A folic acid modified astragaloside iv-hanfangchin a nano microemulsion, a preparation method and application thereof

Abstract

The application belongs to the technical field of nanomedicine, and discloses a folate modified astragaloside-tetrandrine nanomicelle as well as a preparation method and application thereof. The preparation method couples folate and PEG by adopting a DCC and NHS chemical condensation method to prepare a targeting modification material FA-PEG. Then, the FA-PEG is partially replaced with PEG as a co-emulsifier to construct an AS-IV and TET co-loaded nanomicelle system together with Labrafil M1944 CS and EL-35. The obtained FA-AT-MEs can significantly improve the solubility and bioavailability of the two poorly soluble drugs, realize double-targeted delivery through folate ligand-mediated active targeting and EPR effect passive targeting, and synergistically exert anti-tumor activity. The preparation method is simple, convenient to operate, low in cost and easy to scale up.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to a folic acid-modified astragaloside A-tetracycline nanoemulsion, its preparation method, and its application. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, characterized by its insidious onset, high malignancy, high rate of metastasis and recurrence, and low patient survival rate. Currently, treatment methods for liver cancer mainly include surgical resection, radiotherapy, chemotherapy, and targeted therapy. However, most treatments suffer from significant side effects, poor patient tolerance, and high recurrence rates. Therefore, developing efficient, low-toxicity, and targeted drug delivery systems for liver cancer has become an important research direction in the fields of natural medicine and tumor therapy.

[0003] Astragaloside IV (AS-IV) is a triterpenoid saponin extracted from the dried root of Astragalus membranaceus, a legume. It possesses various pharmacological activities, including anti-inflammatory, antioxidant, immunomodulatory, and antitumor effects. Modern research has confirmed that AS-IV can inhibit tumor cell proliferation, migration, and invasion by regulating signaling pathways such as PI3K / Akt and NF-κB, and induce tumor cell apoptosis, demonstrating good inhibitory effects on various solid tumors, including liver cancer and lung cancer. However, AS-IV has extremely poor water solubility, low oral bioavailability, and insufficient stability in physiological environments, severely limiting its clinical translation and widespread application.

[0004] Tetrandrine (TET) is a dibenzylisoquinoline alkaloid extracted from the dried roots of *Stephania tetrandra* S. Moore, a plant in the Menispermaceae family. It possesses pharmacological activities including anti-inflammatory, anti-fibrotic, antitumor, and multidrug resistance reversal. TET exerts its antitumor effects through mechanisms such as blocking calcium ion channels, downregulating P-glycoprotein expression, and regulating the cell cycle, and can significantly enhance the efficacy of chemotherapeutic drugs. It also exhibits significant inhibitory effects on the proliferation and apoptosis-inducing effects on liver cancer cells. However, TET also suffers from drawbacks such as poor water solubility, low oral bioavailability, and numerous adverse reactions from intravenous administration, limiting its widespread clinical application.

[0005] Currently, for the delivery of poorly soluble natural drugs, nanodelivery systems such as liposomes, polymer nanoparticles, nanoemulsions, and microemulsions are commonly used to improve drug solubility, stability, and in vivo absorption efficiency. Among these, nanoemulsions have advantages such as small particle size, good dispersibility, strong drug loading capacity, and ease of preparation, and can effectively improve the solubility and bioavailability of hydrophobic drugs, thus attracting widespread attention in the field of natural drug delivery. Existing technologies often employ single-drug loading to achieve antitumor effects, but single-drug therapy often suffers from problems such as a single mechanism of action, limited therapeutic effect, and easy development of drug resistance. Furthermore, ordinary nanoemulsions lack the ability to actively recognize tumor cells, resulting in high non-specific distribution of drugs in normal tissues, making it difficult to achieve efficient drug accumulation at tumor sites. Summary of the Invention

[0006] This invention provides a folic acid-modified astragaloside A-tetracycline nanoemulsion and its preparation method. The invention employs a DCC and NHS chemical condensation method to couple folic acid with PEG to prepare the targeted modification material FA-PEG. Subsequently, FA-PEG is used to partially replace PEG as a co-emulsifier, and together with Labrafil M1944 CS and EL-35, an AS-IV and TET co-supported nanoemulsion system is constructed. The resulting FA-AT-MEs can significantly improve the solubility and bioavailability of the two poorly soluble drugs.

[0007] This invention provides the application of the FA-AT-MEs in the preparation of antitumor drugs, which can actively target tumor cells overexpressing folate receptors, such as HepG2 liver cancer cells, and can significantly inhibit the proliferation of tumors.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a method for preparing folic acid-modified astragaloside A-tetracycline nanoemulsion, the preparation method comprising the following steps:

[0010] 1) FA, dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS) and polyethylene glycol (PEG) were dispersed in DMSO, followed by a coupling reaction. After the reaction was completed, the mixture was filtered and dialyzed, and dried to obtain folic acid-polyethylene glycol (FA-PEG).

[0011] 2) Place FA-PEG, AS-IV, TET, oil phase carrier, emulsifier and PEG in a container, mix and self-assemble to obtain the folic acid modified astragaloside A-tetracycline nanoemulsion (FA-AT-MEs).

[0012] In steps 1) and 2), the molecular weight of the PEG is 200 to 2000.

[0013] In step 1), the mass ratio of FA, DCC, NHS and PEG is 1:0.2~2:0.3~3:0.2~2.

[0014] In step 1), the coupling reaction is carried out at room temperature, under light-protected conditions, and for 24-48 hours.

[0015] In step 1), the molecular cutoff of the dialysis bag used for dialysis is 500~1000 Da, and the dialysis time is 36~72 h.

[0016] In step 2), the oil phase carrier is of type Labrafil M1944 CS, and the emulsifier is EL-35.

[0017] In step 2), the mass ratio of FA-PEG, AS-IV, TET, oil phase carrier, emulsifier and PEG is 1:0.1~2:0.1~2:23~35:23~38:8~16.

[0018] In step 2), the self-assembly temperature is 35~45℃ and the time is 4~12h.

[0019] This invention provides a folic acid-modified astragaloside A-tetracycline nanoemulsion prepared by the above preparation method.

[0020] The present invention also provides the application of the above-mentioned folic acid-modified astragaloside A-tetracycline nanoemulsion in the preparation of antitumor drugs.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1. This invention uses DCC and NHS chemical condensation to couple folic acid with PEG to prepare FA-PEG targeted modification material, which can stably connect folic acid to the surface of nanoemulsion, improve the folic acid receptor recognition ability, and thus enhance the active targeting effect of nanoemulsion on liver cancer cells.

[0023] 2. In this invention, FA-PEG is used to partially replace PEG 400 as a co-emulsifier. Together with Labrafil M1944 CS and EL-35, a nano-microemulsion system is constructed, which can significantly improve the solubility and dispersion stability of two poorly soluble drugs, AS-IV and TET, and reduce the risk of drug precipitation.

[0024] 3. This invention uses a nano-microemulsion carrier to co-load AS-IV and TET, which can improve the in vivo stability and bioavailability of the two natural active ingredients, prolong the drug circulation time, and thus improve the effective utilization rate of the drug.

[0025] 4. The preparation method of the present invention only requires constant temperature stirring to complete the construction of nanoemulsions, without the need for complex equipment or high energy consumption processes, and has the characteristics of simple process, convenient operation and good repeatability. Attached Figure Description

[0026] Figure 1 The infrared spectrum characterization of FA-PEG 400 prepared in step 1) of Example 1;

[0027] Figure 2 The particle size distribution and zeta potential diagram of the FA-AT-MEs prepared in Example 1 are shown.

[0028] Figure 3 Transmission electron microscope image of the FA-AT-MEs prepared in Example 1;

[0029] Figure 4 The graph shows the cumulative in vitro release curves of AS-IV and TET in the FA-AT-MEs prepared in Example 1 from 0 to 48 h under the conditions of 37°C, pH 7.4 and PBS.

[0030] Figure 5 The graph shows the encapsulation efficiency and drug loading of AS-IV and TET in the FA-AT-MEs prepared in Example 1. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0033] The polyethylene glycol used in the following examples and comparative examples has a molecular weight of 400 and is labeled as PEG400.

[0034] Example 1

[0035] A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, specifically including the following steps:

[0036] 1) Weigh 4.965 g of FA and place it in a 500 mL reaction flask. Add 45 mL of DMSO and stir at room temperature for 2 h in the dark until FA is fully dissolved. Then add 4.575 g of DCC, 2.796 g of NHS and 4.5 g of PEG400 and stir at room temperature for 24 h in the dark to carry out the coupling reaction. After the reaction is completed, mix the reaction solution with deionized water at a volume ratio of 1:5 and filter under reduced pressure in a Buchner funnel to remove insoluble impurities. Transfer the filtrate to a dialysis bag with a molecular weight cutoff of 500 Da and dialyze for 48 h in the dark using flowing deionized water as the dialysis medium. Collect the dialysate and freeze-dry it under vacuum in the dark to obtain a light yellow flocculent powder, namely FA-PEG400, which is stored at -20℃ in the dark for later use.

[0037] 2) Weigh 12.8 mg of FA-PEG400, 5 mg of AS-IV, 5 mg of TET, 400 mg of Labrafil M1944 CS400, 450 mg of EL-35 and 150 mg of PEG400 into a vial. Stir the mixture for 8 hours in a 42°C water bath using a thermostatic magnetic stirrer to ensure complete drug dissolution. Under constant temperature and magnetic stirring at 42°C, slowly add deionized water dropwise to a final volume of 10 mL using a syringe. At this point, the system is clear and transparent, and the FA-AT-MEs are obtained.

[0038] Example 2

[0039] A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, specifically including the following steps:

[0040] The difference between this embodiment and Embodiment 1 is that:

[0041] Replace “FA-PEG400 12.8 mg, AS-IV 5 mg, TET 5 mg, Labrafil M1944 CS400 mg, EL-35 450 mg and PEG400 150 mg” in step 2) with “FA-PEG400 15.0 mg, AS-IV 5 mg, TET 5 mg, Labrafil M1944 CS 350 mg, EL-35 500 mg and PEG400 180 mg”.

[0042] Example 3

[0043] A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, specifically including the following steps:

[0044] The difference between this embodiment and Embodiment 1 is that:

[0045] Replace “DCC 4.575 g, NHS 2.796 g and PEG400 4.5 g, stir at room temperature in the dark for 24 h to carry out the coupling reaction” in step 1) with “DCC 9 g, NHS 5 g and PEG400 9 g, stir at room temperature in the dark for 48 h to carry out the coupling reaction”.

[0046] Example 4

[0047] A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, specifically including the following steps:

[0048] The difference between this embodiment and Embodiment 1 is that:

[0049] Replace “stirring in a 42°C water bath for 8 h” and “slowly add deionized water dropwise to 10 mL with a syringe under constant temperature magnetic stirring at 42°C” in step 2) with “stirring in a 45°C water bath for 10 h” and “slowly add deionized water dropwise to 10 mL with a syringe under constant temperature magnetic stirring at 45°C”. At the same time, adjust the amount of Labrafil M1944 CS in the oil phase to 380 mg and the amount of emulsifier EL-35 to 480 mg.

[0050] Example 5

[0051] A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, specifically including the following steps:

[0052] The difference between this embodiment and Embodiment 1 is that:

[0053] In step 1), replace “PEG400 4.5 g” with “PEG400 6.0 g”, and change the coupling reaction conditions to “stirring at room temperature in the dark for 36 h”; in step 2), replace “FA-PEG400 12.8 mg, AS-IV 5 mg, TET 5 mg, Labrafil M1944CS 400 mg, EL-35 450 mg and PEG400 150 mg” with “FA-PEG400 18.0 mg, AS-IV 6 mg, TET 4 mg, Labrafil M1944 CS 420 mg, EL-35 420 mg and PEG400 160 mg”, and adjust the stirring temperature and time to “stirring in a 40℃ water bath for 12 h”, and control the system temperature at 40℃ when adding deionized water dropwise.

[0054] Comparative Example 1

[0055] An unmodified folic acid-astragaloside A-tetracycline nanoemulsion specifically includes the following steps:

[0056] The difference between Comparative Example 1 and Example 1 is that step 1) is omitted, and FA-PEG400 in step 2) is replaced with an equal mass of PEG400.

[0057] Comparative Example 2

[0058] A folic acid-modified astragaloside A nanoemulsion that does not contain tetrandrine (TET) specifically includes the following steps:

[0059] The difference between Comparative Example 2 and Example 1 is that "TET 5 mg" was not added in step 2), while the other components and preparation conditions remained unchanged, resulting in FA-AS-MEs without TET.

[0060] Comparative Example 3

[0061] A folic acid-modified tetrandrine nanoemulsion without astragaloside IV (AS-IV) was developed, and the synergistic effect of AS-IV was verified. The specific steps included:

[0062] The difference between Comparative Example 3 and Example 1 is that "AS-IV 5 mg" was not added in step 2), while the other components and preparation conditions remained unchanged, resulting in FA-TET-MEs without AS-IV.

[0063] Comparative Example 4

[0064] A physical mixture of astragaloside A and tetrandrine, used to demonstrate that nanoemulsions improve solubility and bioavailability, specifically includes the following steps:

[0065] Weigh 5 mg of AS-IV and 5 mg of TET, add 5 mL of purified water, mix for 30 min, sonicate for 30 min, and bring the volume to 10 mL to obtain a physical mixture of AS-IV and TET.

[0066] Test Example 1

[0067] FTIR structural characterization of FA-PEG 400

[0068] The FT-IR spectrum comparison between FA and FA-PEG 400 from Example 1 showed that FA-PEG 400 exhibited wavelengths from 3000 to 3750 cm⁻¹ corresponding to -OH. -1 and CO 1000~1250 cm -1 The change in characteristic absorption peaks indicates that PEG 400 was successfully introduced into the FA molecule; at the same time, the formation of new ester bond-related characteristic peaks further confirms that an esterification coupling reaction occurred between FA and PEG 400, indicating that the FA-PEG 400 conjugate has been successfully synthesized.

[0069] Test Example 2

[0070] Particle size, Zeta potential

[0071] Take 20 μL of the nanoemulsions prepared in Example 1 and Comparative Example 1, add deionized water to 2 mL, and measure the particle size, PDI, and Zeta potential using dynamic light scattering (DLS) at 25 °C. The results of Example 1 are as follows. Figure 2 As shown, the particle size was measured to be 105.84 ± 0.19 nm, the dispersion index (PDI) was 0.21 ± 0.01, and the zeta potential was -5.00 ± 0.44 mV.

[0072] Morphological characteristics

[0073] One mL of the folic acid-modified astragaloside A-tetracycline nanoemulsion prepared in Example 1 was dropped onto a copper grid with a carbon support film, negatively stained with phosphotungstic acid, and then allowed to air dry. The morphology of the microemulsion was observed under a transmission electron microscope, and the results are as follows: Figure 3 As shown in the TEM image, FA-AT-MEs exhibit a near-spherical structure, consistent with the dynamic light scattering results. The smaller nanoparticle size helps to improve the penetration and accumulation of FA-AT-MEs in tumor tissues. Combined with the specific recognition of folic acid receptors by folic acid ligands, active targeted enrichment of FA-AT-MEs at tumor sites can be further achieved.

[0074] Test Example 3

[0075] In vitro release test

[0076] Weigh 1 mL of the FA-AT-MEs prepared in Example 1 and place it in a dialysis bag with a molecular weight cutoff of 3500 Da. The bag is then placed in 200 mL of PBS solution containing 0.5% Tween 80 at pH 7.4 (37°C). At time points of 0, 15, and 30 min, and 1, 2, 4, 8, 12, 24, and 48 h, 0.2 mL of the release solution is collected and 0.2 mL of fresh medium is added. The concentrations of AS-IV and TET in the release solution are determined by HPLC. An in vitro cumulative release curve from 0 to 48 h is plotted based on the cumulative release at each time point. The results are as follows: Figure 4 As shown in the figure, AS-IV and TET in the FA-AT-MEs prepared in Example 1 exhibited a sustained and slow release characteristic within 0-48 h, indicating that the nanoemulsion has a good encapsulation and sustained-release effect on both drugs, which helps to prolong the in vivo action time of the drugs and reduce the frequency of administration.

[0077] Test Example 4

[0078] Encapsulation efficiency and drug loading determination

[0079] The encapsulation efficiency and drug loading of AS-IV and TET in each example and comparative example were determined by ultrafiltration centrifugation. 1 mL of the nanoemulsion from each example and comparative example was placed in an ultrafiltration tube with a molecular weight cutoff of 10 kDa and centrifuged at 4℃ and 5000 r / min for 30 min. The ultrafiltrate was collected, and 1 mL of the nanoemulsion from each example and comparative example was added to 5 mL of methanol for demulsification. After ultrasonic extraction and centrifugation, the supernatant was collected. The concentrations of AS-IV and TET in the ultrafiltrate and supernatant were determined by HPLC. The encapsulation efficiency (EE%) and drug loading (DL%) were calculated using the following formula:

[0080] EE% = (Total drug content - Free drug content) / Total drug content × 100%;

[0081] DL% = (Total drug content - Free drug content) / Total mass of FA-AT-MEs × 100%.

[0082] The test results of the FA-AT-MEs prepared in Example 1 are as follows: Figure 5 As shown, the encapsulation efficiencies of AS-IV and TET were 99.30 ± 0.35% and 99.30 ± 0.20%, respectively, and the drug loadings were 0.48 ± 0.01% and 0.47 ± 0.02%, respectively.

[0083] The data for each embodiment and comparative example are shown in Table 1 below:

[0084] Table 1 Performance Data Sheet

[0085]

[0086] The above results indicate that, compared with Example 1, replacing FA-PEG 400 with an equal mass of PEG 400 in Comparative Example 1 resulted in changes in the system particle size, Zeta potential, and encapsulation efficiency of both drugs. Specifically, the encapsulation efficiencies of AS-IV and TET decreased from 99.30% and 99.30% to 91.45% and 90.87%, respectively, demonstrating that folic acid modification not only provides active targeting but also helps improve the nanoemulsion interface structure and enhance drug loading stability. In Comparative Examples 2 and 3, removing TET or AS-IV respectively only revealed the encapsulation efficiency and drug loading of the corresponding single drug, indicating that dual-drug co-loading is a necessary prerequisite for achieving simultaneous delivery and synergistic effects of two drugs. Comparative Example 4, a physical mixture of drugs, showed an increased particle size of 215.6 nm and an increase in PDI to 0.48, indicating that the system without nanoemulsion construction exhibited poor dispersibility and uniformity, making it difficult to meet the requirements for efficient and stable delivery.

[0087] The folic acid-modified microemulsion system constructed in this invention can achieve efficient encapsulation of AS-IV and TET, indicating that the drug delivery system has superior drug encapsulation performance and formulation stability, which is beneficial to improving drug delivery efficiency and bioavailability.

[0088] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0089] The above description of the embodiments is intended to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a folic acid-modified astragaloside A-tetracycline nanoemulsion, characterized in that, The preparation method includes the following steps: 1) FA, DCC, NHS and PEG were dispersed in DMSO, followed by a coupling reaction. After the reaction was completed, the mixture was filtered and dialyzed, and then dried to obtain FA-PEG. 2) Place FA-PEG, AS-IV, TET, oil phase carrier, emulsifier and PEG in a container, mix and self-assemble to obtain the folic acid modified astragaloside A-tetracycline nanoemulsion.

2. The preparation method according to claim 1, characterized in that, In steps 1) and 2), the molecular weight of the PEG is 200 to 2000.

3. The preparation method according to claim 1 or 2, characterized in that, In step 1), the mass ratio of FA, DCC, NHS and PEG is 1:0.2~2:0.3~3:0.2~2.

4. The preparation method according to claim 1, characterized in that, In step 1), the coupling reaction is carried out at room temperature, under light-protected conditions, and for 24-48 hours.

5. The preparation method according to claim 1, characterized in that, In step 1), the molecular cutoff of the dialysis bag used for dialysis is 500~1000 Da, and the dialysis time is 36~72 h.

6. The preparation method according to claim 1, characterized in that, In step 2), the oil phase carrier is of type Labrafil M1944 CS, and the emulsifier is EL-35.

7. The preparation method according to claim 1, characterized in that, In step 2), the mass ratio of FA-PEG, AS-IV, TET, oil phase carrier, emulsifier and PEG is 1:0.1~2:0.1~2:23~35:23~38:8~16.

8. The preparation method according to claim 1, characterized in that, In step 2), the self-assembly temperature is 35~45℃ and the time is 4~12h.

9. A folic acid-modified astragaloside A-tetracycline nanoemulsion prepared by the preparation method according to any one of claims 1-8.

10. The application of the folic acid-modified astragaloside A-tetracycline nanoemulsion as described in claim 9 in the preparation of antitumor drugs.

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