A carbamazepine supramolecular hydrogel and a preparation method, characterization method and application thereof

Abstract

The application provides a carbamazepine supramolecular hydrogel and a preparation method, a characterization method and an application thereof. The carbamazepine supramolecular hydrogel is prepared by using Fmoc group modified phenylalanine dipeptide molecules and carbamazepine molecules as raw materials, organic solvents and water as solvents, and has the advantages of simple preparation method, good biocompatibility, crystal form conversion of carbamazepine into amorphous, and slow release of carbamazepine. The infrared spectrum analysis method is used to monitor the change process in situ and in real time, to explore the structure change of the carbamazepine crystal, and to explore the intermolecular interaction mechanism of the peptide-based supramolecular gel on the crystal form conversion, so as to provide a theoretical basis for the structure-activity relationship of the drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomolecular assembly, and particularly relates to a carbamazepine supramolecular hydrogel, a preparation method, a characterization method and an application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] The properties of polymorphic drugs are crucial to the pharmaceutical industry. The solubility, extensibility, melting point and dissolution rate of drug formulations are all affected by the particle size and crystal form of the active pharmaceutical ingredient (API), thereby affecting the bioavailability and clinical efficacy. Carbamazepine (CBZ) is a clinical drug for treating epilepsy and a common polymorphic drug, containing five crystal structures, I, II, III, IV and V, two kinds of dihydrates, monoclinic and orthorhombic, and various solvates. Due to the low solubility of CBZ (about 100 μg / mL, 25℃) and the narrow therapeutic window (4-12 mg / L), patients need to take extremely high doses (>100 mg) and regularly monitor blood drug concentrations, which greatly reduces the medication compliance of patients. CBZ may undergo hydration during wet granulation and long-term storage, and high-dose CBZ in the body can also be converted into dihydrate. Water molecules in the crystal change the intermolecular interactions in the crystal structure, thereby affecting the dissolution rate and bioavailability.

[0004] Gel regulates crystal behavior is a common technique, and the gel matrix can slow down the convection and sedimentation of the drug, thereby reducing the number of nucleation sites. Compared with traditional gels, supramolecular gels have many advantages, including a variety of functional groups, a wide range of gelation solvents, and the ability to redissolve the gel to recover crystals. Therefore, how to prepare carbamazepine supramolecular hydrogel and regulate its crystal structure so as to improve the physicochemical properties of the drug without changing the chemical properties of the drug, thereby improving its bioavailability, is worthy of further study.

[0005] The research on the mechanism of molecular interaction provides a theoretical framework for the structure-activity relationship of drugs. Non-covalent interactions such as hydrogen bonding and π-π stacking interaction are the link for exploring the mechanism of crystal change and accurate regulation of structure. The infrared spectroscopy (IRS) analysis technology is usually used as a preferred process analytical technology (PAT) with the advantages of rapidity, non-destructiveness and non-destruction of samples, and mainly reflects the changes of supramolecular assembly structure through the vibration information of amide band and hydrogen-containing groups. The infrared spectrum of a complex system can obtain the information of the changes of substance structure and hydration, and further processing is carried out through chemometrics method. The infrared spectrum is used to explore the mechanism of intermolecular structural transformation in the crystal structure, so as to promote the effective regulation of drug molecules. However, there is no report on the characterization research on the in-situ real-time monitoring of the process of the change of crystal drug structure based on infrared spectrum. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a carbamazepine supramolecular hydrogel and a preparation method, characterization method and application thereof. The present application uses Fmoc group modified phenylalanine dipeptide molecules and carbamazepine molecules as raw materials, organic solvents and water as solvents to prepare short peptide hydrogel, and the preparation method is simple; the obtained hydrogel has good biocompatibility, the crystal form of carbamazepine is changed into amorphous, and slow release of carbamazepine is realized. The change process is monitored in-situ and in real time by infrared spectroscopy analysis method, the structural change of carbamazepine crystal is explored, and the intermolecular interaction mechanism of peptide-based supramolecular gel on crystal transformation is explored, thereby providing a theoretical basis for the structure-activity relationship of drugs.

[0007] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0008] In a first aspect of the present application, a preparation method of a carbamazepine supramolecular hydrogel is provided, comprising the steps of: fully dissolving N-fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF) and carbamazepine in an organic solvent to obtain a mixed solution, adding water, and then standing to obtain the carbamazepine supramolecular hydrogel;

[0009] The structure of the N-fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF) is as follows:

[0010]

[0011] According to the present application, the organic solvent is deuterated dimethyl sulfoxide (DMSO-d6) or deuterated methanol, preferably deuterated dimethyl sulfoxide (DMSO-d6); and the water is water (H2O) or deuterated water (D2O), preferably deuterated water (D2O).

[0012] According to the application, preferably, the volume ratio of the mixed solution to water is 1:1-49, preferably 1:49.

[0013] According to the application, preferably, the molar ratio of N-fluorenylmethyloxy carbonyl phenylalanine dipeptide (FmocFF) to carbamazepine is 1:0.25-3, preferably 1:0.25-1, and further preferably 1:1.

[0014] According to the application, preferably, the concentration of N-fluorenylmethyloxy carbonyl phenylalanine dipeptide (FmocFF) in the mixed solution is 0.05-5 mmol / L, preferably 0.1 mmol / L.

[0015] According to the application, preferably, the standing temperature is room temperature, and the standing time is 15-60 min.

[0016] In the second aspect of the application, a carbamazepine supramolecular hydrogel is provided, which is prepared by the above method.

[0017] According to the application, preferably, the micro-morphology of the carbamazepine supramolecular hydrogel is nanofiber with a diameter of 10-30 nm.

[0018] In the third aspect of the application, a process analysis characterization method for infrared spectrum of a carbamazepine supramolecular hydrogel is provided, which comprises the following steps:

[0019] (1) N-fluorenylmethyloxy carbonyl phenylalanine dipeptide (FmocFF) and carbamazepine are fully dissolved in deuterated organic solvent, deuterium oxide (D2O) is added and mixed uniformly, and then a carbamazepine supramolecular hydrogel is prepared by standing self-assembly;

[0020] During the above standing process or after the standing is completed, the appearance, micro-morphology, rheological curve, X-ray diffraction, circular dichroism spectrum signal and mid-infrared spectrum data of the sample are collected; the appearance, micro-morphology, rheological curve, X-ray diffraction, circular dichroism spectrum signal and mid-infrared spectrum data of the sample are characterized and the change of intermolecular interaction is judged;

[0021] (2) The mid-infrared spectrum data obtained in step (1) is pretreated, standard normal variable transformation is adopted to eliminate baseline drift, and scattering influence caused by system turbidity is eliminated, so as to improve the spectrum quality;

[0022] (3) The processed spectrum obtained in step (2) is subjected to wavelet denoising processing to eliminate spectrum noise;

[0023] (4) The processed spectrum obtained in step (3) is subjected to second derivative processing to improve the resolution of the amide band waveband;

[0024] (5) using the mid-infrared spectral data obtained in step (3), analyzing the amide band secondary structure characteristic region by principal component analysis, according to the relationship between the principal component score number and the standing time, combining the load spectrum characteristics, analyzing the interaction at different stages;

[0025] (6) processing the mid-infrared spectral data obtained in step (3) by two-dimensional correlation spectroscopy (2DCOS) to judge the group change order and further study the intermolecular interaction.

[0026] According to the application, preferably, in step (1), the mid-infrared spectral data of the sample is collected in the range of 4000-400 cm -1 .

[0027] According to the application, preferably, in step (3), the wavelet base used for wavelet denoising is db4 and the scale is 5.

[0028] According to the application, preferably, in step (4), the smoothing point number in the second derivative processing is 15 points.

[0029] According to the application, preferably, in step (5), the wavelength range corresponding to the amide band secondary structure characteristic region is 1750-1350 cm -1 .

[0030] According to the application, preferably, in step (6), the wavelength range for two-dimensional correlation spectroscopy processing is 1750-1400 cm -1 .

[0031] The fourth aspect of the application provides the application of the above-mentioned carbamazepine supramolecular hydrogel, which is applied to the sustained-release administration of carbamazepine drugs.

[0032] The technical features and beneficial effects of the application are as follows:

[0033] 1. The application provides a method for regulating carbamazepine crystals by dipeptide-based supramolecular gel, which is a novel design strategy for realizing the regulation of polymorphic drugs by using the structural advantages of dipeptide-based supramolecular gel. The dipeptide-based supramolecular hydrogel N-fluorenylmethoxycarbonyl protected diphenylalanine (FmocFF) is one of the derivatives of phenylalanine dipeptide (FF) obtained by chemical modification of N-fluorenylmethoxycarbonyl group (Fmoc). The Fmoc group increases the designability of the FF assembly structure, and due to the change of hydrophobicity, it is easy to form a gel network by rapid self-assembly in aqueous solution. As a gel matrix, FmocFF has good biocompatibility, and under the premise of not changing the chemical properties of the drug, the supramolecular gel improves the physicochemical properties of the drug, thereby enhancing its therapeutic effect. Under the action of the dipeptide-based supramolecular hydrogel, the crystal form of carbamazepine is changed to amorphous, and the effect of sustained release is realized. The method is simple and suitable for mass production.

[0034] 2. In the preparation process of the carbamazepine supramolecular hydrogel of the application, the molar ratio of FmocFF to CBZ molecules affects its performance, and within a certain range, it will cause CBZ to change to amorphous. In the characterization of interaction, the application proves that the π-π stacking interaction plays an important role in the crystal form transition of CBZ, so in the selection of matrix, molecules with aromatic ring structure are preferred. N-fluorenylmethoxycarbonyl protected phenylalanine dipeptide molecule is a relatively ideal gel medium, and if Fmoc is not modified, the crystal form transition effect will not be obvious. The preparation of supramolecular hydrogel is to promote molecular aggregation and then grow and extend by mixing good solvents with poor solvents, and the types of organic solvents can be changed according to the solubility of FmocFF and CBZ, and the solvents used in the application are preferably selected.

[0035] 3. In the infrared spectrum process analysis method of the application, the pretreatment method and the parameters of the second derivative need to be appropriate, otherwise the molecular mechanism of the crystal form transition of carbamazepine cannot be effectively explored; for example, the wavelet base and scale of wavelet denoising pretreatment and the number of smoothing points in the second derivative processing need to be appropriate, otherwise the noise will be too large or the spectral information will be masked. The principal component analysis of the change of the standing assembly time judges the different nodes of the gel formation, and the two-dimensional spectrum is processed by segmenting the different stages to further explain the molecular interaction mechanism. The application explores the molecular mechanism of the crystal form transition of carbamazepine by in-situ monitoring the spectral changes of carbamazepine crystals and co-assembled gel, thereby providing a theoretical basis for the structure-activity relationship of drugs and subsequent applications. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 State photos of the crystal CBZ prepared in Comparative Example 2-3, the hydrogel FmocFF prepared in Comparative Example 1, and the hydrogel FmocFF / CBZ prepared in the example.

[0037] Figure 2 SEM images of the raw material CBZ, the crystal CBZ prepared in Comparative Example 2, the hydrogel FmocFF prepared in Comparative Example 1, and the hydrogel FmocFF / CBZ prepared in Examples.

[0038] Figure 3 Rheology curves of the hydrogel FmocFF prepared in Comparative Example 1 and the hydrogel FmocFF / CBZ prepared in Examples.

[0039] Figure 4 XRD pattern of the crystal CBZ prepared in Comparative Example 2 (a) and CBZ single crystal SC-XRD calculated simulation pattern (b).

[0040] Figure 5 XRD patterns of the crystal CBZ prepared in Comparative Example 2 and the hydrogel FmocFF / CBZ prepared in Examples.

[0041] Figure 6 CD spectra of the crystal CBZ prepared in Comparative Example 2 (a) and the crystal CBZ prepared in Comparative Example 2, the hydrogel FmocFF prepared in Comparative Example 1, and the hydrogel FmocFF / CBZ prepared in Examples (b).

[0042] Figure 7 Infrared raw spectra of the crystal CBZ formation process in the method of Comparative Example 2.

[0043] Figure 8 Infrared raw spectra of the hydrogel FmocFF / CBZ formation process in the method of Example 1 (a), second derivative processing of amide band (b), and principal component analysis of amide band (c, d).

[0044] Figure 9 Two-dimensional correlation synchronous, asynchronous, and power spectra of the hydrogel FmocFF / CBZ in the first stage (a-c) and the second stage (d-f) of gel formation in the method of Example 1.

[0045] Figure 10 In vitro release curves of the crystal CBZ prepared in Comparative Example 2 and the hydrogel FmocFF / CBZ prepared in Example 1.

[0046] Figure 11 Cytotoxicity results of the crystal CBZ prepared in Comparative Example 2, the gel FmocFF prepared in Comparative Example 1, and the gel FmocFF / CBZ prepared in Examples.

[0047] Figure 12 Schematic diagram of the molecular interaction mechanism of the crystal CBZ and the gel FmocFF / CBZ. DETAILED DESCRIPTION

[0048] The application is further described below by way of specific examples and with reference to the accompanying drawings. The raw materials used in the examples are commercially available or prepared according to the prior art unless otherwise specified. The methods used are conventional unless otherwise specified.

[0049] Example 1

[0050] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0051] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF) and carbamazepine (CBZ) were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) at a molar ratio of 1:1, ultrasonically treated for 10-20 s to obtain a transparent concentrate, wherein the concentration of FmocFF was 0.1 mol / L. 490 μL of D2O was immediately added to 10 μL of the above concentrate, mixed uniformly, and the concentrations of FmocFF and CBZ were both 2 mmol / L. Then, the carbamazepine supramolecular hydrogel (abbreviated as FmocFF / CBZ 2mmol / L 1:1 or FmocFF / CBZ 1:1) was obtained by standing at room temperature for 40 min.

[0052] Example 2

[0053] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0054] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF) and carbamazepine (CBZ) were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) at a molar ratio of 1:0.25, ultrasonically treated for 10-20 s to obtain a transparent concentrate, wherein the concentration of FmocFF was 0.1 mol / L. 490 μL of D2O was immediately added to 10 μL of the above concentrate, mixed uniformly, and the concentration of FmocFF was 2 mmol / L, and the concentration of CBZ was 0.5 mmol / L. Then, the carbamazepine supramolecular hydrogel (abbreviated as FmocFF / CBZ 2mM 1:0.25 or FmocFF / CBZ 1:0.25) was obtained by standing at room temperature for 40 min.

[0055] Example 3

[0056] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0057] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF), carbamazepine (CBZ) were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) at a molar ratio of 1:0.5, and ultrasonic treatment was performed for 10-20 s to obtain a transparent concentrate, wherein the concentration of FmocFF was 0.1 mol / L. 490 μL of D2O was immediately added to 10 μL of the above concentrate, and mixed uniformly, wherein the concentration of FmocFF was 2 mmol / L, and the concentration of CBZ was 1 mmol / L. Then, a carbamazepine supramolecular hydrogel (abbreviated as FmocFF / CBZ 2 mmol / L 1:0.5 or FmocFF / CBZ 1:0.5) was obtained by standing at room temperature for 40 min.

[0058] Example 4

[0059] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0060] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF), carbamazepine (CBZ) were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) at a molar ratio of 1:2, and ultrasonic treatment was performed for 10-20 s to obtain a transparent concentrate, wherein the concentration of FmocFF was 0.1 mol / L. 490 μL of D2O was immediately added to 10 μL of the above concentrate, and mixed uniformly, wherein the concentration of FmocFF was 2 mmol / L, and the concentration of CBZ was 4 mmol / L. Then, a carbamazepine supramolecular hydrogel (abbreviated as FmocFF / CBZ 2 mmol / L 1:2 or FmocFF / CBZ 1:2) was obtained by standing at room temperature for 40 min.

[0061] Example 5

[0062] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0063] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF), carbamazepine (CBZ) were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) at a molar ratio of 1:3, and ultrasonic treatment was performed for 10-20 s to obtain a transparent concentrate, wherein the concentration of FmocFF was 0.1 mol / L. 490 μL of D2O was immediately added to 10 μL of the above concentrate, and mixed uniformly, wherein the concentration of FmocFF was 2 mmol / L, and the concentration of CBZ was 6 mmol / L. Then, a carbamazepine supramolecular hydrogel (abbreviated as FmocFF / CBZ 2 mmol / L 1:3 or FmocFF / CBZ 1:3) was obtained by standing at room temperature for 40 min.

[0064] Comparative Example 1

[0065] A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of:

[0066] N-Fluorenylmethoxycarbonyl phenylalanine dipeptide (FmocFF) was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and sonicated for 10-20 s to obtain a clear concentrate with a concentration of 0.1 mol / L FmocFF. 490 μL D2O was immediately added to 10 μL of the above concentrate and mixed well. The mixture was then left to stand at room temperature for 40 min to obtain a hydrogel (abbreviated as FmocFF 2 mmol / L 1:0 or FmocFF).

[0067] Comparative Example 2

[0068] A method for preparing CBZ crystals, comprising the steps of:

[0069] Carbamazepine (CBZ) was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and sonicated for 10-20 s to obtain a clear concentrate with a concentration of 0.1 mol / L CBZ. 490 μL D2O was immediately added to 10 μL of the above concentrate and mixed well. The mixture was then left to stand at room temperature for 20 min to obtain a CBZ crystal solution with a concentration of 2 mmol / L (abbreviated as CBZcrystal 2 mmol / L).

[0070] Comparative Example 3

[0071] A method for preparing CBZ crystals, comprising the steps of:

[0072] Carbamazepine (CBZ) was dissolved in deuterated dimethyl sulfoxide (DMSO-d6) and sonicated for 10-20 s to obtain a clear concentrate with a concentration of 0.05 mol / L CBZ. 490 μL D2O was immediately added to 10 μL of the above concentrate and mixed well. The mixture was then left to stand at room temperature for 20 min to obtain a CBZ crystal solution with a concentration of 1 mmol / L (abbreviated as CBZcrystal 1 mmol / L).

[0073] Test Example 1

[0074] The appearance, micro-morphology, rheological curve, X-ray diffraction, circular dichroism spectrum signal, and mid-infrared spectrum data of the sample were collected.

[0075] i. Appearance: The optical image of the sample was recorded, and the formation of the hydrogel was verified by the inverted test tube method, as shown in Figure 1The CBZ formed visible needle-like crystals. When the concentration increased to 2 mmol / L (Comparative Example 2, CBZ crystal 2 mmol / L), more crystals were formed. After the addition of FmocFF, colorless transparent hydrogels were obtained in the molar ratio range of 1 :0.25-1 :1. When the molar ratio of FmocFF / CBZ increased to 1 :2 and 1 :3, visible white fibers were formed. The above phenomenon indicates that within a certain concentration range, FmocFF inhibits the growth of CBZ crystals, and there is interaction between the two to form nanofibers.

[0076] ii. Micro-morphology characterization

[0077] The samples were placed on a silicon wafer, and the excess liquid was absorbed with filter paper. The samples were dried at room temperature and tested with a JSM-7610FPlus instrument after spraying platinum to obtain SEM images as shown in Figure 2 The electron microscopy results show that the raw material CBZ is blocky, and the CBZ prepared in Comparative Example 2 forms regular needle-like crystals with a size width of about 20 μιη. After the addition of FmocFF, the microstructure in the molar ratio range of 1 :0.25-1 :1 is nanofibers with a width of about 20 nm.

[0078] iii. Rheological curves

[0079] The temperature setting of the rotor was 25.0±0.5 °C (C35 1 ° / Ti). After identifying the linear viscoelastic region through a stress sweep, dynamic frequency sweeps were performed at a fixed stress of 20 Pa using the MARS60 rheometer. The stress sweep curve is shown in Figure 3 a, the storage modulus (G') is greater than the loss modulus (G"), and the yield stress gradually decreases with the increase of the CBZ concentration, indicating that the properties of the gel are affected by CBZ. Dynamic frequency sweeps were performed at a fixed stress of 20 Pa Figure 3 b), in which G' was proved to be an order of magnitude greater than G", and their values were independent of the frequency change, indicating that the FmocFF / CBZ system prepared formed a co-assembled hydrogel with solid-like rheological properties.

[0080] iv. XRD characterization

[0081] The XRD pattern of the sample was tested. The XtaLAB Synergy-S instrument was used to test the sample using CuKa rays The data were collected, and the structure was solved and analyzed using the CDCC Mercury software. In addition, the CBZ crystals prepared in Comparative Example 2 and the FmocFF / CBZ gels prepared in the examples were placed on a silicon wafer, the excess liquid was absorbed with filter paper, and then dried at room temperature. The samples were tested using a SmartLab 9kW instrument with a scanning range of 5-60°. As shown in Figure 4 b, the prepared CBZ single crystal structure was analyzed using SC-XRD technology to be a Cmca orthorhombic dihydrate with a unit cell parameter of α = β = γ = 90°, and its packing mode is shown in Figure 4 b.

[0082] As shown in Figure 5 , in the FmocFF / CBZ gels with a molar ratio of 1:0.25, 1:0.5 and 1:1, there is a broad amorphous peak at about 2θ = 20°. No other significant diffraction peaks were observed for the FmocFF / CBZ gels (1:0.25 and 1:0.5), indicating that CBZ exists in an amorphous state. The FmocFF / CBZ gels (1:1 and 1:2) have diffraction peak signals at 2θ = 6.14°, 8.92°, 18.98° and 24.76°, but the intensity is significantly weaker than that of the CBZ crystals. This indicates that the FmocFF / CBZ gels (1:1 and 1:2) contain a small amount of crystal faces, indicating that within a certain concentration range, FmocFF inhibits the growth of CBZ crystals, resulting in its existence in an amorphous state. However, no needle-shaped crystals were observed in the appearance characterization of the FmocFF / CBZ gel (1:1), and the appearance of partial crystal faces may be due to the drying process during XRD measurement, causing partial crystallization.

[0083] vi. Circular dichroism spectroscopy characterization

[0084] The CD spectra of the CBZ crystals (CBZ-1 and CBZ-2) prepared in Comparative Example 2, FmocFF prepared in Comparative Example 1 and the gels FmocFF / CBZ prepared in the examples were measured in the range of 200-400 nm using a Chirascan V100 CD spectrometer and a 0.5 mm diameter quartz cuvette with a scanning speed of 50 nm / min. As shown in Figure 6 a, the CBZ crystal packing mode has randomness. And Figure 6 b, the peak of FmocFF at 310 nm is attributed to the Fmoc group. When FmocFF and CBZ are co-assembled, a significant negative signal is observed, and the peak intensity increases with the increase of CBZ concentration. The results show that CBZ has a greater impact on the π-π interaction between the aromatic rings of FmocFF.

[0085] vi. Mid-infrared spectroscopy characterization

[0086] During the standing process of preparing carbamazepine supramolecular hydrogel (Example 1) or CBZ crystal (Comparative Example 2), the samples were placed on the ATR accessory for acquisition, and the mid-infrared spectra of 4000-400 cm -1 were recorded by Alpha II spectrometer. The resolution was 4 cm -1 , air as reference, 30 scans, and the ambient and instrument temperatures were maintained at 30.0 °C.

[0087] Table 1 summarizes the characteristic peaks of FmocFF gelation and CBZ crystallization processes. The original infrared spectra of CBZ crystallization process are shown in Figure 7 The spectral changes mainly focused on the first few minutes of CBZ crystal formation, and the peaks of 1428 cm -1 and 1630 cm -1 shifted with the penetration of D2O, which was due to the O-D bending vibration of crystallization water and the hydrogen-bonded C=O stretching vibration of CBZ. The peak intensity of 1492 cm -1 increased sharply, which was attributed to the symmetric stretching vibration of the C=C of the dibenzazepine ring. Therefore, the conversion of CBZ monomer to dihydrate was caused by the π-π stacking interaction between aromatic rings and the hydrogen bond between CBZ and water molecules.

[0088] Table 1

[0089]

[0090] The MIR original spectra of the dynamic process of FmocFF / CBZ gelation are shown in Figure 8 a. The resolution of the amide band was improved by standard normal variate transformation, wavelet denoising (wavelet basis db4, scale 5) and second derivative (15-point smoothing) in turn (processing software MATLAB 2020a, Mathworks, USA), as shown in Figure 8 b. The N-H stretching vibration of CBZ (~3400 cm -1 ) increased with time, indicating that the hydration of N-H group changed. The characteristic peaks of C=O stretching vibration of FmocFF (~1689 cm -1 and ~1645 cm -1 ) and CH2 bending vibration (~1430 cm -1 ) increased significantly with time, which played an important role in gelation. Based on the spectral data after standard normal variate transformation and wavelet denoising, further principal component analysis was performed on the 1750-1350 cm -1 band (processing software MATLAB 2020a, Mathworks, USA) Figure 8c, d), the first principal component (PC 1) and the second principal component (PC 2) explain more than 99% of the spectral variance. For PC1, 1689 cm -1 、1650cm -1 and 1432cm -1 The peak intensity of PC 2 increases with time. -1 The peak is attributed to the bending vibration of NH. -1 The obvious negative peak at is attributed to the CH bending vibration of the CBZ aromatic ring and begins to decrease around 9 min, indicating that the π-π stacking effect plays a dominant role in the second and third stages.

[0091] In order to further study the molecular mechanism of FmocFF / CBZ gelation, 2DCOS was used to analyze the spectral data of 1750-1400 cm-1 after standard normal variable transformation and wavelet denoising. -1 The changes of the main absorption peaks within the range (processing software is 2D Shige software, ver.1.3), such as Figure 9 According to Noda's rule, the order of change of the spectral peaks in the first stage of gel formation is 1541 cm -1 >1689cm -1 >1650cm -1 >1438cm -1 >1495cm -1 The hydration of NH induces the nucleation of aggregates. The π-π stacking interaction of the Fmoc group changes before the C=O group. The π-π stacking between FmocFF and the CBZ aromatic ring forms a steric hindrance, weakening the hydrogen bonding between the NH2 group of CBZ and the water molecule. This may be the main reason for the formation of amorphous. The change order of the spectral peak in the second stage of gel formation is 1689cm -1 >1646cm -1 >1495cm -1 >1438cm -1 1495cm -1 The peak at 1438 cm is attributed to the symmetrical stretching vibration of the C=C of the CBZ dibenzazepine ring. -1 The peak before FmocFF / CBZ at 1438cm -1 The peak intensity at is significantly enhanced, indicating that the π-π stacking interaction of FmocFF / CBZ is stronger, indicating that the π-π stacking interaction mainly affects the growth and extension of the gel fiber.

[0092] Test Example 2

[0093] In vitro release behavior investigation

[0094] FmocFF / CBZ (1:1) gel (prepared by the method of Example 1) and CBZ crystal (prepared by the method of Comparative Example 2) samples were prepared in parallel, left to stand for 72 h, 1.5 mL of PBS buffer solution (pH = 7.4) was added to each sample, and placed on a shaker for 24 h (37.0°C, 100 rpm). Three samples were taken at each time point, and the supernatant was filtered through a 0.22 μm filter membrane. The content of CBZ was determined by HPLC method, so as to characterize the effect of FmocFF gel on the release behavior of CBZ.

[0095] As shown in Figure 10 , the release rate of CBZ crystal reached about 50% within the first 2 h, while the release rate of FmocFF / CBZ gel was close to 20%. CBZ was released at a relatively fast rate, and the maximum cumulative release amount was reached at about 6 h. The release of FmocFF / CBZ was faster at the early stage and slower at the later stage, and the maximum cumulative release amount was reached at 18 h. Subsequently, the release rates of both of them reached about 70% at 24 h. FmocFF hindered the hydration of the amide bond and the π-π stacking interaction between CBZ molecules, and the π-π stacking between FmocFF and CBZ aromatic rings played an important role in the amorphous morphology of CBZ. The co-assembly of FmocFF / CBZ gel prolonged the total release duration of CBZ and reduced its release rate.

[0096] Test Example 3

[0097] In vitro cytotoxicity investigation

[0098] The 24 h in vitro cytotoxicity of the FmocFF / CBZ gel prepared in Example 1 (corresponding to sample C in Figure 11 ), the FmocFF / CBZ gel prepared in Example 2 (corresponding to sample A in Figure 11 ), the FmocFF / CBZ gel prepared in Example 3 (corresponding to sample B in Figure 11 ), the FmocFF gel prepared in Comparative Example 1 and the CBZ crystal prepared in Comparative Example 2 on NIH / 3T3 cells was tested by CCK-8 method, as shown in Figure 11 . The cell survival rate of the CBZ group was about 75%, and the cell survival rates of FmocFF and FmocFF / CBZ 1:0.25, 1:0.5, 1:1 were all greater than 80%, indicating that the hydrogel system had no obvious inhibitory effect on the survival of NIH / 3T3 cells. FmocFF improved the inhibitory activity of CBZ on the growth of NIH / 3T3 cells, and within a certain concentration range, the FmocFF / CBZ gel had biocompatibility.

[0099] The present invention characterizes the intermolecular interaction mechanisms in CBZ crystals and FmocFF / CBZ gels, as shown in Figure 12 FIG. 1.

Claims

1. A method for preparing a carbamazepine supramolecular hydrogel, comprising the steps of: fully dissolving N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) and carbamazepine in an organic solvent to obtain a mixed solution, adding water, and then allowing the mixture to stand to obtain the carbamazepine supramolecular hydrogel; the molar ratio of N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) to carbamazepine is 1:0.25-1; the structure of the N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) is shown below: 。 2. The method for preparing the carbamazepine supramolecular hydrogel according to claim 1, wherein: Include one or more of the following conditions: i. The organic solvent is deuterated dimethyl sulfoxide (DMSO-d6) or deuterated methanol; the water is water H2O or deuterated water (D2O); ii. The volume ratio of the mixed solution to water is 1:1-49; iii. In the mixed solution, the concentration of N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) is 0.05-5 mmol / L; iv. The standing temperature is room temperature and the standing time is 15-60 minutes.

3. The method for preparing the carbamazepine supramolecular hydrogel according to claim 1, wherein: The molar ratio of the N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) to carbamazepine is 1:

1.

4. A carbamazepine supramolecular hydrogel prepared by the method according to any one of claims 1 to 3.

5. The carbamazepine supramolecular hydrogel according to claim 4, characterized in that The microstructure of carbamazepine supramolecular hydrogel is nanofibers with a diameter of 10-30 nm.

6. A method for infrared spectroscopy process analysis and characterization of carbamazepine supramolecular hydrogel, comprising the steps of: (1) N-Fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) and carbamazepine were fully dissolved in a deuterated organic solvent, deuterated water (D2O) was added and mixed evenly, and then static self-assembly was performed to prepare carbamazepine supramolecular hydrogel; During or after the standing period, collecting sample appearance, micromorphology, rheological curve, X-ray diffraction, circular dichroism spectral signal, and mid-infrared spectral data; using the sample appearance, micromorphology, rheological curve, X-ray diffraction, circular dichroism spectral signal, and mid-infrared spectral data to characterize and determine changes in intermolecular interactions; The molar ratio of N-fluorenylmethoxycarbonylphenylalanine dipeptide (FmocFF) to carbamazepine is 1:0.25-1; (2) Preprocessing the mid-infrared spectral data obtained in step (1) by using standard normal variable transformation to eliminate baseline drift and eliminate the scattering effect caused by system turbidity, thereby improving the spectral quality; (3) Perform wavelet denoising on the processed spectrum obtained in step (2) to eliminate spectral noise; (4) performing second-order derivative processing on the processed spectrum obtained in step (3) to improve the resolution of the amide band; (5) Using the mid-infrared spectral data obtained in step (3), the principal component analysis method is used to analyze the secondary structure characteristic region of the amide band. According to the relationship between the principal component score and the standing time, combined with the load spectrum characteristics, the interaction at different stages is analyzed; (6) The mid-infrared spectral data obtained in step (3) are processed by two-dimensional correlation spectroscopy (2DCOS) to determine the order of group changes and further study the intermolecular interactions.

7. The infrared spectroscopy process analysis and characterization method of carbamazepine supramolecular hydrogel according to claim 6, characterized in that: In step (1), collect samples 4000-400 cm -1 Mid-infrared spectral data.

8. The infrared spectroscopy process analysis and characterization method of carbamazepine supramolecular hydrogel according to claim 6, characterized in that: Include one or more of the following conditions: i. In step (3), the wavelet basis used for wavelet denoising is db4 and the scale is 5; ii. In step (4), the number of smoothing points in the second-order derivative processing is 15.

9. The infrared spectroscopy process analysis and characterization method of carbamazepine supramolecular hydrogel according to claim 6, characterized in that: Include one or more of the following conditions: i. In step (5), the wavelength range corresponding to the secondary structure characteristic region of the amide band is 1750-1350 cm -1 ; ii. In step (6), the wavelength range of the two-dimensional correlation spectrum processing is 1750-1400 cm -1 .

10. Use of the carbamazepine supramolecular hydrogel prepared by the method according to any one of claims 1 to 3, characterized in that: Used in the preparation of sustained-release drugs of carbamazepine.

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