Dual-targeting carrier material, preparation method thereof and preparation method of oleanolic acid nanoparticles wrapped by dual-targeting carrier material

Abstract

The invention provides a dual-targeting carrier material, a preparation method thereof and a preparation method of oleanolic acid nanoparticles wrapped by the dual-targeting carrier material, and belongs to the technical field of medicines. The oleanolic acid targeting nanoparticles are prepared by taking a dual-targeting high-molecular copolymer as a carrier material and adopting an emulsification-solvent evaporation method, so that the oleanolic acid can be protected from being quickly degraded, and the in-vivo circulation time of the oleanolic acid can be prolonged. Meanwhile, targeted delivery of oleanolic acid is realized through a targeted group, the concentration of the drug in tumor tissues is improved, and toxic and side effects on normal tissues are reduced. An MTT method is adopted, the inhibition effect of the oleanolic acid nanoparticles on cell growth is researched, and the result shows that the oleanolic acid targeting nanoparticles have a stronger effect of inhibiting tumor cell proliferation compared with an oleanolic acid raw material medicine.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a dual-targeted carrier material, a preparation method of the dual-targeted carrier material, and a preparation method of oleanolic acid nanoparticles encapsulated by the dual-targeted carrier material. In particular, it relates to a dual-targeted polymer carrier material FA-TPGS-PLLA, a preparation method of the dual-targeted polymer carrier material FA-TPGS-PLLA, and a preparation method of oleanolic acid nanoparticles encapsulated by the dual-targeted polymer carrier material FA-TPGS-PLLA. Background Art

[0002] Oleanolic acid (OA) is a pentacyclic triterpenoid compound derived from natural plants, with various pharmacological activities such as anti-inflammatory and anti-tumor effects. It can inhibit the proliferation of tumor cells and induce their apoptosis through multiple mechanisms. However, the low solubility and poor bioavailability of oleanolic acid greatly limit its application in medicinal value. By using synthetic or natural polymer materials to prepare nanoparticles, oleanolic acid can be encapsulated inside the polymer nanoparticles, protecting oleanolic acid from being rapidly degraded and prolonging its circulation time in vivo. At the same time, the polymer can be modified, such as connecting targeting groups, to achieve targeted delivery of oleanolic acid, increase the concentration of the drug in tumor tissues, and reduce the toxic and side effects on normal tissues. Summary of the Invention

[0003] The first object of the present invention is to provide a dual-targeted polymer carrier material FA-TPGS-PLLA.

[0004] The object of the present invention can be achieved by the following technical solutions: A dual-targeted polymer carrier material FA-TPGS-PLLA, whose structure is as follows:

[0005]

[0006] The second object of the present invention is to provide a preparation method of the above dual-targeted polymer carrier material FA-TPGS-PLLA, including the following steps:

[0007] (1) Recrystallization of lactide:

[0008] Add L-lactide (L-LA) to an ethyl acetate solution, heat and stir in a constant temperature water bath at 60 °C until completely dissolved, stop heating, gradually cool down until all the precipitate precipitates out, filter to obtain lactide crystals. Repeat the above steps three times, and vacuum dry the product at 40 °C for 24 h.

[0009] (2) Synthesis of TPGS-PLLA

[0010] The TPGS-PLLA polymer was prepared by the method of ring-opening polymerization. Weigh TPGS, Sn(Oct)2 and recrystallized L-LA into a flask. Under the environmental conditions of nitrogen protection, heat the polymerization reaction in an oil bath, cool it to room temperature, dissolve it with chloroform, transfer it to a beaker, precipitate it in cold anhydrous ether, and dry the precipitate in vacuum until it reaches a constant weight.

[0011] (3) Activate folic acid:

[0012] Weigh the folic acid into a beaker containing DMSO. After dissolution, add EDCI and DMAP in sequence, and stir in the dark at room temperature to obtain a dark yellow solution, which is the folic acid activated ester solution.

[0013] (4) Synthesis of the polymer carrier material FA-TPGS-PLLA:

[0014] Weigh TPGS-PLLA and 20 μL of pyridine and add them to the folic acid activated ester. Stir in the dark at room temperature for 24 h to obtain a yellow solution. Then put it into a dialysis bag with ultrapure water as the dialysis medium for dialysis, change the dialysis solution every 4 h, and then pour the dialyzed solution into a petri dish and freeze-dry to obtain a yellow fluffy powder FA-TPGS-PLLA.

[0015] Furthermore, in the step (1), the molar ratio of L-lactide to ethyl acetate is 1:2.

[0016] Furthermore, in the step (1), when the solution is cooled and crystallized at room temperature, the beaker should not be moved.

[0017] Furthermore, in the step (2), the molar ratio of TPGS, Sn(Oct)2 and L-LA is 100:1:1.

[0018] Furthermore, in the step (2), the temperature of the oil bath heating reaction is 120 °C, and the reaction time is 1 - 10 hours.

[0019] Furthermore, in the step (3), the molar ratio of folic acid, EDCI and DMAP is 1:1:1.

[0020] Furthermore, in the step (3), the reaction time of stirring in the dark at room temperature is 1 - 48 h.

[0021] Furthermore, in the step (4), the dialysis bag M W is 10000.

[0022] The third object of the present invention is to provide a preparation method of oleanolic acid nanoparticles encapsulated by the dual-targeting polymer carrier material FA-TPGS-PLLA, including the following steps:

[0023] 1) Dissolve the carrier material FA-TPGS-PLLA described in claim 1 in dichloromethane, add an oil-phase emulsifier to form phase A;

[0024] 2) Take oleanolic acid and dissolve it in acetone to form phase B;

[0025] 3) While stirring phase A, slowly dropwise add phase B and continue stirring to form a primary emulsion;

[0026] 4) Slowly dropwise add the primary emulsion into an SDS solution and stir to form a multiple emulsion;

[0027] 5) After stirring and curing to volatilize the organic solvent, then perform ultrasonic stirring to obtain the product.

[0028] Furthermore, the dosages of the raw materials in step (1) are as follows: 0.01 g of FA-TPGS-PLLA carrier material, 1 mL of dichloromethane, and 4 drops of oil-phase emulsifier.

[0029] Furthermore, the oil-phase emulsifier in step (1) is selected from one or a mixture of several of Span-80, Span-20, Span-40, and Span-60, and preferably Span-80 is used.

[0030] Furthermore, in step (2), 0.008 g of oleanolic acid and 1 mL of acetone are used.

[0031] Furthermore, in step (4), 5 mL of 1 wt.% SDS solution is used.

[0032] Furthermore, the curing time in step (5) is 4 hours; the ultrasound is provided by an ultrasonic cell disruptor, with a frequency of 195 W and a time of 15 min.

[0033] The oleanolic acid nanoparticles encapsulated by the dual-targeting polymer carrier material FA-TPGS-PLLA can act as a drug carrier.

[0034] The oleanolic acid nanoparticles encapsulated by the dual-targeting polymer carrier material FA-TPGS-PLLA use polylactic acid (PLLA) as the core matrix material, and by introducing folic acid (FA) and vitamin E polyethylene glycol succinate (TPGS) as dual-targeting groups, a class of polymer nanocarrier materials with precise targeting and positioning functions is designed and synthesized. Many tumor cells highly express folate receptors on their surfaces specifically. The introduction of folic acid groups can significantly enhance the active targeting and recognition ability of the nanoparticles to tumor cells, thereby improving drug delivery efficiency, reducing systemic toxicity, and optimizing the therapeutic effect; as a surfactant with excellent biocompatibility and diverse functions, TPGS can not only promote endocytosis by reducing the interfacial tension, but also effectively inhibit the drug efflux mediated by P-glycoprotein (P-gp), significantly reversing the multidrug resistance of tumor cells.

[0035] Beneficial effects of the present invention:

[0036] (1) The present invention uses polylactic acid as the matrix, selects folic acid and TPGS as the positioning groups, and successfully synthesizes a dual-targeted nanoparticle polymer carrier material through precise process design and fine parameter optimization. The carrier material can efficiently encapsulate drugs and achieve precise targeted therapy. It significantly improves the drug concentration at the targeted site and greatly enhances the therapeutic effect; at the same time, it greatly reduces the distribution of drugs in other parts, significantly reduces side effects, and brings patients a safer and more effective treatment experience.

[0037] (2) The method for preparing the dual-targeting polymer carrier material provided by the present invention has great application potential in the medical field due to the excellent targeting and safety advantages of the synthesized carrier material, and has extremely high reference value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the infrared structure of TPGS-PLLA;

[0039] Figure 2 is the H NMR spectrum of TPGS-PLLA;

[0040] Figure 3 is the infrared structure of FA-TPGS-PLLA;

[0041] Figure 4 is the H NMR spectrum of FA-TPGS-PLLA;

[0042] Figure 5 Figure A in the figure is the surface image of OA@FA-TPGS-PLLA nanoparticle solution; Figure 5 Figure B is the particle size distribution diagram of OA@FA-TPGS-PLLA nanoparticles;

[0043] Figure 6 This is the pharmacodynamic evaluation diagram of OA, OA@PLLA, OA@TPGS-PLLA, and OA@FA-TPGS-PLLA nanoparticles. DETAILED DESCRIPTION

[0044] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0045] Embodiment 1

[0046] 1. Preparation of dual-targeting polymer carrier material FA-TPGS-PLLA

[0047] (1) Recrystallization of lactide:

[0048] Take 5 g of L-lactide and add it to 10 mL of ethyl acetate. Heat and stir it in a constant temperature water bath at 60 °C until it is completely dissolved, then stop heating. Cool the solution to room temperature until crystals appear. During the crystallization process, do not move the beaker. Then filter the ethyl acetate. Repeat the above steps three times and dry it in vacuum at 40 °C.

[0049] (2) Synthesis of TPGS-PLLA

[0050] Prepare the TPGS-PLLA polymer by ring-opening polymerization. Weigh 0.4 g of TPGS, 0.1 g of Sn(Oct)₂ and 3.6 g of recrystallized L-LA into a flask. Under the condition of nitrogen protection, heat it in an oil bath at 145 °C for 12 h for the polymerization reaction. Cool it to room temperature, dissolve it with chloroform, transfer it to a beaker, and precipitate it with anhydrous ether 2 - 3 times. Dry it in vacuum at 40 °C until it reaches a constant weight. The infrared and NMR spectra are as Figure 1 、 2 shown.

[0051] (3) Activation of folic acid:

[0052] Weigh 10 mg of folic acid and put it into a beaker containing 5 mL of DMSO. After dissolution, add 4.3 mg of EDCI and 2.8 mg of DMAP in sequence, and stir it in the dark at room temperature to obtain a dark yellow solution, which is the folic acid activated ester solution.

[0053] (4) Synthesis of the polymer carrier material FA-TPGS-PLLA:

[0054] Weigh 101.9 mg of TPGS-PLLA and 20 μL of pyridine and add them to the folic acid activated ester. Stir it in the dark at room temperature for 24 h to obtain a yellow solution. Then put it into a dialysis bag with dialysis medium (Mr = 10000) being ultrapure water and dialyze it. Change the dialysis fluid every 4 h. Then pour the dialyzed solution into a petri dish and freeze-dry it to obtain a yellow fluffy powder FA-TPGS-PLLA. The infrared and NMR spectra are as Figure 3 、 4 shown.

[0055] II. Preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs:

[0056] Preparation of oleanolic acid FA-TPGS-PLLA-NPs nanoparticles by the emulsion-solvent evaporation method. Weigh 0.01 g of FA-TPGS-PLLA carrier material and dissolve it in 1 mL of dichloromethane, then add 2 to 3 drops of Span 80 as oil phase A. Weigh 0.008 g of oleanolic acid and dissolve it in 1 mL of acetone, heat and evaporate to 0.5 mL as inner oil phase B. Stir oil phase A and slowly add inner oil phase B dropwise into oil phase A, then continue stirring for 3 min to form an O / O type primary emulsion. Slowly add the primary emulsion dropwise into 5 mL of 1.5% SDS solution and stir for 5 min to form an O / O / W type multiple emulsion, and then solidify for 4 h on a magnetic stirrer to volatilize the organic solvent. From Figure 5 It can be seen that the nanoparticles have obvious light yellow opalescence, with an average particle size of 87.96 nm, good dispersion, and a Zeta potential of -65.5 mV.

[0057] III. Preliminary study on the anti-tumor effect of oleanolic acid FA-TPGS-PLLA-NPs

[0058] The MTT method was used to evaluate the efficacy of oleanolic acid nanoparticles. Adjust the cell concentration to about 1×10 4 cells per milliliter, and evenly inoculate the cells to be tested into a 96-well plate at a volume of 100 μL of cell suspension per well, and supplement the edge wells with the same amount of PBS. Incubate in a cell culture incubator for 24 h (37 °C, 5% CO2) to allow the cells to adhere and grow. Add 100 μL of drug-containing medium to each well, and repeat each sample in 6 wells. After 48 h of treatment, add 20 μL of MTT solution to each well. After incubating for 4 h, carefully aspirate the supernatant in each well. Then add 150 μL of DMSO to each well, and place the culture plate on a shaker and shake for about 15 min. Detect the absorbance at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell survival rate. The results are as Figure 6 shown. The inhibitory effect of OA@FA-TPGS-PLLA NPs on cell growth is stronger than that of OA@TPGS-PLLA NPs, OA@PLLA NPs, and the raw material oleanolic acid.

[0059] Example 2

[0060] The preparation methods of FA-TPGS-PLLA and FA-TPGS-PLLA-NPs are the same as those in Example 1, except that:

[0061] In the preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs, the dosage of FA-TPGS-PLLA carrier material is 0.02 g, the dosage of oleanolic acid is 0.005 g, the SDS concentration is 1.5 wt%, the dosage is 7 mL, the ultrasonic stirring time is 7 min, and the power is 220 w.

[0062] Example 3

[0063] The preparation methods of FA-TPGS-PLLA and FA-TPGS-PLLA-NPs are the same as those in Example 1, except that:

[0064] In the preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs, the dosage of FA-TPGS-PLLA carrier material is 0.03 g, the dosage of oleanolic acid is 0.004 g, the SDS concentration is 1.5 wt%, the dosage is 7 mL, the ultrasonic stirring time is 10 min, and the power is 210 w.

[0065] Example 4

[0066] The preparation methods of FA-TPGS-PLLA and FA-TPGS-PLLA-NPs are the same as those in Example 1, except that:

[0067] In the preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs, the dosage of FA-TPGS-PLLA carrier material is 0.02 g, the dosage of oleanolic acid is 0.004 g, the SDS concentration is 2 wt%, the dosage is 6 mL, the ultrasonic stirring time is 10 min, and the power is 205 w.

[0068] Example 5

[0069] The preparation methods of FA-TPGS-PLLA and FA-TPGS-PLLA-NPs are the same as those in Example 1, except that:

[0070] In the preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs, the dosage of FA-TPGS-PLLA carrier material is 0.02 g, the dosage of oleanolic acid is 0.003 g, the SDS concentration is 2 wt%, the dosage is 7 mL, the ultrasonic stirring time is 12 min, and the power is 200 w.

[0071] Example 6

[0072] The preparation methods of FA-TPGS-PLLA and FA-TPGS-PLLA-NPs are the same as those in Example 1, except that:

[0073] In the preparation of oleanolic acid nanoparticles FA-TPGS-PLLA-NPs, the dosage of FA-TPGS-PLLA carrier material is 0.01 g, the dosage of oleanolic acid is 0.003 g, the SDS concentration is 2.5 wt%, the dosage is 5 mL, the ultrasonic stirring time is 15 min, and the power is 195 w.

[0074] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar means for substitution, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A dual-targeting polymeric carrier material FA-TPGS-PLLA, characterized in that, The structure is as follows:

2. A method for preparing the dual-targeting polymer carrier material FA-TPGS-PLLA as claimed in claim 1, characterized in that, It includes the following steps: (1) Lactide recrystallization: Add L-lactide (L-LA) into an ethyl acetate solution, heat and stir it in a constant temperature water bath at 60 °C until it is completely dissolved. Stop heating and gradually cool it down until all the precipitates are separated out. Filter to obtain lactide crystals. Repeat the above steps three times, and dry the product in vacuum at 40 °C for 24 h; (2) Synthesis of TPGS-PLLA Prepare the TPGS-PLLA polymer by ring-opening polymerization. Weigh vitamin E polyethylene glycol succinate (TPGS), stannous octoate (Sn(Oct)2) and recrystallized L-LA into a flask. Under the condition of nitrogen protection, heat and polymerize the reaction in an oil bath, cool it to room temperature, dissolve it with chloroform, precipitate it in cold anhydrous ether, and dry the precipitate in vacuum at 40 °C until it reaches a constant weight; (3) Activation of folic acid: Put the weighed folic acid into a beaker containing dimethyl sulfoxide (DMSO). After dissolution, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) and N,N-dimethylaminopyridine (DMAP) in sequence, and stir it in the dark at room temperature to obtain a dark yellow solution, which is the folic acid activated ester solution; (4) Synthesis of the polymer carrier material FA-TPGS-PLLA: Weigh TPGS-PLLA and 20 μL of pyridine and add them to the folic acid activated ester. Stir it in the dark at room temperature for 24 h to obtain a yellow solution. Then put it into a dialysis bag with ultrapure water as the dialysis medium for dialysis, change the dialysis solution every 4 h, and then pour the dialyzed solution into a petri dish and freeze-dry it to obtain a yellow fluffy powder FA-TPGS-PLLA.

3. The preparation method of the dual-targeting polymer carrier material FA-TPGS-PLLA according to claim 2, characterized in that: In the step (1), the molar ratio of lactide to ethyl acetate is 1:

2.

4. The preparation method of the dual-targeting polymer carrier material FA-TPGS-PLLA according to claim 2, wherein: In the step (2), the molar ratio of TPGS, Sn(Oct)2 and L-LA is 1:1:100; the temperature of the oil bath heating reaction is 120 °C, and the reaction time is 1 - 10 h.

5. The preparation method of the dual-targeting polymer carrier material FA-TPGS-PLLA according to claim 2, characterized in that: In the step (3), the molar ratio of folic acid, EDCI and DMAP is 1:1:

1.

6. The preparation method of the dual-targeting polymer carrier material FA-TPGS-PLLA according to claim 2, wherein: In the step (3), the reaction time of stirring in the dark at room temperature is 1 - 48 h.

7. The preparation method of the dual-targeting polymer carrier material FA-TPGS-PLLA according to claim 2, characterized in that: The dialysis bag M in step (4) W is 10,000.

8. A method for preparing oleanolic acid nanoparticles encapsulated by the dual-targeting polymer carrier material FA-TPGS-PLLA described in claim 1, characterized in that, It includes the following steps: 1) Dissolve the double-targeting polymer carrier material FA-TPGS-PLLA in dichloromethane, add an oil-phase emulsifier to form phase A; 2) Dissolve oleanolic acid in acetone to form phase B; 3) While stirring phase A, slowly drip phase B, and continue stirring to form a primary emulsion; 4) Slowly drip the primary emulsion into an SDS solution and stir to form a multiple emulsion; 5) After stirring and solidifying to volatilize the organic solvent, then carry out ultrasonic stirring to obtain the product.

9. The preparation method of oleanolic acid nanoparticles according to claim 8, characterized in that, The dosages of the raw materials are as follows: 0.01 g of the FA-TPGS-PLLA carrier material, 1 mL of dichloromethane, 3 drops of the oil-phase emulsifier, 0.008 g of oleanolic acid, 1 mL of acetone, and 5 mL of 1 wt% SDS solution; the oil-phase emulsifier is selected from one or a mixture of several of Span-80, Span-20, Span-40, and Span-60.

10. Use of oleanolic acid nanoparticles encapsulated by a dual-targeting polymer carrier material FA-TPGS-PLLA, characterized in that, Use the oleanolic acid nanoparticles encapsulated by the double-targeting polymer carrier material FA-TPGS-PLLA described in claim 8 or 9 as the drug carrier.

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