-NH₂ could effectively load 5-FU and that the release of 5-FU against BC cells was promoted in the presence of DHA modification, especially in the weak acid microenvironment of BC tumors. Our findings corroborate prior documentation about pH-sensitive drug delivery systems, where DHA was suggested to be a promising modifier for drug delivery system in cancer treatment due to the low-pH microenvironment in cancer tissues (15,22).

Further, we substantiated that DHA-UIO-66-NH₂ and UIO-66-NH₂ particles could be effectively endocytosed by MCF-7 BC cells, while 5-FU@DHA-UIO-66-NH₂ exhibited stronger inhibiting effects on BC cell viability and stimulative effects on apoptosis as compared with 5-FU@UIO-66-NH₂. It was thus suggested that DHA increased the ability of the MOF to penetrate through the cell membrane, and 5-FU was released more effectively by DHA-modified UIO-66-NH₂. In relation to this, DHA has been identified in significant amounts in the retinal or neuronal cell membranes based on its high fluidity (23). It has also been established that DHA can affect cell or tissue physiology and function through different mechanisms, one of which is the alteration in membrane structure (24). Subsequently, our data revealed that 5-FU@DHA-UIO-66-NH₂ up-regulated the level of miRNA let-7a to reduce the resistance of BC cells to 5-FU. In agreement with our finding, accumulating evidence has recognized let-7a as a biomarker in the management of BC, a tumor-suppressor to inhibit the proliferation of BC cells (25,26). A prior report further illuminated that DHA up-regulated the expression of let-7a and thus increase the sensitivity of BC cells to 5-FU (11). In vivo experiments also validated that 5-FU@DHA-UIO-66-NH₂ effectively suppressed the tumorigenic ability of BC cells and enhanced the chemosensitivity in BC mice. Moreover, we examined the biosafety of 5-FU@DHA-UIO-66-NH₂. It was revealed that 5-FU@DHA-UIO-66-NH₂ could be metabolized by BC mice and exerted minimized toxic effects on major organs and normal tissues of the mice.

Conclusions

Based on the evidence acquired, the present study elucidated that 5-FU could be effectively loaded and released by DHA-modified UIO-66-NH₂, and that 5-FU@DHA-UIO-66-NH₂, with validated biocompatibility and biosafety, up-regulated the level of let-7a to reduce the resistance of BC cells to 5-FU (Figure 8). By constructing 5-FU@DHA-UIO-66-NH₂ and investigating the role of this novel drug delivery system in BC, this study characterized DHA-UIO-66-NH₂ as a promising MOF for the development of nano-targeted therapy for BC. Due to the ultrahigh surface area and porosity of MOFs, the 5-FU drug loading capacity can be greatly increased; in addition, MOFs have well-defined structure, tunable pore size, and easy chemical functionalization. When MOFs were modified with DHA, the terminal carboxyl functional group of DHA can improve the biocompatibility of MOFs and enhance the synergy with anticancer drugs (27-29). However, the MOFs used in this study still have the common problems of MOFs, such as cumbersome synthesis steps, toxic reagents needed and difficult storage.

Figure 8 The mechanism graph of the regulatory role of 5-FU@DHA-UIO-66-NH₂ in BC. 5-FU@DHA-UIO-66-NH₂ up-regulated the level of let-7a to reduce the resistance of BC cells to 5-FU though inhibiting BC cell viability and promoting the apoptosis. 5-FU, 5-fluorouracil; DHA, docosahexaenoic acid; BC, breast cancer.

Acknowledgments

Funding: This study was supported by Heilongjiang Provincial Natural Science Foundation of China (No. H2017048).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://dx.doi.org/10.21037/atm-21-5978

Data Sharing Statement: Available at https://dx.doi.org/10.21037/atm-21-5978

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/atm-21-5978). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were approved by the Animal Ethics Committee of Harbin Medical University Cancer Hospital (approval number: 2019-185) and performed according to the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health. Extensive efforts were made to ensure minimal suffering of the animals used in the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: B. Meiser)