To sum up, Zn2(EBNB)2(BPY)2·2H2O can be synthesized by solvothermal method via(E)-bis(p-3-nitrobenzoic acid) ethylene ligand (C16H10N2O8). After drying and activation treatment, Zn2(EBNB)2(BPY)2·2H2O was loaded into Methadone with high drug loading. And the drug release curve shows that Zn2(EBNB)2(BPY)2·2H2O has slow-release function and can prolong Methadone's work. In addition, Zn2(EBNB)2(BPY)2·2H2O has good biocompatibility and is expected to become an excellent drug carrier.
In this study, MOFs was introduced into the field of drug release. Some features of MOFs, such as high specific surface area, tailorable, designable, adjustable channel size and channel surface functionalization, are used to study the sustained-release mechanism of MOFs as a new drug delivery form, and provide reference information for the research and development of new dosage forms of anti-drug addiction drugs.
Experimental section
Chemistry
Synthesis of (E) - bis (p-3-nitrobenzoic acid) ethylene ligand (C16H10N2O8)
200ml concentrated sulfuric acid was pouring into a 400ml flask, and 100ml fuming nitric acid was added under 0℃ ice water bath while stirring constantly. Then 10g of p-chloromethylbenzoic acid was add in small parts. After 90 minutes of reaction, p-chloromethylbenzoic acid was completely dissolved into the mixed acid. All substances in the bottle was pouring into 600ml of ice water, a large amount of white solid is precipitated immediately. The residual mix-acid was filtered and washed, and then the product was recrystallized in toluene solvent. The white crystal of 3-nitro-p-chloromethylbenzoic acid was obtained and dried in an oven (Yield: 89%). 1HNMR(200MHz DMSO-d6)δ8.76(s,1H);8.36(d,1H);7.87(d,1H);5.03(s,2H)
50ml anhydrous ethanol was poured into a 300ml beaker, and 5.7g KOH was dissolved in it. Then 5.00g of 3-nitro-p-chloromethylbenzoic acid was poured in to form brown precipitate, which is potassium salt of (E) - di (p-3-nitrobenzoic acid) ethylene. After reaction at room temperature for 45 min, the solid was vacuumed and dissolved in 70ml water. Adding HCl to the aqueous solution to adjust pH=1 then form a precipitate. The solid was collected and recrystallized with tetrahydrofuran solvent. The yellow crystal (E)-di(p-3-nitrobenzoic acid) vinyl (C16H10N2O8) compound was obtained and dried in an oven (Yield: 78%). 1HNMR(200MHz DMSO-d6)δ7.62(s,2H);7.89(d,2H);8.52(d,2H);9.06(s,2H)
Synthesis of Zn2(EBNB)2(BPY)2·2H2O
The (E)-di(p-3-nitrobenzoic acid) ethylene ligand (C16H10N2O8) (0.15mmol), 4,4'-bipyridine ligand (BPY,0.15mmol), zinc nitrate (Zn(NO3)2·6H2O,0.15mmol), ethanol 2ml and H2O 10ml were put into a 50ml high-pressure reactor, and the pH was adjusted to 9, ultrasound, 150 ℃ for two days. The rhombic yellow crystal (E)-di(p-3-nitrobenzoic acid) ethylene can be obtained after filtration and washing. Element analysis: C 52.45, n 9.34, H 3.11, Zn 10.89%; theoretical value: C 52.37, n 9.40, H 3.02, Zn 10.98%. The product is composed of C26H18N4O9Zn (595.81).
Determination of structure
The single crystal data of Zn2(EBNB)2(BPY)2·2H2O were measured by Bruker smart-1000 CCD single crystal X-ray diffraction analyzer. Crystal structure analysis and structure refinement use SHELXL-2014 software[22]. The main crystallographic data are listed in the table 2, 3, 4.
CCDC: 1545065 for 3-nitro-p-chloromethylbenzoic acid
CCDC: 912099 for (E)-di(p-3-nitrobenzoic acid) vinyl
CCDC: 910300 for Zn2(EBNB)2(BPY)2·2H2O
Table 2 Crystallographic data and structural correction conditions of 3-nitro-p-chloromethylbenzoic acid
Empirical formula C7H5ClN2O4
Fomular weight 216.58
Crystalsystem Monoclinic
Space group P2(1)/c
Wavelength(Å) 0.71073
Temperature(K) 293(2)
a(Å) 7.5316(7)
b(Å) 17.0508(15)
c(Å) 14.1080(13)
α(°) 90.00
β(°) 100.6050(10)
γ(°) 90.00
V(Å3) 1780.8(3)
Z 8
Density (g/cm3) 1.515
μ(mm-1) 0.418
F(000) 880
θ range (°) 2.389 to 23.956
GOF on F2 1.036
R1, wR2 [I>2σ(I)] R1=0.0661
wR2=0.1719
R1, wR2 (all data) R1=0.1127
wR2=0.2159
Table 3 Crystallographic data and structural correction conditions of (E)-di(p-3-nitrobenzoic acid) vinyl
Empirical formula C16H13N2O9
Fomular weight 385.28
Crystalsystem Triclinic
Space group P-1
Wavelength(Å) 0.71073
Temperature(K) 293(2)
a(Å) 7.3757(4)
b(Å) 7.7827(5)
c(Å) 15.2903(10)
α(°) 79.473(4)
β(°) 82.9270(10)
γ(°) 72.069(4)
V(Å3) 818.92(9)
Z 2
Density (g/cm3) 1.545
μ(mm-1) 0.132
F(000) 398
θ range (°) 2.717 to 28.347
GOF on F2 1.029
R1, wR2 [I>2σ(I)] R1=0.0444
wR2=0.1161
R1, wR2 (all data) R1=0.0600
wR2=0.1321
Table 4 Crystallographic data and structural correction conditions of Zn2(EBNB)2(BPY)2·2H2O
Empirical formula C26H18N4O9Zn
Fomular weight 595.81
Crystalsystem Triclinic
Space group P-1
Wavelength(Å) 0.71073
Temperature(K) 293(2)
a(Å) 8.1862(9)
b(Å) 11.4220(14)
c(Å) 14.2863(17)
α(°) 96.8030(10)
β(°) 93.0450(10)
γ(°) 102.472(2)
V(Å3) 1290.8(3)
Z 2
Density (g/cm3) 1.533
μ(mm-1) 1.013
F(000) 608
θ range (°) 2.557 to 26.079
GOF on F2 1.008
R1, wR2 [I>2σ(I)] R1=0.0499
wR2=0.1067
R1, wR2 (all data) R1=0.0750
wR2=0.1153
Methadone loaded into Zn2(EBNB)2(BPY)2·2H2O
Accurately weighing proper amount of Methadone, and prepare the solution with methanol solvent. The maximum absorption peak was 292nm. Then the concentration gradient of the standard solution is prepared, and the standard curves of absorbance (A) and concentration (c) at this wave length are established. The standard curve is suitable for drug loading environment.
In the same way as the above operation, the appropriate concentration of Methadone solution was prepared with pH = 7.4 phosphate buffer (PBS) as the solvent, and the standard curves of absorbance (A) and concentration (c) were established with PBS as the blank control. The standard curve is suitable for the release environment.
Accurately weighing 2mg of dried and activated Zn2(EBNB)2(BPY)2·2H2O, add in 1ml of methanol solution which contain 10mg of Methadone, mixing under ultrasound, acting for 24h. Then centrifugate the reaction solution (10000rpm, 10min), take 100 μl of the supernatant, diluted to 10ml (100 times), measure its absorbance at 292 nm, and calculate the drug loading of Zn2(EBNB)2(BPY)2·2H2O.
Drug loading = (amount of drug in MOF / total mass of MOF)×100%
In vitro release of Methadone from Zn2(EBNB)2(BPY)2·2H2O
Take 2mg of Zn2(EBNB)2(BPY)2·2H2O loaded with Methadone and put it into 20 ml PBS buffer solution, which is the experimental group. Take another 1.16 mg of Zn2(EBNB)2(BPY)2·2H2O and put it into 20 ml PBS buffer, which is the blank group. In (37±1℃) constant temperature oscillator (oscillation speed: 100r·Min-1), take 1ml of solution every 12h and add in equal amount of fresh PBS buffer, then centrifugate the solution (12000rpm, 20 minutes), after that take the supernatant to determine its absorbance by ultraviolet spectrum. The content of Methadone in the solution was calculated according to the established Methadone standard curve, and the relationship curve between cumulative release and time was drawn. The in vitro release performance of Methadone from Zn2(EBNB)2(BPY)2·2H2O was investigated accordingly.
Cytotoxicity of Zn2(EBNB)2(BPY)2·2H2O
HeLa cells were cultured in RPMI 1640 medium, which contains 10% fetal bovine serum. HeLa cells were used only at logarithmic growth stage and good growth state. Cells were digested with 0.25% trypsin and centrifuged it to precipitate, then RPMI 1640 culture medium which contains 10% fetal bovine serum was used to prepare cell suspension with a cell density of 1×105 cells·ml-1. 104 cells per well were inoculated into 96 well culture plate (six multiple holes), each hole was 100 μ L.
Then the culture plate was transferred to the incubator, and 100-μ L RPMI 1640 culture medium was replaced at 37 ℃, 5% CO2 and saturated humidity for 24 hours. The experiment was divided into three groups: blank control (without cells), negative control (without cells, without Zn2(EBNB)2(BPY)2·2H2O), Zn2(EBNB)2(BPY)2·2H2O (with cells, with Zn2(EBNB)2(BPY)2·2H2O). Then,100 μl solution of each group was added in to make the mass concentration of 200,100, 50, 25, 12, 6 μg·ml-1 respectively, continue to culture for 48h. Then change 100 μl RPMI 1640 culture solution for each hole and 20 μl MTT solution (5mg·ML-1) was added. Shake it on a micro oscillator for 20 min, continue to culture for 24h, remove the culture solution, add 150 μl DMSO for each hole, absorbance (A) at 490 nm was determined by enzyme-linked immunosorbent assay, then the cell survival rate can be calculated by accordingly.
Survival rate% = (drug group - blank group) / (negative control group - blank group) × 100%