A hand-functionalized nanomicelle, its preparation method and application

Abstract

The present invention provides a hand-functionalized nanomicelle and its preparation method and application, relating to the technical field of biomaterials. In this application, a novel nanomicelle material is prepared by using CM-β-CD as the hydrophilic group and long-chain fatty amine as the hydrophobic group. This novel amide compound forms nanomicelles through self-assembly in a buffer solution, serving both as a chiral selector and as a pseudo-stationary phase. Compared with the separate CM-β-CD system and the CM-β-CD / sodium dodecyl sulfate micellar electrokinetic capillary chromatography system, the separation of the model drug is significantly improved. The nanomicelles are comprehensively characterized by methods such as infrared spectroscopy, nuclear magnetic resonance spectroscopy, particle size determination, and TEM. In addition, amlodipine is selected as the model analyte, and the drug loading amounts in the separate CM-β-CD system and the nanomicelle system are determined by high-performance liquid chromatography. The research results show that the nanomicelles have excellent drug loading capacity. The nanomicelles show great promise in the field of chiral separation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a chiral-functionalized nanomicelle, a preparation method thereof, and an application thereof. Background Art

[0002] Chirality is a fundamental feature of biological systems. Enantiomers of chiral drugs usually exhibit different metabolic pathways and pharmacological effects. However, the differences in physical and chemical properties between enantiomers are only manifested in optical activity, making the separation of chiral drugs a long-standing challenge in pharmaceutical analysis. Among several chiral separation methods, capillary electrophoresis (CE) has the advantages of high speed, high efficiency, low consumption of samples and reagents, and flexible separation modes, and thus occupies an important position in chiral separation.

[0003] Regardless of the separation mode, chiral selectors are crucial in chiral capillary electrophoresis. A variety of compounds have been successfully developed as effective chiral selectors, including cyclodextrins and their derivatives, antibiotics, proteins, crown ethers, saccharides, surfactants, and metal complexes. However, the drug separation range of most chiral selectors is limited, and the enantioselectivity of unmodified separation systems is also limited. Cyclodextrins and their derivatives are the most commonly used class of chiral selectors, among which carboxymethyl-β-cyclodextrin (CM-β-CD) is favored due to its high water solubility, strong enantioselectivity, and low price. However, in some cases, the separation using only CM-β-CD as the sole chiral selector is still not ideal. To solve this problem, researchers are committed to developing new separation systems (such as monolithic columns, coated columns, transfersomes, ionic liquids, deep eutectic solvent systems, etc.) to improve enantioselectivity.

[0004] Micellar electrokinetic capillary chromatography (MEKC) is a separation mode in capillary electrophoresis. It requires adding an appropriate amount of surfactant to the capillary electrophoresis buffer solution. Then, the surfactant polymerizes to form micelles as a pseudo-stationary phase, thus combining capillary electrophoresis with electrochromatography technology. MEKC is essentially a liquid-liquid partition chromatography, which does not rely on a solid support to immobilize the liquid stationary phase, so micelles are usually called pseudo-stationary phases. During the separation process, the analyte is partitioned between the buffer mobile phase and the micellar pseudo-stationary phase. The separation mechanism of MEKC involves principles such as partitioning, electrostatic interaction, and electrophoretic migration. Although MEKC is widely used in chiral separation, most studies add chiral selectors and micelle-forming surfactants to the buffer solution separately. There are still few studies on using chiral micelles directly as chiral selectors. Currently, chiral micelles are composed of natural chiral surfactants such as cholates and saponins, as well as synthetic chiral surfactants such as alkyl amino acid derivatives, alkyl glucosides, and alkyl tartramides. However, the enantioselectivity of most chiral micelles is limited. In many cases, when using only chiral micelles as the sole chiral selector, the separation effect is not satisfactory. It is necessary to develop new chiral micelles with excellent performance as the sole chiral selector. Summary of the Invention

[0005] The purpose of this application is to solve the technical problem that the enantioselectivity of chiral micelles in the prior art is limited, and in many cases, when using only chiral micelles as the sole chiral selector, the separation effect is not satisfactory.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A method for preparing nano-micelles, comprising the following steps:

[0008] S1: Weigh an appropriate amount of CM-β-CD and place it in a flask. Add anhydrous formamide that has passed through a molecular sieve overnight to the flask, heat to 50 °C and stir. After CM-β-CD is dissolved, cool the reaction solution to room temperature.

[0009] S2: Add a catalyst and stir for 2 h under ice bath and nitrogen protection to activate the carboxyl group.

[0010] S3: Weigh long-chain fatty amine and dissolve it in DMF. Slowly add it dropwise to the above reaction solution, then heat to 50 °C and stir and react for 24 h under nitrogen protection in the dark.

[0011] S4: After the reaction is completed, add cold ethanol with a volume 5 times that of the reaction solution to precipitate the product; stir for 10 min to fully precipitate the product, then centrifuge to remove the supernatant; dissolve the remaining precipitate with an appropriate amount of water and dialyze for 2 days to remove small molecule impurities.

[0012] S5: Finally, the product is obtained by freeze-drying. An appropriate amount of the product is dissolved in water, and amphiphilic micelles can be self-assembled.

[0013] Preferably, in S1, the weighed amount of CM-β-CD is 0.2 mmol, and the added amount of anhydrous formamide is 10 mL.

[0014] Preferably, the catalyst is EDC and HNS, and the added amounts are 1.2 eq and 0.8 eq respectively.

[0015] Preferably, the long-chain fatty amine is one of DDA, ODA, and OCA.

[0016] This application also provides a kind of nano-micelles, which are prepared by using the above-mentioned preparation method.

[0017] Application of the above-mentioned nano-micelles as chiral functional materials.

[0018] Preferably, the conditions for the application of the nano-micelles as chiral functional materials are: capillary temperature, 25 °C; separation voltage, 14 - 20 kV; BGE, 30 mM NaH2PO4 buffer solution containing 0.66 - 1.33% CM-β-CD-DDA (m / v); buffer solution pH value, 2.8 - 3.5.

[0019] This application also provides a preparation method of drug-loaded nano-micelles, using the nano-micelles prepared by the above-mentioned preparation method.

[0020] Preferably, the preparation method includes the following steps:

[0021] S1: Weigh 10 mg of blank micelles and dissolve them in 5 mL of distilled water to prepare a blank micelle solution.

[0022] S2: Dissolve 2 mg of the model drug in 0.5 mL of methanol and drop it into the above blank micelle solution.

[0023] S3: Ultrasonically treat the obtained mixed solution in an ice bath for 30 min; ultrasonic power 200 W, work for 2 s, interval 3 s.

[0024] S4: After the ultrasonic treatment is completed, centrifuge the mixed solution, and then filter the supernatant through a 0.8 μm microporous membrane to remove insoluble substances and impurities.

[0025] S5: Dialyze the remaining solution in distilled water to remove organic solvents, and then perform freeze-drying treatment on the dialyzed solution to obtain the freeze-dried powder of drug-loaded micelles.

[0026] Preferably, the model drug is one of AML, HOM, PHE, TER, MIA, and MIR.

[0027] In this application, a chiral selector is combined with a micellar pseudo-stationary phase to develop a new capillary electrophoresis chiral separation method. By using carboxymethyl-β-cyclodextrin (CM-β-CD) as the hydrophilic group and long-chain fatty amine as the hydrophobic group, a new type of nano-micellar material is prepared. This new amide compound forms nano-micelles through self-assembly in the buffer solution, acting as both a chiral selector and a pseudo-stationary phase. Compared with the separate CM-β-CD system and the CM-β-CD / sodium dodecyl sulfate micellar electrokinetic chromatography system, the separation of model drugs is significantly improved. This study optimized several separation parameters and comprehensively characterized the nano-micelles by methods such as infrared spectroscopy, nuclear magnetic resonance spectroscopy, particle size determination, and transmission electron microscopy (TEM). In addition, amlodipine was selected as the model analyte, and the drug loading capacity in the separate CM-β-CD system and the nano-micellar system was determined by high performance liquid chromatography. The research results show that the nano-micelles have excellent drug loading capacity. Particle size and TEM analysis visually confirmed the distribution of enantiomers in the hydrophobic core of the nano-micelles. The nano-micelles show great promise in the field of chiral separation, and this strategy opens up a new way for the development of functional chiral materials. Brief Description of the Drawings

[0028] Figure 1 It is the synthesis route diagram of CM-β-CD-DDA nano-micelles in one embodiment of the present invention;

[0029] Figure 2 It is the chemical structures of six model drugs in one embodiment of the present invention;

[0030] Figure 3 It is the FT-IR spectra of CM-β-CD and CM-β-CD-DDA in one embodiment of the present invention;

[0031] Figure 4 It is of CM-β-CD and CM-β-CD-DDA in one embodiment of the present invention 1 HNMR spectra (500 MHz, deuterium oxide);

[0032] Figure 5 It is the Tyndall effect of several solutions in one embodiment of the present invention (A) blank solution, (B)

[0033] CM-β-CD, (C) CM-β-CD-DDA nano-micelles and (D) CM-β-CD-DDA nano-micelles in the dark. Conditions: 30 mM NaH2PO4 buffer containing 1.0% CM-β-CD or CM-β-CD-DDA (m / v); buffer pH value is 3.0;

[0034] Figure 6Particle size distribution diagrams of (A) CM-β-CD-DDA blank nanomicelles and (B) nanomicelles loaded with AML; transmission electron microscope images of (C) CM-β-CD-DDA blank nanomicelles and (D) nanomicelles loaded with AML. Conditions: 2 mg / mL blank nanomicelles or nanomicelles loaded with AML;

[0035] Figure 7 Effect of the concentration of CM-β-CD-DDA nanomicelles on enantiomeric separation in one embodiment of the present invention. Conditions: Capillary temperature 25 °C; separation voltage 16 kV (TER is 18 kV); BGE,

[0036] 30 mM NaH2PO4 buffer containing 0.66%-1.33% CM-β-CD-DDA (m / v); buffer pH value 3.0 (PHE is 3.2);

[0037] Figure 8 Effect of buffer pH value on chiral separation in one embodiment of the present invention. Conditions: Capillary temperature, 25 °C; separation voltage, 16 kV (TER is 18 kV); BGE, containing 1% CM-β-CD-DDA

[0038] (m / v) in 30 mM NaH2PO4 buffer; buffer pH value, 2.8 - 3.5;

[0039] Figure 9 Effect of separation voltage on chiral resolution in one embodiment of the present invention. Conditions: Capillary temperature, 25 °C; separation voltage, 14 - 20 kV; BGE, 30 mM NaH2PO4 buffer containing 1% CM-β-CD-DDA (m / v); buffer pH value, 3.0 (PHE is 3.2);

[0040] Figure 10 Typical electrophoretograms of chiral separation in different systems in one embodiment of the present invention. Magenta, single CM-β-CD system; cyan, CM-β-CD-DDA nanomicelle system. Conditions: Capillary temperature, 25 °C; separation voltage, 16 kV (TER is 18 kV); BGE, 30 mM NaH2PO4 buffer containing 1% CM-β-CD-DDA (m / v) or 1% CM-β-CD (m / v); buffer pH,

[0041] 3.0 (PHE is 3.2);

[0042] Figure 11 Typical chromatogram of the determination of AML by HPLC method for calculating drug loading in one embodiment of the present invention. Detailed implementation manners

[0043] The present invention will be further described in detail below in conjunction with specific embodiments.

[0044] A preparation method of nano micelles, the synthetic route of the micelles is as Figure 1 shown, and the preparation method includes the following steps:

[0045] S1: Weigh an appropriate amount of CM-β-CD and place it in a flask. Add anhydrous formamide that has passed through a molecular sieve overnight to the flask, heat it to 50 °C and stir. After CM-β-CD is dissolved, the reaction solution is cooled to room temperature.

[0046] In one embodiment, the weighed amount of CM-β-CD is 0.2 mmol, and the added amount of anhydrous formamide is 10 mL.

[0047] S2: Subsequently, add a catalyst and stir for 2 h under an ice bath and nitrogen protection to activate the carboxyl group.

[0048] In one embodiment, the catalyst is EDC and HNS, and the added amounts are 1.2 eq and 0.8 eq respectively.

[0049] S3: Weigh a long-chain fatty amine and dissolve it in DMF. Slowly add it drop by drop to the above reaction solution, then heat it to 50 °C and stir and react for 24 h under nitrogen protection in the dark.

[0050] In one embodiment, the long-chain fatty amine is one of dodecylamine (DDA), octadecylamine (ODA), and octylamine (OCA), and preferably DDA.

[0051] S4: After the reaction is completed, add cold ethanol with a volume 5 times that of the reaction solution to precipitate the product; stir for 10 min to fully precipitate the product, then centrifuge to remove the supernatant; dissolve the remaining precipitate in an appropriate amount of water and dialyze for 2 days to remove small molecule impurities;

[0052] S5: Finally, obtain the product by freeze-drying. Take an appropriate amount of the product and dissolve it in water to form amphiphilic micelles by self-assembly.

[0053] Based on the above-mentioned preparation method of nano micelles, the present application also provides a drug-loaded nano micelle. Specifically, the preparation method of the drug-loaded nano micelle is as follows:

[0054] First, weigh 10 mg of blank micelles and dissolve them in 5 mL of distilled water to prepare a blank micelle solution;

[0055] Dissolve 2 mg of the model drug in 0.5 mL of methanol and drop it into the above blank micelle solution;

[0056] The model drug is one of AML, HOM, PHE, TER, MIA, and MIR.

[0057] The obtained mixed solution was ultrasonically treated in an ice bath for 30 min with an ultrasonic power of 200 W, working for 2 s and interval for 3 s.

[0058] After the ultrasonic treatment was completed, the mixed solution was centrifuged, and then the supernatant was filtered through a 0.8-μm microporous membrane to remove insoluble substances and impurities.

[0059] In one embodiment, the centrifugation was carried out at 5000 revolutions per minute for 15 min;

[0060] The remaining solution was dialyzed in distilled water to remove organic solvents, and then the dialyzed solution was freeze-dried to obtain the freeze-dried powder of the drug-loaded micelles.

[0061] Based on the above preparation method, the present application also provides the application of the nanomicelles as chiral functional materials.

[0062] The above content is elaborated below in combination with specific experiments:

[0063] Reagents:

[0064] CM-β-CD (average degree of substitution = 5.6), pheniramine maleate (PHE, pKa 9.32), sodium dodecyl sulfate (SDS), formamide, sodium dihydrogen phosphate (NaH2PO4), amlodipine (AML, pKa 8.6) were all purchased from Shanghai Bide Pharmaceutical Co., Ltd.

[0065] Methanol, ethanol, acetonitrile, N,N-dimethylformamide (DMF) and phosphoric acid (85 wt.%) were all purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.

[0066] Dodecylamine (DDA), octadecylamine (ODA), octylamine (OCA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (HNS) were all purchased from Shanghai Aladdin Reagent Co., Ltd.

[0067] Mianserin hydrochloride (MIA, pKa 7.50), terbutaline sulfate (TER, pKa 8.70) were respectively purchased from Yuanye Biotechnology Co., Ltd.

[0068] Mirtazapine (MIR, pKa 8.10) was purchased from Macklin Biochemical Co., Ltd.

[0069] Homatropine hydrobromide (HOM, pKa 9.9) was purchased from Leyan Biochemical Technology Co., Ltd.

[0070] Sodium hydroxide was purchased from Xilong Chemical Co., Ltd.

[0071] Ammonium acetate was purchased from Sins Biotechnology Co., Ltd.

[0072] Borax was purchased from Energy Chemical Co., Ltd.

[0073] Please refer to Figure 2 , Figure 2 which shows the structure of the model drug.

[0074] Example 1: Preparation of nanomicelles:

[0075] An appropriate amount of CM-β-CD (0.2 mmol) was accurately weighed and placed in a flask, and 10 mL of anhydrous formamide that had passed through a molecular sieve overnight was added. It was heated to 50 °C and stirred. After CM-β-CD was dissolved, the reaction solution was cooled to room temperature.

[0076] Subsequently, two catalysts, EDC (1.2 eq) and HNS (0.8 eq), were added. It was stirred for 2 h under an ice bath and nitrogen protection to activate the carboxyl group.

[0077] Then, an appropriate amount of dodecylamine (1.0 eq) was accurately weighed and dissolved in 13 mL of DMF, and it was slowly added dropwise to the above reaction solution. It was heated to 50 °C and stirred and reacted for 24 h under nitrogen protection in the dark.

[0078] After the reaction was completed, cold ethanol with a volume 5 times that of the reaction solution was added to precipitate the product. It was stirred for 10 min. After the product was fully precipitated, the supernatant was removed by centrifugation. The remaining precipitate was dissolved in an appropriate amount of water and dialyzed for 2 days to remove small molecule impurities. Finally, the product was obtained after freeze-drying. It should be noted that the entire reaction requires anhydrous conditions. An appropriate amount of the product was dissolved in water, and amphiphilic micelles could be self-assembled.

[0079] Example 2: Preparation of drug-loaded nanomicelles

[0080] In this example, amlodipine (AML) was used as the model drug.

[0081] 10 mg of blank micelles (CM-β-CD-DDA) was dissolved in 5 mL of distilled water to prepare a blank micelle solution. 2 mg of AML was dissolved in 0.5 mL of methanol and dropped into the above blank micelle solution. The mixed solution was ultrasonically treated in an ice bath for 30 min (ultrasonic power 200 W, working for 2 s, interval 3 s). After centrifuging at 5000 revolutions per minute for 15 min, the supernatant was filtered through a 0.8 μm microporous membrane to remove insoluble substances and impurities. The remaining solution was dialyzed in distilled water to remove organic solvents. Then, it was freeze-dried to obtain the freeze-dried powder of the drug-loaded micelles. The preparation method of the drug-loaded CM-β-CD is similar to the above, and the only difference is that the blank micelles are replaced by CM-β-CD.

[0082] Example 3: Characterization of nanomicelles:

[0083] Please refer to Figure 3 ,Figure 3 The FT-IR spectra of CM-β-CD and CM-β-CD-DDA are shown. As Figure 3 shown, the broad peak at 3388 cm -1 is attributed to the hydroxyl group (νO-H), while the absorption peak at 1031 cm -1 is ascribed to the stretching vibration of the hydroxyl group (νC-OH). The symmetric stretching vibration peak of the methyl group (ν -1 C-H3) is observed at 2924 cm as . The absorption peak at 1416 cm -1 is caused by the bending vibration of the methyl group (δC-H3). The out-of-plane bending vibration (γC-C) absorption peak is at 709 cm -1 . In addition, the characteristic peak at 1603 cm -1 is attributed to the carboxyl group (νC=O). Compared with the spectrum of CM-β-CD, the characteristic peak of the corresponding amide (νC=O) is observed at 1691 cm -1 . Only one carboxyl group in the CM-β-CD molecule participated in the acylation reaction. Therefore, two carbonyl peaks (νC=O) are observed in the product, indicating the successful amide modification of CM-β-CD.

[0084] Figure 4 The 1 1H NMR spectra of CM-β-CD and CM-β-CD-DDA are shown. Compared with the raw materials, the chemical shift values of CM-β-CD in the product did not change significantly. However, the proton signals of DDA are observed in the product, and the peak assignments of these protons are successfully completed. 1 1H NMR analysis further confirmed the successful synthesis of CM-β-CD-DDA.

[0085] The Tyndall effect is an important optical property of colloidal dispersions and is commonly used for colloidal identification. When the size of the dispersed particles is smaller than the wavelength of the incident light, light scattering occurs and this phenomenon appears. When a red laser beam is vertically irradiated on the blank solution and the CM-β-CD solution ( Figure 5 ), no bright light path can be observed. This lack of brightness is attributed to the high uniformity of the molecular solution, which cancels out the scattered light through mutual interference. When a red laser beam is vertically irradiated on the CM-β-CD-DDA solution, an obvious light column is observed. This result indicates the successful formation of nano micelles.

[0086] The particle size distribution of the blank nano micelles was analyzed using a ZETa potential and particle size analyzer (NanoBrook90Plus, USA). Figure 6A shows a relatively uniform distribution with an average size of 150 nm. Finally, in this example, blank nanomicelles were directly observed by transmission electron microscopy (TEM, Hitachi HT-7800). Figure 6 C shows that according to the scale bar, the particle size of the nanomicelles is about 130 nm and presents a spherical shape. The particle size measured by TEM is generally smaller than that measured by a particle size analyzer. This difference is attributed to the fact that the particle size analyzer takes into account the movement of particles in the solution, and the measured result is generally the hydrodynamic particle size of the particles.

[0087] Example 4: Electrophoresis experiment and optimization of electrophoresis conditions

[0088] The separation experiment was carried out on a CL1030 CE system (Beijing Huayang Limin Instrument Co., Ltd.), which includes a UV absorption detector (190 - 700 nm), a high-voltage power supply (±30 kV), and a data processor. A new capillary (365 μm O.D. and 50 μm I.D.) was purchased from Yongnian Optical Fiber Factory (Hebei, China). A new capillary (50 cm total length and 41 cm effective length) was activated by flushing with 1 M NaOH, 0.1 M NaOH, and H2O for 20 minutes respectively. Between consecutive injections, the capillary was flushed with 0.1 M NaOH, H2O, and the running buffer for 3 minutes. Sample injection was carried out in hydrodynamic mode with an injection time of 10 seconds and a height difference of 10 cm. Samples were monitored at different wavelengths (210 nm for TER and HOM, 237 nm for AML, 240 nm for MIR and MIA, 265 nm for PHE). The temperature of the capillary and the sample tray was maintained at 25 °C.

[0089] The sample solution (0.5 mg / mL) was freshly prepared by dissolving the racemic drug in methanol / water (1:1, v / v). The optimized buffer solution was 30 mM NaH2PO4 buffer containing 1% (m / v) CM-β-CD-DDA. Before use, the pH value of the running buffer was adjusted to 3.0 using 20% (v / v) H3PO4.

[0090] To achieve better separation effects, several key separation parameters were optimized in this application:

[0091] First: The types and concentrations of the buffer solution and organic additives were optimized. The concentration of the buffer was set to 30 mM, and the effects of three buffer systems (ammonium acetate, borax, and NaH2PO4) on enantiomeric separation were studied. According to their respective pH buffer ranges, running baselines, and separation effects, the NaH2PO4 buffer system was considered the most ideal.

[0092] Subsequently, in this example, the effects of buffer salts at different concentrations (20 mM, 30 mM, and 40 mM) on enantiomeric separation were studied. It was observed that the concentration of NaH2PO4 had little effect on the separation. Therefore, a medium concentration (30 mM) of NaH2PO4 buffer solution was used in this example for further analysis. On this basis, the effects of three organic solvents (methanol, ethanol, and acetonitrile) as additives on enantiomeric separation were investigated in this example. No matter which organic additive was used, they all had a negative impact on enantiomeric separation. This is because the hydrophobic long chains of CM-β-CD-DDA have a relatively high solubility in organic solvents, which will disrupt the micelle structure.

[0093] In this example, the effects of the hydrophobic chain length on enantiomeric separation were studied by synthesizing nano-micelles based on three long-chain fatty amines (OCA, DDA, and ODA). The results showed that CM-β-CD-ODA (CM-β-CD octadecanamide) had low water solubility and formed a white turbid solution in the buffer. Although the water solubility of CM-β-CD-OCA (CM-β-CD octanamide) was improved, a higher concentration was required to form micelles. Even at a concentration of 2% (m / v), CM-β-CD-OCA still could not effectively form micelles. Therefore, CM-β-CD-DDA (CM-β-CD dodecanamide) was used as the nano-micelles in this study.

[0094] CM-β-CD-DDA nano-micelles, acting as both a chiral selector and a pseudo-stationary phase, significantly affect chiral separation. When the concentration of CM-β-CD-DDA is low, it cannot provide sufficient chiral recognition interactions. A high concentration of CM-β-CD-DDA will also reduce the enantioselectivity and prolong the migration time, resulting in peak broadening and affecting the separation efficiency. After investigation ( Figure 7 ), the best separation of the model drug was achieved when the concentration of CM-β-CD-DDA was 1% (m / v).

[0095] The pH value of the buffer affects the charging properties of the capillary inner wall, thus affecting the electroosmotic flow. It also affects the charging states of the chiral selector and the enantiomers, thus affecting the interaction between them. In the pH range of 2.8 - 3.5, the effects of the buffer pH value on chiral separation were studied in this example ( Figure 8) The results showed that the resolution (Rs) value of the model drugs initially increased with the increase of pH value and then decreased. Most model drugs showed the maximum Rs value at pH 3.0, under which the interaction between the nanomicelles and the enantiomers was saturated. For PHE, its Rs value reached the maximum at pH 3.2. It is worth mentioning that at pH 3.5, no enantiomeric peaks of MIA and MIR were detected within 60 minutes. This was due to the high lipophilicity of MIA and MIR, and the increase in pH led to the dissociation of enantiomers and the reduction of charged molecules. More MIA and MIR were encapsulated into the nanomicelles, resulting in an extended migration time. Therefore, pH 3.0 or 3.2 (for PHE) was selected as the optimal pH value in this example.

[0096] In addition, in capillary electrophoresis, the separation voltage is one of the key factors affecting chiral separation. On the one hand, a higher voltage can improve the separation efficiency and shorten the analysis time, but it may also reduce the contact between the chiral selector and the enantiomers. On the other hand, a lower voltage can extend the migration time and increase the interaction opportunity between the chiral selector and the enantiomers. However, this may also lead to peak broadening and affect the Rs value. Therefore, it is necessary to select an appropriate separation voltage. To determine the optimal separation voltage, relevant experiments were carried out in this example. As Figure 9 shown, the Rs of most drugs increased as the separation voltage increased from 14 kV to 16 kV. Further increasing the separation voltage, the Rs of the model drugs began to decrease. As for TER, the best separation was achieved at a separation voltage of 18 kV. Finally, 16 kV (18 kV was selected for TER) was chosen as the optimal separation voltage for this system in this example.

[0097] Example 4: Establish a separation system:

[0098] Based on the separation parameters in Example 3, six chiral drugs including AML, HOM, PHE, TER, MIA, and MIR were selected as model analytes in this example. Three different separation systems were established, including the single CM-β-CD system, the CM-β-CD / SDS system, and the CM-β-CD-DDA nanomicelle system. The separation results of the model drugs in each system are as Figure 10 shown in Table 1.

[0099] Table 1 Chiral separation results of six model drugs in different systems

[0100]

[0101] In the CM-β-CD-DDA nanomicelle system, the separation effect of the model drug is significantly better than that in the CM-β-CD system alone. At the same time, the migration times of the enantiomers all increase to varying degrees. This is attributed to the combined effect of the inclusion effect of CM-β-CD and the distribution effect of the nanomicelle enantiomers. To further highlight the superiority of CM-β-CD-DDA nanomicelles, in this example, it was compared with the classical MEKC separation system constructed by CM-β-CD and SDS. Although the separation effect of the model drug in the CM-β-CD / SDS system has been improved, it is far from the level of the CM-β-CD-DDA nanomicelle system. In addition, in the CM-β-CD / SDS system, neither MIA nor MIR was detected within 60 minutes.

[0102] In addition, in this example, the relative standard deviations (RSDs) of Rs and migration time of the model drug in the CM-β-CD-DDA nanomicelle system were calculated to evaluate the repeatability of the method. The analysis data were obtained by continuously injecting the sample 5 times under the optimal separation conditions. The intra-day RSDs of Rs and migration time were respectively lower than 3.5% and 3.8%. The inter-day RSDs of Rs and migration time were respectively lower than 3.7% and 4.1%. The results show that the developed method has good repeatability. The stability of CM-β-CD-DDA nanomicelles was also studied in this example. Before sample analysis, the buffer solution was allowed to stand for 12 hours, 24 hours, 48 hours, 72 hours, and 96 hours. It was found that the RSDs of Rs and migration time were respectively kept below 4.4% and 4.5%, indicating that CM-β-CD-DDA nanomicelles have good stability.

[0103] Example 5: Chiral recognition mechanism

[0104] The chiral recognition of cyclodextrin mainly stems from two aspects: the inclusion effect of the hydrophobic cavity on the enantiomers and the hydrogen bond interaction of the peripheral chiral hydroxyl groups on the enantiomers.

[0105] CM-β-CD not only has the above interactions, but also has ionizable carboxyl groups, carries a negative charge in the solution, and can produce electrostatic attraction with many positively charged alkaline drugs. In the traditional MEKC separation system, a pseudo-stationary phase in the form of micelles and a buffered aqueous phase containing a chiral selector coexist. Enantiomers are constantly distributed between the two phases. Due to the different interactions with the pseudo-stationary phase and the aqueous phase, differential migration occurs, resulting in the separation of drugs with different properties. In this study, CM-β-CD is not free in the buffer solution, but is only located on the hydrophilic outer surface of the nanomicelles. The enantiomer needs to pass through the hydrophilic outer layer made of CM-β-CD to reach the core of the nanomicelle. CM-β-CD itself has an inclusion effect on the enantiomer, so whether the enantiomer is distributed in the core of the nanomicelle and whether the nanomicelle acts as a pseudo-stationary phase remains to be verified.

[0106] To address this issue, in this example, AML was used as a model analyte using a particle size analyzer to determine the particle size distribution of drug-loaded nanomicelles. Figure 6 B) shows that the average particle size of the nanomicelles encapsulating AML is 226nm. Compared with blank nanomicelles (150nm), the particle size of the nanomicelles encapsulating AML is significantly increased. In this embodiment, the morphology of the nanomicelles encapsulating AML was also observed by transmission electron microscopy. The nanomicelles encapsulating AML also show a uniform spherical distribution, and according to the scale, the particle size is about 175nm. TEM analysis shows that the particle size (175nm) of the nanomicelles encapsulating AML is significantly larger than that of the blank nanomicelles (130nm). The above results show that the enantiomer has indeed entered the core of the nanomicelles, resulting in an increase in particle size after the nanomicelles are loaded with drugs.

[0107] In addition, in this example, AML was used as a model analyte to quantitatively evaluate the drug loading capacity of CM-β-CD and nanomicelles by HPLC. Drug loading is an important parameter in micelle evaluation. In this example, the drug loading was calculated as follows:

[0108] Accurately weigh 8 mg of drug-loaded micelles and drug-loaded CM-β-CD, and dissolve them separately in 4 mL of distilled water. The two solutions are ultrasonicated for 30 minutes and then filtered (0.8 μm microporous membrane). Take 200 μL of the supernatant, add 800 μL of methanol. The mixed solution is ultrasonicated for 30 minutes and centrifuged at 12,000 revolutions per minute for 10 minutes. Take the supernatant and inject 10 μL into the HPLC device for detection, and record the peak area. Accurately prepare a methanol solution of AML standard and dilute it to 0.02 mg / mL. Take 10 μL and inject it into the HPLC device, and record the peak area. The external standard method is used to calculate the content of AML in the nanomicelles. The formula for calculating the drug loading is as follows: Drug loading (%) = mass of drug in nanomicelles (CM-β-CD) / mass of total drug-loaded nanomicelles (CM-β-CD) × 100%.

[0109] In this example, the drug loading experiment was carried out using a high-performance liquid chromatograph (Agilent 1200 type, USA). In this study, a Phenomenon Gemini C18 column (250×4.6 mm, 5 μm) was used. Mobile phase A is a 30 mM ammonium acetate solution, and mobile phase B is methanol. The separation was completed by isocratic elution (40% phase A and 60% phase B). The detection wavelength was 237 nm, and the column temperature was controlled at 35 °C. The flow rate was 1.0 mL / min, and the sample injection volume was 10 μL.

[0110] The results showed that the drug loading of CM-β-CD was 1.28%, while the drug loading of CM-β-CD-DDA nanomicelles was 1.61%( Figure 11 and Table 2).

[0111] Table 2 Calculation results of drug loading in CM-β-CD and CM-β-DDA systems

[0112]

[0113] The higher drug loading of the nanomicelles indicates that the enantiomers are not only encapsulated in the hydrophobic cavity of CM-β-CD but also penetrate into the hydrophobic core of the nanomicelles. Therefore, the nanomicelles can act as a pseudo-stationary phase. In this system, CM-β-CD aggregates on the periphery of the nanomicelles, increasing the local concentration of the chiral selector and improving the enantioselectivity of chiral separation. In addition, the enantiomers are continuously distributed between CM-β-CD and the nanomicelle core, and this distance is shortened. These factors lead to a significant improvement in the separation of the model drug.

[0114] In summary, in this application, CM-β-CD and three long-chain fatty amines are used as raw materials to synthesize nanomicelle precursors through amidation reactions, namely CM-β-CD octanamide (CM-β-CD-OCA), CM-β-CD dodecanamide (CM-β-CD-DDA), and CM-β-CD octadecanamide (CM-β-CD-ODA). The prepared materials can self-assemble into nanomicelles in buffer solutions and simultaneously serve as chiral selectors and pseudo-stationary phases. In this constructed system, the separation of model drugs is significantly improved. In this application, various methods are used to characterize the nanomicelles, and the drug loading is determined by high-performance liquid chromatography (HPLC). On this basis, the chiral recognition mechanism of the nanomicelles is further discussed. Such nanomicelles are proven to be promising chiral functional materials.

[0115] In this application, a unique chiral separation system is provided, which uses CM-β-CD-DDA nanomicelles as chiral selectors and pseudo-stationary phases in CE. Compared with the individual CM-β-CD system and the CM-β-CD / SDS MEKC system, the nanomicelle system exhibits enhanced enantioselectivity and improved separation of model drugs. In this application, various methods are used to characterize the nanomicelles, and the HPLC method also proves an increase in the drug loading of the nanomicelles. After the formation of CM-β-CD-DDA nanomicelles, the local concentration of CM-β-CD increases, resulting in enhanced enantioselectivity. It also shortens the distance between the inclusion of enantiomers in CM-β-CD and their distribution in the nanomicelle core. Such micellar materials are easy to synthesize, have excellent properties, and have great potential in enantiomer separation.

Claims

1. A method for preparing nano micelles, characterized in that: The following steps are involved: S1: Weigh 0.2 mmol of CM-β-CD and place it in a flask. Add 10 mL of anhydrous formamide that has been passed through a molecular sieve overnight into the flask. Heat to 50 °C and stir. After the CM-β-CD dissolves, cool the reaction solution to room temperature. S2: adding catalyst p-toluenesulfonyl imide hydrochloride and N-hydroxysuccinimide, the catalyst addition amounts are 1.2 eq and 0.8 eq, respectively, and stirring for 2 h in an ice bath under nitrogen protection to activate the carboxyl group; S3: Weigh a long-chain fatty amine and dissolve it in DMF, slowly add it dropwise to the above reaction solution, then heat it to 50°C, and stir and react for 24 hours under the protection of nitrogen and away from light; the long-chain fatty amine is one of DDA, ODA, and OCA; S4: After the reaction is completed, add 5 times the volume of the reaction solution of cold ethanol to precipitate the product; stir for 10 min to allow the product to precipitate fully, then centrifuge to remove the supernatant; dissolve the remaining precipitate with an appropriate amount of water and dialyze for 2 days to remove small molecule impurities; S5: Finally, the product is obtained by freeze-drying, and an appropriate amount of the product is dissolved in water to self-assemble into amphiphilic micelles.

2. A nano micelle, characterized in that: The preparation method according to claim 1 is used for preparation.

3. Application of the nanomicelles prepared by the preparation method according to claim 1 as chiral functional materials.

4. The use of the nano micelles according to claim 3 as chiral functional materials, characterized in that: The conditions for applying the nanomicelles as chiral functional materials are as follows: capillary temperature, 25°C; separation voltage, 14-20 kV; BGE, 30 mM NaH2PO4 buffer containing 0.66-1.33% CM-β-CD-DDA; buffer pH, 2.8-3.

5.

5. A drug-loaded nanomicelle, characterized in that: The invention comprises nano micelles prepared by the preparation method according to claim 1 and a model drug.

6. A method for preparing drug-loaded nanomicelles, characterized in that: For preparing the drug-loaded nanomicelles according to claim 5, the preparation method comprises the following steps: S1: Weigh 10 mg of blank nanomicelles and dissolve them in 5 mL of distilled water to prepare a blank nanomicelle solution; S2: 2 mg of the model drug was dissolved in 0.5 mL of methanol and dropped into the blank nanomicelle solution; S3: The obtained mixed solution was ultrasonically treated in an ice bath for 30 min; the ultrasonic power was 200 W, the working time was 2 s, and the interval was 3 s. S4: After the ultrasonic treatment, the mixed solution was centrifuged, and then the supernatant was filtered through a 0.8 μm microporous membrane to remove insoluble matter and impurities; S5: The remaining solution is dialyzed in distilled water to remove the organic solvent, and then the dialyzed solution is freeze-dried to obtain drug-loaded nanomicelle freeze-dried powder.

7. The method for preparing drug-loaded nanomicelles according to claim 6, characterized in that: The model drug is one of amlodipine, homatropine hydrobromide, pheniramine maleate, terbutaline sulfate, mianserin hydrochloride and mirtazapine.

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