CN113893355A - Metal organic framework modified by sulfhydryl chitosan surface as oral drug carrier, preparation method and application thereof - Google Patents
Metal organic framework modified by sulfhydryl chitosan surface as oral drug carrier, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of preparation of oral administration systems, in particular to a metal organic framework modified by a sulfhydryl chitosan surface as a medicine oral carrier, a preparation method and application thereof. The metal organic framework takes Zr as metal ions and 2, 6-naphthalene diacid as an organic ligand, then a medicine is loaded in the metal organic framework to obtain Zr-NDC, and then the surface of the metal organic framework is wrapped by sulfhydryl chitosan to obtain TCS-Zr-NDC @ Drug. The preparation process is simple and convenient, and the obtained drug carrier material has excellent performance. The TCS-Zr-NDC @ Drug can prevent the Zr-NDC from degrading in the stomach, improve the stability of the Zr-NDC in an acidic environment, form a disulfide bond with a cysteine-rich region in the glycoprotein of intestinal mucus layer, remarkably increase the adhesion and permeability of the Drug delivery system to the intestinal mucosa, and provide guarantee for a metal organic framework as a Drug oral carrier.
Description
Technical Field
The invention relates to the field of preparation of oral administration systems, in particular to a metal organic framework modified by a sulfhydryl chitosan surface as a medicine oral carrier, a preparation method and application thereof.
Background
Metal-Organic Frameworks (MOFs), which are a very promising class of crystalline microporous materials, are emerging solid materials with periodic network structures constructed by coordination of Metal ions or Metal clusters and Organic bridging ligands. MOFs have a wide range of applications including gas separation and storage, catalysis, sensing, fuel cells, electronics, drug delivery, and many others. The metals which currently constitute the MOFs include main group elements, transition elements, lanthanide elements and the like, and in addition, the metal elements have multiple valence states and can be subjected to different coordination and combination. The organic ligands are various in variety, the nitrogen-containing organic ligands are generally used for organizing the MOFs in the early stage, and the MOFs constructed by the carboxylic acid ligands are found to have better stability in the later stage and are easier to perform surface modification on the MOFs. These unique advantages have led to the extensive study of MOFs materials as a good drug carrier.
However, most of the MOFs themselves have weak coordination bonds, are easily damaged by gastric acid, and have poor stability in gastrointestinal conditions, thereby limiting the application of the MOFs in oral administration. In 2016, researchers modified the MOFs with modified polycaprolactone to achieve oral administration of paclitaxel and cisplatin. In 2017, Hidalgo and the like adopt chitosan modified MIL-100 to load ibuprofen, and prepare a nano MOFs carrier suitable for oral administration. As previously explored, we can see that surface modification can both retain the advantages of MOFs and provide stability to oral drug delivery systems.
According to the data, sulfhydryl chitosan (TCS) is a biocompatible polysaccharide which can prevent the degradation of MOFs in the stomach, and can form a disulfide bond with a cysteine-rich region in the intestinal mucus layer glycoprotein, and the covalent bond is much stronger, so that the adhesiveness and the permeability of TCS to the intestinal mucosa are remarkably increased. Therefore, the surface modification of TCS on MOFs can prevent the MOFs from being degraded outside the plan of the stomach, improve the stability of the MOFs, improve the permeability of intestinal mucosa through TCS and provide guarantee for the MOFs as a drug oral carrier. In addition, MOFs (Zr-MOFs) using zirconium as a metal cluster have excellent thermal stability and chemical stability, and have a wide application range. Zirconium is a biocompatible metal, and humans typically contain about 300mg of zirconium, with a daily zirconium intake of 4.15mg being recommended, and among the existing MOFs, Zr-NDC is particularly suitable for biological applications due to its optimal stability to hydrolysis and low toxicity. Based on the structure, the invention provides a preparation method and application of a metal organic framework drug delivery system for oral administration.
Disclosure of Invention
The invention aims to provide a sulfhydryl chitosan modified metal organic framework oral administration carrier. Zr-MOFs with high safety and biocompatibility are adopted, and are subjected to surface modification by using sulfhydryl chitosan, so that the advantages of the MOFs are retained, meanwhile, the MOFs can be prevented from being degraded in the stomach, the biological adhesion to the intestine can be improved, the permeability of the intestinal mucosa is improved, and the MOFs oral administration carrier is obtained.
In order to achieve the purpose, the technical scheme of the invention is as follows: a surface-modified metal organic framework drug carrier material is characterized in that Zr is used as metal ions, 2, 6-naphthalene diacid is used as an organic ligand to obtain Zr-NDC, and sulfhydryl chitosan is used for carrying out surface modification on the Zr-NDC to obtain the drug carrier material.
The preparation method of the surface modified metal organic framework drug carrier material is characterized by comprising the following steps:
1) sequentially adding ZrCl into a container4Uniformly mixing 2, 6-naphthalene diacid, N-dimethylformamide, acetic acid and deionized water, reacting, cooling, centrifuging, washing and drying to obtain Zr-NDC;
2) dissolving a Drug in ethanol, dissolving Zr-NDC in the ethanol, ultrasonically dispersing and mixing the two, stirring for a certain time, performing rotary evaporation, washing, centrifuging and drying to obtain a Drug-loaded metal organic framework Zr-NDC @ Drug;
3) mixing the ethanol dispersion solution of the metal organic framework Zr-NDC @ Drug-carrying system with the acetic acid aqueous solution of the sulfhydryl chitosan, stirring, centrifuging, washing and drying to obtain the metal organic framework Drug-carrying system TCS-Zr-NDC @ Drug.
The preparation method of the surface-modified metal organic framework drug carrier material is characterized by comprising the following steps of: in the step 1), the reaction temperature is 100-130 ℃, and the reaction time is 12-36 h.
The preparation method of the surface-modified metal organic framework drug carrier material is characterized by comprising the following steps of: in the step 2), the mass ratio of the medicament to the Zr-NDC is 2-5: 2.
The preparation method of the surface-modified metal organic framework drug carrier material is characterized by comprising the following steps of: in the step 2), the ultrasonic temperature is 25-75 ℃; the stirring time is 1-12 h.
The preparation method of the surface-modified metal organic framework drug carrier material is characterized by comprising the following steps of: in the step 3), according to the mass ratio, Zr-NDC @ Drug: mercaptochitosan ═ 1: 1 to 2.5.
The preparation method of the surface-modified metal organic framework drug carrier material is characterized by comprising the following steps of: in the step 3), the stirring time is 0.5-2 h.
The application of any surface modified metal organic framework drug carrier material in the preparation of oral administration systems.
The invention has the following beneficial effects:
1. the preparation method is simple, easy to operate, low in cost and suitable for large-scale production.
2. The drug carrier TCS-Zr-NDC prepared by the invention not only can load 5-FU, but also can load other oral drugs, thereby improving the compliance of patients and widening the application space of drugs.
3. The sulfhydryl chitosan adopted by the invention can form a disulfide bond with a cysteine-rich region in the intestinal mucus layer glycoprotein, and the adhesiveness and permeability of TCS to the intestinal mucosa are obviously improved. Therefore, by modifying the surface of the Zr-NDC with TCS, the Zr-NDC can be prevented from being degraded outside the plan of the stomach, the stability of the Zr-NDC is improved, the permeability of the intestinal mucosa can be improved through the TCS, and powerful guarantee is provided for the Zr-NDC as a medicine oral carrier.
4. The invention adopts Zr-NDC with higher biocompatibility and takes 5-FU as a model drug, and the huge specific surface area of the Zr-NDC can obviously improve the drug loading of the 5-FU. Meanwhile, TCS is adopted to carry out surface modification on the Zr-NDC @5-FU, an oral administration system is constructed, the Zr-NDC @5-FU is prevented from being degraded and damaged in the digestive tract, and the problems that the Zr-NDC stays in the digestive tract for a short time after being taken orally, cannot pass through intestinal mucosa easily, and is removed rapidly in vivo and the like are solved.
Drawings
FIG. 1 is an IR spectrum of TCS, Zr-NDC, TCS-Zr-NDC;
FIG. 2 Zeta potential diagram of Zr-NDC;
FIG. 3 Zeta potential diagram of TCS-Zr-NDC;
FIG. 4 DSC spectra of TCS, Zr-NDC, TCS-Zr-NDC;
FIG. 5 XRD spectra of TCS, Zr-NDC, TCS-Zr-NDC;
FIG. 6 TGA spectra of TCS, Zr-NDC, TCS-Zr-NDC;
FIG. 7 SEM chromatogram of TCS-Zr-NDC;
FIG. 8 is a graph showing the release profiles of TCS-Zr-NDC @5-FU and Zr-NDC @5-FU in artificial gastric fluid and artificial intestinal fluid;
FIG. 9 wherein (a) is the inhibitory effect of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU on HEK293 cells; wherein (b) is the inhibitory effect of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU on HepG2 cells; wherein (c) is the inhibition of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU on A549 cells; wherein (d) is the inhibition of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU on DLD cells;
FIG. 10 absorption rate constants (Ka) for 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU in different intestinal sections;
FIG. 11 apparent absorption coefficients (P) of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU in different intestinal sectionsapp)。
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention but are merely illustrative thereof.
The following examples zirconium tetrachloride, 2, 6-naphthalenediamine and chitosan were obtained from Allandine reagent (Shanghai) Co., Ltd. 5-FU (> 99.9%) was purchased from Shanghai Michelin Biotech, Inc. The various reagents and procedures used in the examples are conventional in the art, unless otherwise indicated.
The drug described in the examples below is 5-fluorouracil (5-FU).
The drug loading rate calculation formula of the Zr-NDC @5-FU is shown as (1) and (2)
Wherein:
EE% is the encapsulation efficiency;
DL is drug loading (g.g)-1)；
W5-FUInitial dosage (mg);
W′5-FUis the unloaded dose (mg);
WMOFas a carrier of Zr-NDC mass (mg)
Calculating the cumulative release rate Q by the following formula:
wherein:
Ctthe drug concentration in the release medium (mg/mL) was measured for each time point;
w is the total amount of the drug (mg) added;
V0to release the total volume of the medium
The cell inhibition rate is calculated as shown in (4)
The drug absorption rate constant (Ka) and the apparent drug absorption coefficient (Papp), and the formulas (5), (6) are calculated:
wherein:
Vinvolume of test solution (mL) to be filled;
Voutvolume of test solution collected (mL);
v is perfusion speed (mL. min)-1)；
CinThe mass concentration (mu g. mL) of the drug in the perfusion fluid at the entrance of the intestinal tract-1)；
CoutThe mass concentration (μ g. mL) of the drug in the perfusion solution at the outlet of the intestinal tract-1)；
L is the length (cm) of the perfused intestinal segment;
r is the cross-sectional radius (cm) of the perfused intestinal segment.
EXAMPLE 1 preparation of a Metal-organic framework Zr-NDC
Weighing 0.4g of zirconium chloride, placing the zirconium chloride in a 100mL reaction kettle, adding 20mL of DMF, performing ultrasonic dispersion and dissolution at 50-60 ℃, and adding 2.85mL of glacial acetic acid for later use. Weighing 0.368g of 2, 6-naphthalenedicarboxylic acid, placing the 2, 6-naphthalenedicarboxylic acid into a 50mL centrifuge tube, adding 20mL of DMF, performing ultrasonic dissolution, adding the mixture into the reaction kettle, adding 0.125mL of deionized water, covering the kettle cover tightly, performing ultrasonic treatment at 50-60 ℃ for 30min, placing the reaction kettle into a 120 ℃ blast drying oven, heating for 24h, cooling to room temperature, and centrifuging (5000 r.min)-130min), discarding supernatant, adding 10mL of DMF, mixing, standing at room temperatureCentrifuging for 2 hours, removing supernatant, washing with absolute ethyl alcohol for three times, and drying in a drying oven at 80 ℃ to obtain the Zr-NDC.
EXAMPLE 2 preparation of Zr-NDC @5-FU
Weighing 40.0mg of 5-fluorouracil, placing the 5-fluorouracil in a 50mL flask, adding 10mL of ethanol, ultrasonically dissolving for later use, weighing 20.0mg of Zr-NDC, ultrasonically dissolving in 10mL of ethanol at the ultrasonic frequency of 90Hz and the ultrasonic temperature of 55 ℃. Mixing the two, ultrasonically dispersing for 20min, stirring at normal temperature for 9h, placing the flask in a rotary evaporator for evaporation and concentration, adding 10mL of absolute ethanol after the solvent is evaporated to dryness, washing off 5-fluorouracil adsorbed on the surface, and subjecting the product to 12000 r.min-1Centrifuging for 5min, centrifuging to remove the upper solution layer, repeating for three times, and drying the obtained solid in a freeze dryer for 24h for later use.
EXAMPLE 3 preparation of Zr-NDC @5-FU
Weighing 80.0mg of 5-fluorouracil and 40.0mg of Zr-NDC, placing the 5-fluorouracil and 40.0mg of Zr-NDC into a 100mL flask, adding 40mL of ethanol for ultrasonic dissolution, stirring at normal temperature for 9 hours, placing the flask into a rotary evaporator for evaporation and concentration, adding 10mL of absolute ethanol after the solvent is evaporated to dryness, washing off 5-fluorouracil adsorbed on the surface, and passing the product through 12000 r.min-1Centrifuging for 5min, centrifuging to remove the upper solution layer, repeating for three times, and drying the obtained solid in a freeze dryer for 24h for later use.
Example 4 preparation of Zr-NDC @5-FU
Weighing 40.0mg of 5-fluorouracil, placing the 5-fluorouracil in a 50mL flask, adding 10mL of ethanol, stirring at normal temperature until the 5-fluorouracil is dissolved for later use, weighing 20.0mg of Zr-NDC in 10mL of ethanol, and stirring at normal temperature until the Zr-NDC is dissolved. Mixing the two solutions uniformly, stirring for 12h, placing the flask in a rotary evaporator for evaporation and concentration, adding 10mL of absolute ethanol after the solvent is evaporated to dryness, washing off 5-fluorouracil adsorbed on the surface, and subjecting the product to 12000 r.min-1Centrifuging for 5min, centrifuging to remove the supernatant, repeating for three times, and drying the obtained solid in a freeze dryer for 24h for later use.
EXAMPLE 5 preparation of Zr-NDC @5-FU
Weighing 50.0mg of 5-fluorouracil, placing the 5-fluorouracil in a 50mL flask, adding 10mL of ethanol, performing ultrasonic dissolution for later use, weighing 20.0mg of Zr-NDC in 10mL of ethanolMedium ultrasonic dissolution is carried out, the ultrasonic frequency is 90Hz, and the ultrasonic temperature is 65 ℃. Mixing the two, stirring at normal temperature for 12h, placing the flask in a rotary evaporator for evaporation and concentration, adding 10mL of absolute ethanol after the solvent is evaporated to dryness, washing off 5-fluorouracil adsorbed on the surface, and subjecting the product to 12000 r.min-1Centrifuging for 5min, centrifuging to remove the upper solution layer, repeating for three times, and drying the obtained solid in a freeze dryer for 24h for later use.
EXAMPLE 6 preparation of Zr-NDC @5-FU
Weighing 30.0mg of 5-fluorouracil, placing the 5-fluorouracil in a 50mL flask, adding 10mL of ethanol, ultrasonically dissolving for later use, weighing 20.0mg of Zr-NDC, ultrasonically dissolving in 10mL of ethanol at the ultrasonic frequency of 90Hz and the ultrasonic temperature of 55 ℃. Mixing the two solutions, dispersing in ultrasound for 20min, stirring in water bath at 65 deg.C for 12h, placing the flask in a rotary evaporator for evaporation and concentration, adding 10mL of anhydrous ethanol after solvent evaporation, washing off 5-fluorouracil adsorbed on surface, and subjecting the product to 12000 r.min-1Centrifuging for 5min, centrifuging to remove the upper solution layer, repeating for three times, and drying the obtained solid in a freeze dryer for 24h for later use.
EXAMPLE 7 preparation of TCS
500mg of CS was dissolved in 50mL of a 1% acetic acid solution, and then a 125mM EDAC solution was prepared, and after mixing the two solutions, 500mg of TGA was added to adjust the pH to 5.0. Stirring at room temperature in the dark for 3h, dialyzing with dialysis bag (12kDa) at 5 mmol. L-1Dialyzing with HCl medium for 3 days, and adding 5 mmol. multidot.L solution containing 1.0% NaCl-1HCl for a second dialysis. Finally using 1.0 mmol. L-1HCl was dialyzed for two days, adjusting the pH of the solution to 4.0. And (5) freeze-drying to obtain the sulfhydryl chitosan TCS.
EXAMPLE 8 preparation of TCS-Zr-NDC @5-FU
30mg of TCS was weighed into a 100mL round-bottomed flask, 10mL of a 0.2% aqueous solution of acetic acid was added, and the mixture was stirred overnight. 30mg of Zr-NDC @5-FU (prepared in example 2) was added to ethanol for ultrasonic dispersion, and then added to the above TCS solution and stirred for 30min, and the product was subjected to 10000 r.min-1Centrifuge for 5 min. Washing with 1% acetic acid solution, washing with water for three times, and lyophilizing to obtain TCS-Zr-NDC @ 5-FU.
Example 9 preparation of TCS-Zr-NDC @5-FU
45mg of TCS was weighed into a 100mL round-bottomed flask, 10mL of a 0.2% aqueous solution of acetic acid was added, and the mixture was stirred overnight. 30mg of Zr-NDC @5-FU (prepared in example 2) was added to ethanol for ultrasonic dispersion, and then added to the above TCS solution and stirred for 30min, and the product was subjected to 10000 r.min-1Centrifuge for 5 min. Washing with 1% acetic acid solution, washing with water for three times, and lyophilizing to obtain TCS-Zr-NDC @ 5-FU.
EXAMPLE 10 preparation of TCS-Zr-NDC @5-FU
30mg of TCS was weighed into a 100mL round-bottomed flask, 10mL of a 0.2% aqueous solution of acetic acid was added, and the mixture was stirred overnight. 30mg of Zr-NDC @5-FU (prepared in example 2) was added to ethanol for ultrasonic dispersion, and then added to the above TCS solution and stirred for 1h, and the product was subjected to 10000 r.min-1Centrifuge for 5 min. Washing with 1% acetic acid solution, washing with water for three times, and lyophilizing to obtain TCS-Zr-NDC @ 5-FU.
Example 11 preparation of TCS-Zr-NDC @5-FU
50mg of TCS was weighed into a 100mL round-bottomed flask, 10mL of a 0.2% aqueous solution of acetic acid was added, and the mixture was stirred overnight. 30mg of Zr-NDC @5-FU (prepared in example 2) was added to ethanol for ultrasonic dispersion, and then added to the above TCS solution and stirred for 1h, and the product was subjected to 10000 r.min-1Centrifuge for 5 min. Washing with 1% acetic acid solution, washing with water for three times, and lyophilizing to obtain TCS-Zr-NDC @ 5-FU.
EXAMPLE 12 preparation of TCS-Zr-NDC @5-FU
30mg of TCS was weighed into a 100mL round-bottomed flask, 10mL of a 0.2% aqueous solution of acetic acid was added, and the mixture was stirred overnight. 30mg of Zr-NDC @5-FU (prepared in example 2) was added to ethanol for ultrasonic dispersion, and then added to the above TCS solution and stirred for 2 hours, and the product was subjected to 10000 r.min-1Centrifuge for 5 min. Washing with 1% acetic acid solution, washing with water for three times, and lyophilizing to obtain TCS-Zr-NDC @ 5-FU.
EXAMPLE 13 characterization of TCS-Zr-NDC
FIG. 1 shows the IR spectra of TCS, Zr-NDC and TCS-Zr-NDC. In the infrared spectrum of TCS: 3400cm-1The broad peaks at the left and right are O-HAnd multiple absorption peaks formed by overlapping the stretching vibration absorption peak of N-H. 2800 to 2900cm-1Is the C-H stretching vibration peak of the methyl and methine on the chitosan ring. 2600cm-1Is a characteristic peak of sulfydryl, and is 1600-1500 cm-1The peaks of C ═ O and N-H flexural vibrations in the amide. IR spectrum of Zr-NDC: 3500cm-1～3000cm-1Broad peak region corresponding to O-H stretching vibration peak, 1654cm-1～1585cm-11398cm corresponding to OCO asymmetric stretching vibration peak-1Corresponding to the symmetric telescopic vibration peak of OCO, 1650cm-1～1640cm-1Corresponding to the peak of stretching vibration of C ═ O. The comparison of the IR spectrums of the Zr-NDC and the TCS-Zr-NDC shows that the IR spectrum of the TCS-Zr-NDC is similar to that of the Zr-NDC, but after the TCS is modified, the surface of the Zr-NDC is covered, the IR peak part of the Zr-NDC is covered by the TCS, the TCS-Zr-NDC shows the characteristic peak of the Zr-NDC in general and the characteristic peak of the TCS in partial area, and the TCS-Zr-NDC is successfully synthesized.
FIGS. 2 and 3 show the Zeta potentials of Zr-NDC and TCS-Zr-NDC, which were 35.4. + -. 0.16mV and 53.7. + -. 0.21mV, respectively. After the mercapto chitosan is modified, the Zeta potential of the Zr-NDC is increased, which indicates that the TCS-Zr-NDC is successfully synthesized.
FIG. 4 shows DSC spectra of TCS, Zr-NDC, and TCS-Zr-NDC. Comparing the three heat absorption curves in the figure, it can be seen that: at 250 ℃, TCS has a distinct endothermic peak. The curve of TCS-Zr-NDC is closer to that of Zr-NDC, but at 250 ℃ an endothermic peak appears, whereas Zr-NDC shows no endothermic peak at 250 ℃. The successful modification of TCS onto the vector Zr-NDC was demonstrated.
FIG. 5 shows XRD patterns of TCS, Zr-NDC and TCS-Zr-NDC. It can be seen that: the Zr-NDC has a crystal form structure similar to that of the TCS-Zr-NDC, has a sharp diffraction peak with high intensity, and is stable in morphological structure, but after the TCS is modified, the surface of the Zr-NDC is covered, the diffraction peak of the Zr-NDC in the TCS-Zr-NDC is covered, and the TCS-Zr-NDC shows a certain diffraction peak of the TCS.
FIG. 6 is a TGA spectrum of TCS, Zr-NDC, TCS-Zr-NDC. The weight loss interval of TCS is 200-400 ℃, and the weight loss rate of the corresponding TCS-Zr-NDC in the interval is 5.04%.
FIG. 7 is a scanning electron micrograph of TCS-Zr-NDC. It can be found that: after the modification by sulfydryl chitosan, the Zr-NDC still keeps the regular octahedral structure. Due to the viscosity of mercaptochitosan, the Zr-NDC was coherent.
EXAMPLE 14 in vitro drug Release study
In order to simulate the gastrointestinal conditions, in an in vitro release test, a sample is usually placed in artificial gastric juice and artificial intestinal juice in sequence for test exploration, and the test method is more practical. Respectively placing Zr-NDC @5-FU or TCS-Zr-NDC @5-FU in a dialysis bag containing 3mL of dialysate, fastening two ends, placing in a 50mL centrifuge tube containing 20mL of dialysate, and vibrating at 37 deg.C with a constant temperature oscillator (120r min)-1) In vitro release assays were performed. The dialysis medium is artificial gastric juice in 1-2 h, and is artificial intestinal juice in 2-6 h, and in the in-vitro release process, 0.5mL of release medium is taken every 0.5h, and meanwhile, 0.5mL of fresh release medium solution is supplemented. The sample was filtered through a 0.45 μm microporous membrane, the initial filtrate was discarded, 20 μ L of the subsequent filtrate was taken, the 5-FU content was measured by HPLC, and the cumulative release rate at each time point was calculated according to the formula (3).
FIG. 8 shows the release of Zr-NDC @5-FU and TCS-Zr-NDC @5-FU in artificial gastric and intestinal fluids. From the release profile of Zr-NDC @5-FU it can be found that: in artificial gastric juice, the release amount of Zr-NDC @5-FU is 72 percent; in the environment of artificial intestinal juice, the release rate of 5-FU in Zr-NDC @5-FU is obviously reduced, and the release amount of 5-FU is 78% after 6 h. The Zr-NDC @5-FU can be obtained to release a large amount of medicaments in the artificial gastric juice environment, and only a small amount of medicaments can reach the intestinal tract. From the release profile of TCS-Zr-NDC @5-FU it can be found that: the release of 5-FU goes through a slow release phase (initial 2 hours), then a fast release phase (from 2 to 4 hours), and finally the release becomes slow again (from 4 to 6 hours). In artificial gastric juice, the release rate of 5-FU is slow; and in the environment of artificial intestinal juice, the release speed of the 5-FU from the carrier is increased. For example, the cumulative release rate of the TCS-Zr-NDC @5-FU group in the artificial gastric juice environment is about 2.3%, and the stability of the drug delivery system can be maintained so as to reach the small intestine. When entering the small intestine, the accumulative release rate of TCS-Zr-NDC @5-FU is increased continuously, and reaches 60 percent at 6 h. The result shows that the TCS-Zr-NDC @5-FU drug delivery system can protect 5-FU from smoothly passing through the stomach to reach the intestinal tract, thereby releasing the drug and further playing the drug effect.
Example 15 in vitro antitumor Activity Studies
Collecting cells in log phase, adjusting cell density to 100000 cells/mL, adding 100 μ L of the solution to each well of 96-well plate, and placing in a container containing 5% CO2Incubating in a constant-temperature incubator at 37 ℃, culturing for 24h, adding 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU with concentration gradient, setting 6 gradients which are respectively 1, 10, 20, 40, 80 and 160 mu mol/L, setting a group of blank controls, adding 100 mu L of DMEM culture solution without fetal calf serum into each hole of each blank control, and setting 6 multiple holes. The incubation was continued for 24h, 20. mu.L of MTT solution was added, the incubation was continued for 4h, the culture medium was discarded, 200. mu.L of DMSO was added, the mixture was shaken on a shaker, and the absorbance of each well was measured at OD 490nm in a microplate reader. Finally, each group of ICs is calculated according to the formula (4)50The value is obtained. The MTT assay was repeated five times and the best results were screened.
TABLE 15 IC of FU, Zr-NDC @5-FU, TCS-Zr-NDC @5-FU50Value of
Note: p <0.05 compared to 5-FU group
FIG. 9 shows the inhibition rate of four cells by three drugs, and Table 1 shows the IC of the three drugs acting on four cells50The value is obtained. As can be seen from FIG. 9, 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU at the same concentration showed different inhibitory effects on cancer cells, which was substantially represented by TCS-Zr-NDC @5-FU > 5-FU. Taking HepG2 cell as an example, when the drug concentration is 160. mu. mol. L-1When the drug is used, the inhibition rates of 5-FU, Zr-NDC @5-FU and TCS-Zr-NDC @5-FU are respectively 78%, 80% and 83%, and the inhibition effect of TCS-Zr-NDC @5-FU on HepG2 cells is stronger than that of the other two groups of drugs, but the difference of the inhibition rates is not too large, and the drug substance is released from the carrier after being cultured for 24 hours. In addition, as can also be seen from Table 1,IC of TCS-Zr-NDC @5-FU group50The values were significantly lower than the 5-FU group (p < 0.05). These results indicate that TCS-Zr-NDC @5-FU has a higher inhibitory effect on cancer cells. In addition, it can be seen that: after HEK293, HepG2, A549 and DLD cells are incubated for 24h by TCS-Zr-NDC @5-FU, the number of living cells is reduced, the larger the medicament dosage is, the stronger the inhibition effect is, and the lower the cell survival rate is. In addition, the drug has stronger inhibiting effect on three cancer cells of HepG2, A549 and DLD than HEK 293. For example, the drug concentration of the TCS-Zr-NDC @5-FU group is 160. mu. mol. L-1The inhibition rates of the HEK293, the HepG2, the A549 and the DLD four cells are respectively 67%, 82%, 80% and 87%, the inhibition rates of the HEK293, the HepG2, the A549 and the DLD four cells reach 80%, but the inhibition rate of the HEK293, the HepG2, the A549 and the DLD four cells only reaches 67% to normal cells, so that the drug delivery system has better safety.
Example 16 rat in vivo intestinal perfusion study
SD rats were fasted before the test, but were assured of drinking water and were randomly assigned 3 groups, 5-FU group, Zr-NDC @5-FU group, TCS-Zr-NDC @5-FU, 5 per group. During the test, the rat needs to be weighed, the use amount of the urethane is determined, after anesthesia, the urethane is fixed on an operating table, the abdomen of the rat is opened, an intestinal section to be investigated is found out and is connected with a peristaltic pump, the content of the intestine is discharged by using normal saline, then K-R liquid is rapidly pumped into the intestinal section, the flow rate is reduced, and timing is started after a stable state is reached. Vials were placed at the access ports, and the collection vials were replaced every 15min, each time the vial was weighed until the end of the test. Measuring the length (L) and inner radius (r) of the test intestine section, placing the perfusion liquid at inlet and outlet into a centrifuge at 10000 r.min-1Centrifuging for 5min, filtering with 0.45 μm filter membrane, and performing liquid phase measurement to obtain inlet and outlet 5-FU concentration (C)inAnd Cout). In addition, the volume change of the intestinal fluid and the intestinal fluid needs to be calculated, and the mass of the inlet and outlet small bottles is m before perfusiona0And mb0The mass of the inlet and outlet vials at 15min is ma1And mb1Thus, the quality of the intestinal entering liquid can be obtained as follows: m isin＝mao-ma1Intestinal juiceMass of the body is mout＝mb1-mbo. Weighing 500 mu L of perfusion liquid, and weighing to obtain the density rho of the liquid entering the intestineinWeighing 500 μ L of the uniformly mixed receiving liquid to obtain the density ρ of the intestinal juiceout. Volume V of intestinal fluidin＝min/ρinVolume V of intestinal fluidout＝mout/ρout. The drug absorption rate constant (Ka) and the apparent drug absorption coefficient (Papp) were calculated according to formula (5) and formula (6).
As can be seen from FIGS. 10 and 11, in the four intestinal segments, the Ka of the TCS-Zr-NDC @5-FU group drug is significantly higher than that of the 5-FU group (p < 0.05), and the Ka value of the Zr-NDC @5-FU group is not significantly different from that of the 5-FU group (p > 0.05). As can be seen from FIG. 11, the apparent absorption coefficient of the drug in each intestinal segment was also significantly increased (p < 0.05) by the carrier, wherein TCS-Zr-NDC @5-FU group (p < 0.01). It can be seen that the absorption of TCS-Zr-NDC @5-FU is best in the rat intestine. In addition, the TCS-Zr-NDC @5-FU groups Ka and P in the duodenumappThe larger values are (1.82. + -. 0.04). times.102、(2.19±0.07)×103The jejunum is smaller than the duodenum, larger than the ileum, and the colon is smallest.
Thus, Ka, Papp for each segment of the intestine, expressed as duodenum > jejunum > ileum > colon, was found to be the major site of absorption. The best absorption of TCS-Zr-NDC @5-FU by rat intestinal tract indicates that the drug delivery system can better realize the oral administration of 5-FU.
Claims (8)
1. The metal organic framework is characterized in that Zr is used as metal ions, 2, 6-naphthalene diacid is used as an organic ligand to obtain Zr-NDC, and sulfhydryl chitosan is used for carrying out surface modification on the Zr-NDC to obtain the drug carrier material.
2. The method for preparing the metal-organic framework modified by the surface of the sulfhydryl chitosan, as recited in claim 1, is characterized by comprising the following steps:
1) sequentially adding ZrCl into a container42, 6-naphthalenedicarboxylic acidUniformly mixing acid, N-dimethylformamide, acetic acid and deionized water, reacting, cooling, centrifuging, washing and drying to obtain Zr-NDC;
2) dissolving a Drug in ethanol, dissolving Zr-NDC in the ethanol, ultrasonically dispersing and mixing the two, stirring for a certain time, performing rotary evaporation, washing, centrifuging and drying to obtain a Drug-loaded metal organic framework Zr-NDC @ Drug;
3) mixing the ethanol dispersion solution of the Drug-loaded metal organic framework Zr-NDC @ Drug with the acetic acid aqueous solution of the sulfhydryl chitosan, stirring, centrifuging, washing and drying to obtain the TCS-Zr-NDC @ Drug of the metal organic framework Drug-loaded system material.
3. The method for preparing the metal-organic framework modified by the surface of mercaptochitosan according to claim 2, wherein: in the step 1), the reaction temperature is 100-130 ℃, and the reaction time is 12-36 h.
4. The method for preparing the metal-organic framework modified by the surface of mercaptochitosan according to claim 3, wherein: in the step 2), the mass ratio of the medicament to the Zr-NDC is 2-5: 2.
5. The method for preparing the metal-organic framework modified by the surface of mercaptochitosan according to claim 4, wherein: in the step 2), the ultrasonic temperature is 25-75 ℃; the stirring time is 1-12 h.
6. The method for preparing the metal-organic framework modified by the surface of mercaptochitosan according to claim 5, wherein: in the step 3), according to the mass ratio, Zr-NDC @ Drug: mercaptochitosan ═ 1: 1 to 2.5.
7. The method for preparing the metal-organic framework material with the surface modified by the mercapto chitosan according to claim 6, wherein the method comprises the following steps: in the step 3), the stirring time is 0.5-2 h.
8. Use of the mercaptochitosan surface-modified metal organic framework of claim 1 for the preparation of an oral delivery system.
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| CN105769817A (en) * | 2016-03-09 | 2016-07-20 | 辽宁科技学院 | NLC (Nanostructured lipid carrier) with surface modified by TCS (thiolated chitosan) and preparation method of NLC |
| CN111671736A (en) * | 2020-06-18 | 2020-09-18 | 辽宁大学 | Metal organic framework-based drug carrier, preparation method thereof and application thereof in oral drug carrier |
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| CN105754016A (en) * | 2016-03-09 | 2016-07-13 | 沈阳药科大学 | Novel bioadhesive thiolated chitosan synthesis method |
| CN105769817A (en) * | 2016-03-09 | 2016-07-20 | 辽宁科技学院 | NLC (Nanostructured lipid carrier) with surface modified by TCS (thiolated chitosan) and preparation method of NLC |
| CN111671736A (en) * | 2020-06-18 | 2020-09-18 | 辽宁大学 | Metal organic framework-based drug carrier, preparation method thereof and application thereof in oral drug carrier |
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