Y-shaped chiral nano-micelle as well as preparation method and application thereof

Abstract

The invention provides a Y-type chiral nano-micelle as well as a preparation method and application thereof, and relates to the technical field of biomedicine, the technical key points are as follows: the preparation method of the Y-type chiral nano-micelle comprises the following steps: preparing and dissolving gamma-cyclodextrin in anhydrous formamide (a molecular sieve is overnight), then stirring at 60 DEG C to dissolve the gamma-cyclodextrin, and then cooling to room temperature; adding catalysts EDC and NHS into the S1 solution, and after addition is completed, magnetically stirring under ice bath nitrogen charging conditions; the preparation method comprises the following steps: weighing long-chain carboxylic acid, dissolving the long-chain carboxylic acid in anhydrous formamide, adding DMAP, dropwise adding the solution into a gamma-cyclodextrin solution, and magnetically stirring at 80 DEG C under the dark nitrogen charging condition; after the reaction is finished, diluting the reaction liquid by using ultrapure water, and dialyzing for 24 hours by using a dialysis bag; and taking out dialysate, and freeze-drying to obtain the Y-type chiral nano-micelle. According to the invention, the superiority of the Y-type nano-micelle in CE chiral separation is proved through a verification test, and an important step is made for developing a novel chiral recognition material.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a Y-type chiral nanomicelle, its preparation method, and its application. Background Technology

[0002] Approximately half of the drugs used worldwide are chiral, and different enantiomers of chiral drugs often possess different pharmacological activities and side effects. Therefore, rapid and efficient chiral separation methods are a research hotspot and also a challenging area in the field of pharmaceutical analysis. Capillary electrophoresis (CE) is highly favored in the field of chiral separation due to its low operating cost, multiple separation modes, short analysis time, and high separation efficiency.

[0003] In chiral separation in CE (eclectic cyclodextrin) studies, the most common method is to directly add a suitable chiral selector to the buffer solution. A wide variety of chiral selectors exist, with cyclodextrins and their derivatives being the most widely used due to their good water solubility, extremely low UV absorption, strong enantioselectivity, and moderate cost. Compared to α-cyclodextrins and γ-cyclodextrins and their derivatives, β-cyclodextrins and their derivatives have been studied more extensively due to their cavity size matching that of most drugs. Besides the original β-cyclodextrin, developed β-cyclodextrin chiral selectors include methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, glucosyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, sulfobutyl-β-cyclodextrin, and various ammonium-cationically modified β-cyclodextrins. Research reports on the use of original γ-cyclodextrins in chiral separation in CE studies are relatively few, even though they possess outstanding water solubility. In many studies, satisfactory separation results were not achieved using original γ-cyclodextrins alone. Summary of the Invention

[0004] The purpose of this invention is to solve the problem that satisfactory separation results cannot be obtained by using raw γ-cyclodextrin alone in the prior art.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing Y-type chiral nanomicelles includes the following steps:

[0007] S1: Dissolve γ-cyclodextrin in anhydrous formamide (molecular sieve overnight), then stir at 60 ℃ to dissolve, and then cool to room temperature;

[0008] S2: Add catalysts EDC and NHS to the S1 solution. After the addition is complete, magnetically stir for 2 hours under ice bath nitrogen purging to obtain an activated γ-cyclodextrin solution.

[0009] S3: Weigh out long-chain carboxylic acids and dissolve them in anhydrous formamide, add DMAP, and then add the solution dropwise to the activated γ-cyclodextrin solution. Stir magnetically for 24 h at 80 °C under light-proof and nitrogen-filled conditions.

[0010] S4: After the reaction in S3 is completed, dilute the reaction solution with ultrapure water and then dialyze using a dialysis bag for 24 hours.

[0011] S5: Take out the dialysate from S4 and freeze-dry it to obtain Y-type chiral nanomicelles.

[0012] Preferably, the ratio of γ-cyclodextrin to anhydrous formamide in S1 is 1.297 g: 20 mL.

[0013] Preferably, the molar ratio of γ-cyclodextrin in S1 to EDC and NHS in S2 is 1:1.2:0.8.

[0014] Preferably, the long-chain carboxylic acid in S3 is any one of octanoic acid (OA), 2-butyloctanoic acid, and 4-ethyloctanoic acid.

[0015] Preferably, the molar ratio of γ-cyclodextrin in S1 to long-chain carboxylic acid and DMAP in S3 is 1:1:1.2.

[0016] This application also provides a Y-type chiral nanomicelle, which is prepared using the preparation method described above.

[0017] This application also provides the application of Y-type chiral nanomicelles in the preparation of drug-loaded nanomicelles.

[0018] This application also provides a drug-loaded nanomicelle using the Y-type chiral nanomicelles described above.

[0019] Preferably, the drug loaded in the drug-loaded nanomicelles is a racemic drug.

[0020] Compared with the prior art, this application has the following beneficial effects:

[0021] In this application, nanomicelle precursors were prepared via esterification using pristine γ-cyclodextrin as the hydrophilic group and Y-type long-chain carboxylic acid as the hydrophobic group. These precursors can self-assemble into nanomicelles in buffer solution, simultaneously serving as chiral selectors and pseudo-stationary phases. Compared to ordinary nanomicelles, Y-type nanomicelles exhibit greater drug loading and superior separation performance. The corresponding mechanism of action has been thoroughly verified and discussed through various research methods. This research further expands the application of nanomicelles in chiral separation of CE (Cellular Electrode Extraction), laying a solid foundation for the development of novel chiral recognition materials. Attached Figure Description

[0022] Figure 1This is a synthetic route for Y-type chiral nanomicelles according to one embodiment of the present invention;

[0023] Figure 2 The chemical structures of the reagents used in this application are shown, wherein A is the chemical structure of γ-cyclodextrin and B is the chemical structure of a long-chain carboxylic acid.

[0024] Figure 3 The structural formula of the racemic drug in this application;

[0025] Figure 4 The 1H NMR spectra (500 MHz, heavy water) of γ-CD-EOA nanomicelles and their synthetic raw materials are shown.

[0026] Figure 5 FT-IR spectra of γ-CD-EOA nanomicelles and their synthetic raw materials

[0027] Figure 6 SEM images of γ-CD-OA (A and B), γ-CD-EOA (C and D), and γ-CD-EOA (E and F) nanomicelles.

[0028] Figure 7 The particle size distribution and TEM images of the nanomicelles are shown. (A and D) Blank γ-CD-OA nanomicelles; (B and E) Blank γ-CD-EOA nanomicelles; (C and F) PRO-loaded γ-CD-EOA nanomicelles.

[0029] Figure 8 The foaming phenomenon of solutions (A) γ-CD, (B) γ-CD-OA, and (C) γ-CD-EOA. Conditions: Concentration, 5 mg / mL in water; Temperature, 25℃.

[0030] Figure 9 The Tyndall effect was observed in solutions of (A) γ-CD, (B) γ-CD-OA, and (C) γ-CD-EOA. Conditions: 27 mg / mL concentration in 20 mM borax buffer, pH 9.0.

[0031] Figure 10 The effect of γ-CD-EOA nanomicelle concentration on SOT enantiomer separation was investigated. Conditions: capillary temperature, 25℃; voltage, 12 kV; BGE, 20 mM borax buffer containing 20-30 mg / mL γ-CD-EOA nanomicelles; buffer pH 9.0.

[0032] Figure 11The effect of buffer pH on SOT enantiomer separation was investigated. Conditions: capillary temperature, 25℃; voltage, 12kV; BGE, 20mM borax buffer containing 27 mg / mL γ-CD-EOA nanomicelles; buffer pH range 8.5-9.5.

[0033] Figure 12 The effect of separation voltage on SOT enantiomer separation was investigated. Conditions: capillary temperature, 25℃; voltage, 10-16 kV; BGE, 20 mM borax buffer containing 27 mg / mL γ-CD-EOA nanomicelles; buffer pH 9.0.

[0034] Figure 13 Typical electrophoretic images of enantiomer separation in different systems. Green, γ-CD system alone; magenta, γ-CD-OA nanomicelle system; orange, γ-CD-EOA nanomicelle system. Conditions: capillary temperature, 25℃; voltage, 12 kV; BGE, 20 mM borax buffer containing 27 mg / mL γ-CD-OA or γ-CD-EOA nanomicelles or 27 mg / mL γ-CD; buffer pH 9.0.

[0035] Figure 14 The 1H NMR spectra of (A) PRO and (B) γ-CD-EOA / PRO are shown. Conditions: 500 MHz, 5 mg γ-CD-EOA and / or 3 mg PRO in 0.5 mL D2O.

[0036] Figure 15 The ROESY NMR spectrum of the γ-CD-EOA / PRO system. Conditions: 500 MHz, 5 mg of γ-CD-EOA and 3 mg of PRO in 0.5 mL D2O at pH 9.0.

[0037] Figure 16 Typical HPLC chromatograms for determining PRO content when calculating drug loading in different systems. (A) PRO standard; (B) γ-CD system; (C) γ-CD-OA nanomicelle system; (D) γ-CD-EOA nanomicelle system.

[0038] Figure 17 (A) Thermogravimetric curves of blank γ-CD-EOA, γ-CD-EOA loaded with PRO, and γ-CD-OA loaded with PRO. (B) Differential scanning calorimetry curves of blank γ-CD-EOA, γ-CD-EOA loaded with PRO, and γ-CD-OA loaded with PRO. Magenta, γ-CD-EOA; orange, γ-CD-EOA loaded with PRO; green, γ-CD-OA loaded with PRO. Temperature range: 30-1000℃. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to specific embodiments.

[0040] This application provides a method for preparing Y-type chiral nanomicelles, the synthesis route of which is as follows: Figure 1 As shown, the specific steps are as follows:

[0041] S1: Prepare 1 eq of γ-cyclodextrin (γ-CD) dissolved in anhydrous formamide (molecular sieve overnight), then stir at 60 ℃ to dissolve, and then cool to room temperature. The structure of γ-cyclodextrin is as follows. Figure 2 As shown in Figure A.

[0042] In one embodiment, the ratio of γ-cyclodextrin to anhydrous formamide is 1.297 g: 20 mL.

[0043] S2: Add catalyst EDC and NHS to the S1 solution. The amount of EDC added is 1.2 eq and the amount of NHS added is 0.8 eq. After the addition is completed, the solution is magnetically stirred for 2 hours under nitrogen-filled ice bath conditions to activate the hydroxyl groups.

[0044] S3: Accurately weigh 1 eq of long-chain carboxylic acid and dissolve it in anhydrous formamide, then add 1.2 eq of DMAP. Add the above solution dropwise to the γ-cyclodextrin solution in S2 and stir magnetically for 24 h at 80 °C under light-protected nitrogen purging.

[0045] The long-chain carboxylic acid, such as Figure 2 As shown in B, it is any one of octanoic acid (OA), 2-butyloctanoic acid, and 4-ethyloctanoic acid.

[0046] S4: After the reaction in S3 is completed, dilute the reaction solution with ultrapure water and dialyze it for 24 hours using a dialysis bag. Dialyze the solution to remove the organic solvent from the solution by removing the molecular weight cutoff of 1000 Da through the dialysis bag.

[0047] S5: Take out the dialysate from S4 and freeze-dry it to obtain Y-type chiral nanomicelles.

[0048] This application also provides a Y-type chiral nanomicelle, which is prepared by the above preparation method. The Y-type chiral nanomicelle is one of γ-cyclodextrin-octyl ester (γ-CD-OA), γ-cyclodextrin-2-butyloctyl ester (γ-CD-BOA), or γ-cyclodextrin-4-ethyloctyl ester (γ-CD-EOA).

[0049] Furthermore, based on the aforementioned Y-type chiral nanomicelles, this application provides the application of Y-type chiral nanomicelles in the preparation of drug-loaded nanomicelles.

[0050] Furthermore, this application also provides a drug-loaded nanomicelle, which includes the aforementioned Y-type chiral nanomicelles. The drug loaded in the drug-loaded nanomicelle is a racemic drug, and the racemic drug is one of SOT, SYN, ANI, ORC, ATR, SAL, HOM, PRO, and OCT. Nine model drugs are also provided. Figure 3 As shown.

[0051] The method for preparing the drug-loaded nanomicelles is as follows:

[0052] Weigh Y-type chiral nanomicelles and place them in a round-bottom flask, and add distilled water. The ratio of Y-type chiral nanomicelles to distilled water is 100 mg / 50 mL. Stir magnetically at room temperature until completely dissolved to form a transparent or translucent blank micelle solution.

[0053] Weigh out promethazine hydrochloride and dissolve it in water at a ratio of 25 mg / 10 mL. Add the drug solution dropwise to a blank micelle solution under magnetic stirring. The mass ratio of promethazine hydrochloride to Y-type chiral micelles is 1:4.

[0054] After the addition was complete, the mixture was stirred at room temperature for 12 hours, followed by intermittent sonication at 250W using a probe sonicator (2 seconds on, 3 seconds off). This process lasted for 30 minutes to promote drug encapsulation. After sonication, the solution was centrifuged (12000 rpm, 20 min) and the supernatant was collected. The supernatant was filtered through a 0.8 μm microporous membrane and transferred to a dialysis bag for dialysis (molecular weight cutoff 1000 Da, 24 h). Finally, the dialysate was freeze-dried to obtain drug-loaded micelle lyophilized powder.

[0055] The above content will be explained in conjunction with specific verification experiments:

[0056] 1. Experimental reagents and their sources:

[0057] γ-Cyclodextrin (98%), 2-Butyloctanoic acid (BOA), synephrine hydrochloride (SYN), racemic scopolamine (ANI), salbutamol sulfate (SAL), and octamethasone hydrochloride (OCT) were all purchased from Shanghai Bid Pharmaceutical Co., Ltd. (Shanghai, China). Atropine sulfate (ATR), 4-ethyloctanoic acid (EOA), N-(3-dimethylaminopropyl)-N'-ethylcarbamate hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 4-dimethylaminopyridine (DMAP) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Octanoic acid (OA), sotalol hydrochloride (SOT), and promethazine hydrochloride (PRO) were purchased from Maclean Biochemical Co., Ltd. (Shanghai, China). Homatropine hydrobromide (HOM) was purchased from Leyan Biotechnology Co., Ltd. (Shanghai, China). Osinaline sulfate (ORC) was purchased from Taoshu Biotechnology Co., Ltd. (Shanghai, China). Sodium borate decahydrate (borax) was purchased from Anaiji Chemical Co., Ltd. (Shanghai, China). Formamide (FA) was purchased from Xinsheng Biochemical Technology Co., Ltd. (Tianjin, China). Sodium hydroxide was purchased from Xilong Chemical Co., Ltd. (Guangdong, China).

[0058] 2. Example 1: Synthesis of nanomicelles:

[0059] A. Synthesis of nanomicelles:

[0060] Weigh 1.297 g of γ-cyclodextrin (γ-CD) (1 mmol, 1 eq) and dissolve it in 20 mL of anhydrous formamide (molecular sieve overnight). Stir at 60 °C until dissolved and cool to room temperature.

[0061] Add catalysts EDC (230 mg; 1.2 mmol; 1.2 eq) and NHS (92 mg; 0.8 mmol; 0.8 eq), and stir magnetically for 2 h under ice bath nitrogen purging conditions (to activate carboxyl groups).

[0062] Accurately weigh 144 mg of octanoic acid (OA) (or 200 mg of 2-butyloctanoic acid, or 172 mg of 4-ethyloctanoic acid, 1 mmol, 1 eq) and dissolve it in 5 mL of anhydrous formamide. Add DMAP (146 mg; 1.2 mmol; 1.2 eq) and add it dropwise to the γ-cyclodextrin solution. Stir magnetically for 24 h at 80 °C under light-protected and nitrogen-filled conditions.

[0063] After the reaction was complete, the reaction solution was diluted with 50 mL of ultrapure water and then dialyzed for 24 h using a dialysis bag (molecular weight cutoff 1000 Da) to remove the organic solvent. The dialysate was then removed and freeze-dried to obtain γ-CD-EOA nanomicelles, wherein the γ-CD-EOA nanomicelles are one of γ-cyclodextrin-octyl ester (γ-CD-OA), γ-cyclodextrin-2-butyloctyl ester (γ-CD-BOA), or γ-cyclodextrin-4-ethyloctyl ester (γ-CD-EOA).

[0064] B. Characterization of nanomicelles:

[0065] This application provides a detailed characterization of γ-CD-EOA nanomicelles. Figure 4 The γ-CD-EOA nanomicelles and their reactants were demonstrated. 1 ¹H NMR spectra. Corresponding proton signals from both starting materials were observed in the products, and their assignments were completed. Compared to the EOA starting material, the chemical shifts of the EOA portion in the products did not change significantly, but some signals were weaker.

[0066] In addition, infrared spectral characterization was performed in this application; please refer to [link / reference]. Figure 5 3414 cm -1 The broad and strong peak belongs to the hydroxyl group, which red-shifts to 3408 cm⁻¹ in the product. -1 At this location, a change in the hydroxyl state is observed. In the EOA molecule, at 1713 cm⁻¹, this change is observed. -1 A distinct stretching vibration peak of the carboxyl group (νC=O) can be observed at [location missing]. In the product, the stretching vibration peak of the ester (νC=O) can be observed, but its intensity is weakened and its position changes to 1726 cm⁻¹. -1 This characteristic peak indicates the successful conduction of the esterification reaction.

[0067] This application also observed the morphology of two types of lyophilized nanomicelle powders, γ-CD-OA and γ-CD-EOA. Figure 6 γ-CD-OA exhibits a smooth, flat, sheet-like structure, while γ-CD-EOA displays a wavy, wrinkled surface and is thicker. When γ-CD-EOA encapsulates the drug (PRO), its overall morphology does not change significantly from that of blank γ-CD-EOA, but the surface wavy texture becomes wider. Figure 7 (A and B) show the particle size analysis of γ-CD-OA and γ-CD-EOA, respectively. Their particle sizes are not significantly different, at 112 nm and 119 nm, respectively. Figure 7(D and E) are transmission electron micrographs of γ-CD-OA and γ-CD-EOA, which clearly show the morphology of the nanomicelles, which are distributed in a spherical shape. According to the scale bar, their particle size is approximately 80 nm. The results obtained by the particle size analyzer are the hydrated particle size, so they are usually larger than the results observed in TEM.

[0068] Furthermore, this application also verifies the surfactant through foaming phenomena; the amphiphilic surfactant structure can oriented at the gas-liquid interface, reducing the surface tension of water. For example... Figure 8 As shown, the γ-CD solution exhibits no foaming effect regardless of agitation. However, γ-CD-OA and γ-CD-EOA solutions show obvious foaming upon slight agitation. The Tyndall effect is another simple and effective testing method. When a sufficient number of tiny particles (with particle sizes close to or slightly larger than the wavelength of light) are present in the medium, the light beam is scattered and dispersed in all directions, forming a bright light path. This phenomenon does not occur when the particle size of the dispersed particles is too large or too small. Figure 9 As shown, no obvious light column was observed when the γ-CD solution was irradiated with a red laser. However, a clear Tyndall effect was observed in the γ-CD-OA and γ-CD-EOA solutions. These observations all demonstrate the successful synthesis of nanomicelles.

[0069] 3. Example 2: Preparation of drug-loaded nanomicelles:

[0070] Accurately weigh 100 mg each of γ-CD-OA (or γ-CD-EOA or γ-CD) nanomicelles and place them in a 100 mL round-bottom flask, then add 50 mL of distilled water. Stir magnetically at room temperature until completely dissolved, forming a transparent or translucent blank micelle solution. Weigh 25 mg of promethazine hydrochloride and dissolve it in 10 mL of water. Add the drug solution dropwise to the blank micelle solution under magnetic stirring. After addition, stir at room temperature for 12 h. Intermittently sonicate the solution using a probe sonicator at 250 W (2 s on, 3 s off). This process lasts for 30 minutes to promote drug encapsulation. After sonication, centrifuge the solution (12000 r / min, 20 min) and collect the supernatant. Filter the supernatant through a 0.8 μm microporous membrane and transfer it to a dialysis bag for dialysis (molecular weight cutoff 1000 Da, 24 h). Finally, freeze-dry the dialysate to obtain the drug-loaded micelle lyophilized powder.

[0071] 4. Example 3: Electrophoresis and Optimization

[0072] This study used a CL1030 CE system (Beijing Huayang Limin Instrument Co., Ltd., Beijing, China) equipped with a sample injector, UV detector, power supply, and data processor for enantiomeric separation experiments. Capillaries with an outer diameter of 365 μm and an inner diameter of 50 μm were purchased from Yongnian Fiber Optic Factory (Hebei, China). The total length and effective length of the new capillaries used for enantiomeric separation were 50 cm and 41 cm, respectively. The capillaries were activated by rinsing with NaOH solution (1 M) and water for 30 minutes each. Before each sample injection, the capillaries were rinsed with NaOH solution (0.1 M), water, and a buffer solution for 3 minutes each. Model drugs were separated at 25 °C and monitored at different wavelengths (200 nm for HOM; 210 nm for ATR, SAL, and ANI; 220 nm for SOT; 230 nm for OCT and SYN; 254 nm for PRO; and 280 nm for ORC). The injection method was siphon injection with a height difference of 10 cm and an injection time of 15 seconds.

[0073] The optimized running buffer in this study consisted of 20 mM borax and 27 mg / mL of γ-CD-OA nanomicelles, γ-CD-EOA nanomicelles, or γ-CD. The pH of the running buffer was adjusted to 9.0. All buffer solutions were freshly prepared. The model drug was dissolved in a mixture of methanol and water (1:1, v / v) to prepare the sample solution (0.5 mg / mL). Both γ-CD-OA and γ-CD-EOA could self-assemble into nanomicelles in the buffer solution.

[0074] To achieve better separation, this application systematically investigated several separation conditions using SOT as a model drug. First, the effect of γ-CD-EOA nanomicelle concentration on chiral separation was investigated. In this study, γ-CD-EOA nanomicelles acted as both a chiral selector and a pseudo-stationary phase, and their concentration was crucial to the construction of the separation system. For example... Figure 10 As shown, higher concentrations of nanomicelles resulted in longer migration times for the SOT enantiomers, indicating stronger interactions between the nanomicelles and their enantiomers. The separation efficiency (Rs) of SOT improved when the concentration of nanomicelles increased from 20 mg / mL to 27 mg / mL. However, when the concentration continued to increase to 30 mg / mL, the Rs of SOT began to decrease. Excessively high concentrations of nanomicelles reduced the differential interactions with the two enantiomers, thus affecting the Rs. After investigation, 27 mg / mL was determined to be the optimal concentration of γ-CD-EOA nanomicelles.

[0075] Next, this application investigated the effect of buffer pH on chiral separation. pH not only affects the dissociation state of silanol groups on the capillary inner wall, thus affecting electroosmotic flow, but also the dissociation state of enantiomers or chiral selective agents, thus affecting their interactions. For example... Figure 11 As shown, pH has a more significant effect on the migration time of SOT enantiomers. It can be observed that within the pH range of 8.5 to 9.5, higher pH values ​​lead to longer migration times. This is because SOT is a basic drug (pKa = 9.8). Within the studied pH range, as the pH value increases, the dissociation of SOT weakens, enhancing its interaction with nanomicelles and thus prolonging the migration time. Within this pH range, the peak shapes of the SOT enantiomers continuously change, reflecting to some extent the changes in their interaction with nanomicelles. The Rs of SOT reaches its maximum at pH 9.0. Therefore, pH 9.0 was chosen for further analysis.

[0076] Finally, the effect of separation voltage on chiral separation was also investigated in this application. Generally, higher voltages lead to shorter migration times and improved column efficiency; however, this also tends to induce Joule heating and result in insufficient interaction between the chiral selector and the enantiomer. Conversely, lower voltages can provide an opportunity for sufficient interaction between the chiral selector and the enantiomer, but excessively long migration times can lead to peak broadening. Figure 12 As shown, the migration time of the SOT enantiomer decreases with increasing separation voltage. The Rs value of the SOT reaches its maximum when the separation voltage reaches 12 kV. Therefore, 12 kV is selected as the optimal separation voltage for this system in this application.

[0077] 5. Example 4: Establishment of the separation system

[0078] This application selected nine racemic drugs, including ANI, as model analytes and constructed three separation systems: a single γ-CD system, a γ-CD-OA system, and a γ-CD-EOA nanomicelle system. Notably, two types of Y-type nanomicelles, γ-CD-EOA and γ-CD-BOA, were prepared in this application. Due to the strong hydrophobicity of the hydrophobic chain in γ-CD-BOA, the γ-CD-BOA molecule exhibits poor water solubility, forming a milky white turbidity in water. Therefore, no further research was conducted on γ-CD-BOA. Both γ-CD-OA and γ-CD-EOA exhibit good water solubility in aqueous solutions and can self-assemble into nanomicelles in buffer solutions. Figure 13Table 1 shows typical chromatograms of nine model drugs in different separation systems. It can be seen that some drugs, such as SYN, ORC, OCT, and SAL, could not be separated or showed only slight separation in the γ-CD system alone, while they were well separated in both nanomicelle systems. ATR and PRO showed poor peak shapes in the γ-CD system alone, resulting in unsatisfactory separation. This situation improved in the γ-CD-EOA nanomicelle system. For SOT, ANI, and HOM, their separation was also improved to varying degrees in both nanomicelle systems.

[0079] Table 1. Chiral separation results of model drugs in different systems

[0080]

[0081] Conditions: Capillary temperature, 25℃; Voltage, 12 kV; BGE, 20 mM borax buffer containing 27 mg / mL γ-CD-OA or γ-CD-EOA nanomicelles or 27 mg / mL γ-CD; Buffer pH 9.0.

[0082] Furthermore, this application investigated the method reproducibility of the γ-CD-EOA nanomicelle system by calculating the relative standard deviations (RSDs) of the migration time and resolution of the SOT enantiomers. The data were obtained through five repeated injections under optimal separation conditions. The results (Table 2) showed that the intraday RSDs for Rs and migration time (first peak) were 3.7% and 4.4%, respectively. The interday RSDs for Rs and migration time (first peak) were 4.1% and 4.8%, respectively. This application also calculated the stability of the γ-CD-EOA nanomicelle system under different pH conditions (8.5–9.5) by allowing the buffer to stand for 6 h, 24 h, 48 h, 72 h, and 96 h. The results showed that the system maintained good stability under the investigated pH conditions. When the pH reached 9.5, the separation stability was slightly affected, but still within an acceptable range. This may be because higher pH values ​​tend to cause hydrolysis of ester compounds, thus affecting the formation of nanomicelles.

[0083] Table 2. Reproducibility and stability results of SOT enantiomers in the γ-CD-EOA nanomicelle system.

[0084]

[0085] Conditions: Capillary temperature, 25℃; Voltage, 12 kV; BGE, 20 mM borax buffer containing 27 mg / mL γ-CD-EOA nanomicelles; Buffer pH was 9.0 in repeatability tests and 8.5-9.5 in stability tests; Standing time: 6 h, 24 h, 48 h, 72 h and 96 h.

[0086] 6. Example 5: Chiral Recognition Mechanism

[0087] For nanomicelles, the stronger the hydrophobicity of the hydrophobic chain, the stronger its encapsulation effect on drugs. However, this effect cannot be achieved by simply increasing the hydrophobicity of the hydrophobic chain. This is because chiral separation generally requires a high concentration of chiral selector, and increasing the hydrophobicity of the hydrophobic chain directly leads to its insolubility in water. Therefore, the structure and properties of the hydrophobic chain need to be cleverly designed. In this study, γ-CD aggregates on the surface of the nanomicelles, playing a chiral recognition role. The micelle core encapsulates the enantiomers, acting as a pseudo-stationary phase. To demonstrate the enantiomers entering the micelle core, several experiments were designed in this application.

[0088] ROESY two-dimensional NMR is a powerful tool for studying interactions by examining the spatial proximity of atoms or groups. For the γ-CD-EOA / PRO system, the chemical shift assignments of relevant components are shown in... Figure 4 and Figure 14 From Figure 15 As can be seen, there are cross-peaks between the aromatic protons of PRO and the hydrophobic cavity protons of γ-CD, indicated by blue circles. Furthermore, the aromatic protons of PRO also interact with the hydrophobic core of the nanomicelles, indicated by pink circles. This result reveals that enantiomers are not only distributed within the hydrophobic cavity of γ-CD but also permeate into the hydrophobic core of the nanomicelles. Figure 7 (C and F) show the particle size distribution and TEM images of the drug-loaded γ-CD-EOA nanomicelles, respectively, with particle sizes of approximately 145 nm and 110 nm. Regardless of the method used, the particle size of the drug-loaded γ-CD-EOA nanomicelles is significantly increased compared to the unloaded γ-CD-EOA nanomicelles. This is precisely because the enantiomers enter the micelle core, leading to the increased particle size.

[0089] Y-shaped nanomicelles have a stronger ability to encapsulate drugs, thereby further promoting chiral separation. To verify this hypothesis, a series of experiments were designed in this application.

[0090] In this application, PRO was used as a model analyte, and the drug loading capacity of γ-CD and two types of nanomicelles, γ-CD-OA and γ-CD-EOA, was quantitatively evaluated by HPLC.

[0091] The HPLC steps are as follows:

[0092] Drug loading experiments were performed using a single HPLC system (Shimadzu, 20AD) with an Agilent C18 column (250 × 4.6 mm, 5 μm). The flow rate was 1.0 mL / min, and the injection volume was 10 μL. Mobile phase A was a 2 mM KH₂PO₄ solution (pH 2.5) containing 0.4% triethylamine and 10% acetonitrile, and mobile phase B was acetonitrile. The ratio of mobile phase A to mobile phase B was 70% to 30%. The detection wavelength was 254 nm, and the column temperature was maintained at 35 °C.

[0093] The drug loading was calculated as follows: Accurately weigh 10 mg of lyophilized γ-CD, γ-CD-OA, or γ-CD-EOA powder loaded with promethazine, add 5 mL of water, and vortex to form a micelle solution. Accurately measure 2 mL of the micelle solution, add 8 mL of methanol, and sonicate under ice bath conditions (250 W, 2 s on, 3 s off) for 30 min. After sonication, centrifuge (12000 rpm, 20 min), and inject 10 μL of the supernatant into the liquid chromatograph for detection, recording the peak area. Separately, accurately weigh 10 mg of promethazine reference standard, place it in a 10 mL volumetric flask, add an appropriate amount of 80% methanol aqueous solution, sonicate to dissolve, and dilute to the mark to prepare the promethazine stock solution. Take an appropriate amount of the promethazine stock solution, dilute it with 80% methanol aqueous solution to 0.025 mg / mL, inject 10 μL into the liquid chromatograph for detection, and record the peak area. Calculate the amount of drug in the micelles using the external standard method. The formula for calculating drug loading is as follows: Drug loading (LD%) = (Total amount of drug in micelles) / Mass of drug-loaded nanomicelles × 100%.

[0094] The results are as follows Figure 16 As shown in Table 3, the drug loading of γ-CD was 1.9%, that of γ-CD-OA nanomicelles was 4.3%, and that of Y-type nanomicelles (γ-CD-EOA) reached 6.1%. This result directly confirms the drug loading capacity of Y-type nanomicelles.

[0095] Table 3. Calculation results of drug loading in different systems

[0096]

[0097] Elemental analysis is also an effective method to solve this problem. Since PRO contains the characteristic element S, the CHNS model was used in this application to perform elemental analysis on the two drug-loaded nanomicelles. The content of S can directly reflect the content of PRO (Table 4).

[0098] Table 4. Elemental analysis results of two types of PRO-loaded nanomicelles

[0099]

[0100] The results showed that the sulfur content of drug-loaded γ-CD-OA was approximately 0.50%, while the sulfur content of drug-loaded γ-CD-EOA was approximately 0.67%. This directly demonstrates the stronger drug loading capacity of Y-type nanomicelles.

[0101] Finally, thermogravimetric analysis was performed on the two types of drug-loaded nanomicelles in this application, and the results are as follows: Figure 17 As shown, the thermogravimetric curves changed significantly after PRO was loaded onto γ-CD-EOA. The blank γ-CD-EOA began to lose a significant amount of mass at approximately 290℃, while the two PRO-loaded nanomicelles began to lose a significant amount of mass at approximately 260℃. This may be because PRO has poorer stability compared to other components. Within the 300-800℃ range, the PRO-loaded γ-CD-EOA had less remaining mass than the PRO-loaded γ-CD-OA, suggesting that γ-CD-EOA loaded with more PRO.

[0102] In summary, this application synthesizes an improved novel Y-type nanomicelles via esterification, which utilize γ-CD as the hydrophilic group and branched long-chain carboxylic acids as the hydrophobic group. These ester compounds can self-assemble into nanomicelles in aqueous solution, thus simultaneously serving as chiral selectors and pseudo-stationary phases in CE chiral separation. The Y-type nanomicelles were systematically characterized using infrared spectroscopy, nuclear magnetic resonance, particle size analysis, scanning electron microscopy, transmission electron microscopy, and methods for analyzing bubbling and Tyndall effects. Due to the presence of branched chains, the hydrophobic chains of the Y-type nanomicelles exhibit stronger hydrophobicity, and the interactions between their hydrophobic cores are also enhanced. Therefore, the Y-type nanomicelles are more stable, less prone to depolymerization, and have a stronger encapsulation effect on drugs, thus better fulfilling their role as pseudo-stationary phases. The results show that the separation of nine model drugs in the Y-type nanomicelle system is significantly improved compared to the γ-CD system alone and the ordinary nanomicelle system. This application uses promethazine as a model drug and investigates the drug loading of γ-CD, ordinary nanomicelles, and Y-type nanomicelles using HPLC. Furthermore, this application studies the interaction between Y-type nanomicelles and enantiomers using particle size analysis, transmission electron microscopy, elemental analysis, nuclear magnetic resonance, and thermogravimetric analysis. These results demonstrate that enantiomers are more abundantly distributed in Y-type nanomicelles. This research demonstrates the superiority of Y-type nanomicelles in chiral separation of CE (cell echogenicity), representing a significant step forward in the development of novel chiral recognition materials.

Claims

1. A method for preparing Y-type chiral nanomicelles, characterized in that: Includes the following steps: S1: Dissolve γ-cyclodextrin in anhydrous formamide (molecular sieve overnight), then stir at 60 ℃ to dissolve, and then cool to room temperature; S2: Add catalysts EDC and NHS to the S1 solution. After the addition is complete, magnetically stir for 2 hours under ice bath nitrogen purging to obtain an activated γ-cyclodextrin solution. S3: Weigh out long-chain carboxylic acids and dissolve them in anhydrous formamide, add DMAP, and then add the solution dropwise to the activated γ-cyclodextrin solution. Stir magnetically for 24 h at 80 °C under light-proof and nitrogen-filled conditions. S4: After the reaction in S3 is completed, dilute the reaction solution with ultrapure water and then dialyze using a dialysis bag for 24 hours. S5: Take out the dialysate from S4 and freeze-dry it to obtain Y-type chiral nanomicelles.

2. The method for preparing Y-type chiral nanomicelles according to claim 1, characterized in that: The ratio of γ-cyclodextrin to anhydrous formamide in S1 is 1.297 g: 20 mL.

3. The method for preparing Y-type chiral nanomicelles according to claim 2, characterized in that: The molar ratio of γ-cyclodextrin in S1 to EDC and NHS in S2 is 1:1.2:0.

8.

4. The method for preparing Y-type chiral nanomicelles according to claim 3, characterized in that: The long-chain carboxylic acid in S3 is any one of octanoic acid (OA), 2-butyloctanoic acid, and 4-ethyloctanoic acid.

5. The method for preparing Y-type chiral nanomicelles according to claim 4, characterized in that: The molar ratio of γ-cyclodextrin in S1 to long-chain carboxylic acid and DMAP in S3 is 1:1:1.

2.

6. A Y-type chiral nanomicelle, characterized in that: It is prepared using the preparation method described in any one of claims 1-5.

7. The application of the Y-type chiral nanomicelles as described in claim 6 in the preparation of drug-loaded nanomicelles.

8. A drug-loaded nanomicelle, characterized in that: Use the Y-type chiral nanomicelles according to any one of claims 1-5.

9. The drug-loaded nanomicelles according to claim 8, characterized in that: The drug loaded in the drug-loaded nanomicelles is a racemic drug.

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