Drug delivery carriers with a high drug loading capacity and biocompatibility, especially for controlled drug release, are urgently needed due to the side effects and frequent dose in the traditional therapeutic method. Guided by nanomaterials, we have successfully synthesized zirconium-based metal−organic frameworks, Zr-TCPP (TCPP: tetrakis (4-carboxyphenyl) porphyrin), namely, PCN-222, which is synthesized by solvothermal method. And it has been designed as a drug delivery system (DDS) with a high drug loading of 38.77 wt%. In our work, PCN-222 has achieved pH-sensitive drug release and showed comprehensive SEM, TEM, PXRD, DSC, FTIR, and N2 adsorption-desorption. The low cytotoxicity and good biocompatibility of PCN-222 were certificated by the in vitro results from an MTT assay, DAPI staining, and Annexin V/PI double-staining even cultivated L02 cells and HepG2 cells for 48h. Furthermore, Oridonin, a commonly used cancer chemotherapy drug, is adsorbed into PCN-222 via the solvent diffusion technique. Based on an analysis of the Oridonin release profile, results suggest that it can last for more than 7 days in vitro. And cumulative release rate of Ori at the 7 d was about 86.29% and 63.23% in PBS (pH 5.5 and pH 7.2, respectively) at 37°C. HepG2 cells were chosen to research the cytotoxicity of PCN-222@Ori and free Oridonin. The results demonstrated that the PCN-222@Ori nanocarrier shows higher cytotoxicity in HepG2 cells compared to Oridonin.
In the past two decades, microporous metal−organic frameworks (MOFs), combined by different metal ions or clusters and organic ligands [
The Zr6 cluster is thought to be a perfect building block to synthesize the mixed-linker MOFs, because of their intrinsic open frameworks, superior stability, and adjustable connectivity [
Structural diagram. (a) schematic illustration for the construction of PCN-222 and (b) the chemical structure of Oridonin.
Oridonin (Ori) is a well-known ent-kaurene tetracyclic diterpenoid compound (Figure
Based on the above thought, we have successfully synthesized a biocompatible Zr-based nanoscale MOFs (PCN-222) with unique advantage, high load dose, nontoxicity, biocompatibility, pH-sensitive release, etc. We aim to prepare a PCN-222@Ori sustained-release and controlled-release drug delivery system by using PCN-222 load Ori. To the best of our knowledge, PCN-222 is the first MOF that carries Oridonin.
The synthesis of PCN-222 was realized by dissolving meso-tetra (4-carboxyphenyl) porphyrin (TCPP, 0.4g) and zirconium chloride octahydrate (ZrOCl2·8H2O, 2.0g) in 500 mL of dry N, N-dimethylformamide (DMF), and 300 mL of formic acid. The mixture was placed in a round bottom flask equipped with a condenser and was kept stirring and heated for three days at 408 K under air. The mixture was returned to room temperature. Dark red solid was recovered by filtration. In order to remove unreacted starting ligands, inorganic species, as-synthesized PCN-222 (~100mg) samples were immersed into 100mL DMF with 3mL of 4M HCl at 120°C for 12 h. After cooling to room temperature, the supernatant was carefully decanted and washed with DMF and acetone for three times. Fresh acetone was subsequently added, and the sample had a right to stay for 8h to exchange and remove the nonvolatile solvates (DMF) and this process was repeated six times. After removal of acetone by decanting, the sample was activated by drying under vacuum for 6h.
The synthesized PCN-222 was characterized by scanning electron microscopy (the samples that need checking were fixed on the aluminum sample column with carbon conductive tape and observed after the gold sputtering treatment 2.5min, SEM, Hitachi S-4800, Japan), transmission electron microscopy (the samples were mixed by anhydrous alcohol, a drop of the prepared solution was transferred to the carbon film coated grids for overnight drying, TEM, JEM-1230, Japan), N2 adsorption-desorption at 77K (Micromeritics ASAP 2020, USA), powder X-ray diffraction (Cu-K
Human hepatoma cells (HepG2, Jenniobio Biotechnology, China) and human normal hepatocyte cells (L02, Jenniobio Biotechnology, China) were incubated in a humidified incubator (37°C, 5 % CO2) for two or three days employing DMEM (Coning, USA) with 10 % fetal bovine serum (FBS, China) and 1 % penicillin-streptomycin solution (USA). The cytotoxicity of PCN-222 at various concentrations (0, 10, 20, 40, 80, and 160
The PCN-222@Ori pH-sensitive nanoparticles were prepared by the solvent diffusion technique (QESD). Ori (400.48 mg, Nantong Feiyu Biological Technology Co., Ltd., China) was dissolved in methanol and made into 8.0 mg/mL standard solutions. The dried PCN-222 (3 mg) with a different amount of the standard solution in Xilin bottles was subject to magnetic stirring at room temperature. The orthogonal design L9 (33) was implemented to ensure the optimum process. And we studied how the ratio of drug to PCN-222 (A), mixing time (B), and the amount of solvent (C) influenced the process of loading. SEM, TEM, XRD, Fourier transform infrared spectroscopy (studied by the KBr method, FTIR, Thermo Fisher Nicolet6700, USA), and differential scanning calorimetry (the samples were placed on a flat-bottomed aluminum plate and tested at 40-300°C, heating rate of 10°C/min, and nitrogen flow rate of 50 mL/min, DSC, Mettler-Toledo Stare, Switzerland) were used to describe its characteristics.
PCN-222@Ori was precipitated through methanol cleaning twice and centrifuging (9500 rpm, 15 min) after 2d, 3d, or 4d. The supernatant of methanol was collected and its concentration of Ori was determined by HPLC (Methanol: Water = 55: 45,
For the determination of the impregnated drug molecule release from the PCN-222@Ori, an in vitro cumulative Ori release study was performed in various pH environments. A semipermeable dialysis bag diffusion technique (dialysis bag, MW3400, MD34 mm, USA) was implemented to evaluate the cumulative drug release. Firstly, approximately 10mg of PCN-222@Ori was dispersed at pH7.2 (100mL) followed by incubation at 37°C, and then exactly the same experimental procedure was executed at pH5.5 (100mL). At the given time, 3 mL of the digestion liquor was collected, and the equivalent volume of fresh PBS was added. And the digestion liquor was filtered through a 0.45
As large pore kind of Zr-MOF, PCN-222 can be synthesized in aqueous phase under mild conditions and is a promising candidate for encapsulating anticancer drugs. Dark violet rod-like crystals of PCN-222 were obtained via solvothermal reactions. SEM (Figure
Surface topography and structural analysis of PCN-222: (a) SEM, (b) TEM, (c) PXRD spectra, (d) the N2 adsorption-desorption isotherms at 77 K for PCN-222, (e) emission wavelength spectrum, and (f) excitation wavelength spectrum of PCN-222 (dispersion in high purity water).
For the delivery of nanomaterials with large specific surface area, it easy to enter and deposit into the liver cells. The cytotoxicity of the PCN-222 in L02 cells was determined by the MTT assay. Cells were treated with different concentrations (0–160
Effects of PCN-222 on cell viability and morphology. (a) MTT assay data were presented as mean ± SD of viability % of three independent experiments. (b) LDH assay was used to assess cell membrane damage and results were presented as mean ± SD of three independent experiments. (
Effects of PCN-222 on apoptosis in L02 cells. (a) Flow cytometry detection of apoptosis with FITC-Annexin V/PI double staining. (b) The percentages of viable, early apoptosis, and necrosis cells of L02 cells after incubation with different concentrations of PCN-222 for 48 h. The data are expressed as means ± SD from three independent experiments (
By using the L9(34) orthogonal table, three factors such as rate of Ori to PCN-222 (A), mixing time (B), and the amount of solvent (C) were selected to be optimized. The drug loading rate reached up to 38.77 wt%, under optimized conditions: PCN-222: Ori: Methanol (1: 3: 1), with magnetic stirring for 4 days. To further confirm the existence of the drug Ori in PCN-222@Ori, the 1H-NMR spectra of alkaline-digested PCN-222@Ori were analyzed in KOH/D2O solution, as shown in Figure
1H-NMR spectra of (a) alkaline-digested PCN-222@Ori, (b) alkaline-digested Ori, and (c) PCN-222 in KOH/D2O solution.
Surface topography and structural analysis of PCN-222@Ori: (a) SEM, (b) TEM, (c) FTIR, (d) the N2 adsorption-desorption isotherms at 77 K, (e) XPRD, and (f) DSC.
The pH value in tumor and inflammatory tissues tends to be more acidic (pH 6.0–7.0) than that in blood and healthy tissue (pH 7.4), just as was said by Valeria De Matteis [
Fitting curve by different mathematical model under different pH values (Rt: accumulated release rate).
pH=5.5 | pH=7.2 | |||
---|---|---|---|---|
Equation | r2 | Equation | r2 | |
zero-order | | 0.811 | | 0.850 |
First-order | Ln(1-Rt) = -0.0118 t - 0.2627 | 0.944 | Ln(1-Rt) = - 0.0065t - 0.1379 | 0.897 |
Higuchi | | 0.952 | Rt= 0.055t1/2 + 0.0079 | 0.963 |
Weibull | lnln[1/(1- | 0.977 | lnln[1/(1- | 0.987 |
Ritger-Peppas | ln ( | 0.933 | ln ( | 0.981 |
In vitro Ori release profiles of the PCN-222@Ori at different pH values. (a) The release curve and (b) the total amount of drug release.
Free Ori and PCN-222@Ori were also investigated for the cancer therapy. PCN-222@Ori showed excellent therapeutic efficacy for HepG2 cells as the dosage increased with time prolonged, compared with free Ori. As is shown in Figure
Comparison of the antitumor activity of PCN-222@Ori and free Ori by incubating various concentrations of samples for 24 h, 48 h, and 72 h with HepG2 cells.
The inorganic cluster of PCN-222 (Zr6(
In summary, we have successfully fabricated a PCN-222, which can be used as an ideal drug delivery system. Frameworks with high surface area and suitable pore size are good candidates for Ori loading. What is more, it provides opportunity to reduce the adverse effects of Ori because the system showed a release mechanism. Low cytotoxic activity, efficient drug loading capacity (about 38.77 wt %), and controlled release with a Weibull distribution drug release under different pH values of PCN-222 prove its practical value as a drug carrier. In particular, the pH-responsive release of material confirms the potential applications of PCN-222 as smart drug carriers.
The original data used to support the findings of this study are available from the corresponding author upon request.
The authors have declared that there are no conflicts of interest.
Xin Leng, Xingbin Yin, and Jian Ni designed the research. Xin Leng, Longtai You, and Wenping Wang performed the experiments. Hongliang Huang and Na Sai conducted the data analyses. Leng Xin wrote the paper. All authors reviewed the manuscript.
This work was financially supported by the National Natural Science Foundation of China (No. 81703715, 21536001, and 21606007); the Training Programme Foundation for the Beijing Municipal Excellent Talents (No. 2017000020124G295); and the Fundamental Research Funds for the Central Universities (Beijing University of Chinese Medicine Scientific Research Project for Distinguished Young Scholar (No. 2018-JYBXJQ005). The test was assisted by BioNMR Facility, Tsinghua University, Branch of China, National Center for Protein Sciences (Beijing).