CN104774322B - Pluronic F87-containing polymer and preparation method and application thereof - Google Patents

Abstract

The invention discloses a polymer containing Pluronic F87 and its preparation method and application, belonging to the field of biomedicine, and discloses a polyoxyethylene-oxypropylene-oxyethylene as a hydrophilic segment, polyoxyethylene-oxypropylene-oxyethylene, polypropylene Lactide is a polymer folic acid-polyoxyethylene-polyoxypropylene-polyoxyethylene-polylactic acid (FA-F87-PLA) with novel chemical structure as the amphiphilic block of the hydrophobic segment, and a preparation method of the compound. The invention has good application prospect.

Description

A kind of polymer containing Pluronic F87 and its preparation method and application

technical field

The invention belongs to the field of biomedicine and relates to a targeted polymer drug carrier containing folic acid and a preparation method thereof.

Background technique

Amphiphilic polymers, especially biocompatible amphiphilic polymers (that is, polymers containing both hydrophilic and hydrophobic segments) have been extensively studied because they can pass through the interactions between hydrophobic segments in water. Self-aggregation such as hydrophobic interaction forms nanoparticles with various morphologies. This property makes amphiphilic polymers have great application prospects in drug delivery systems, such as controllable release systems and targeted release systems. We know that most of the current drugs (such as anticancer drugs) are hydrophobic, that is, insoluble in water, and are easily excreted by a series of rejection reactions in the human body, such as drug resistance, enzyme degradation, etc., which greatly Limiting the effectiveness of treatments for diseases such as cancer. Nanoparticles formed by amphiphilic polymers can be used as drug carriers, embedding the drug in the hydrophobic core, and the surface is protected by the hydrophilic layer of the nanoparticle, so that the drug can be delivered to the lesion (such as a tumor, etc.), thereby play an effective role in the treatment of cancer.

The modification of targeting groups on the surface of polymer nanoparticles can improve the selectivity of drug delivery and the effectiveness of disease treatment. Because the targeted nanoparticles transport the embedded drug to tumors and other lesions, it can not only reduce the damage of the drug to normal cells, but also reduce the dosage of the drug by improving the utilization rate of the drug, thereby reducing the impact of the drug on the human body. side effect. Therefore, targeted polymer nanoparticles have great application prospects in drug delivery systems.

Folic acid is an essential vitamin for cells (especially proliferating cells), and it participates in the one-carbon transfer reaction of various metabolic pathways. The cellular transport of folate is accomplished by two transmembrane proteins, the low-affinity reducing folate carrier and the high-affinity folate receptor (FR). It has been confirmed that FR is overexpressed on the surface of various tumor cells, while the expression in most normal tissues is limited to the apical membrane of some epithelial cells that are difficult to enter the blood circulation. Because of the characteristics of FR expression, the natural ligand of FR—folic acid (FA) has become an important molecule for targeting drugs to tumor cells. Folic acid has a high affinity with folic acid receptors (K _d =l×10 ^-10 mol · L ^-1 ), low immunogenicity, easy modification, small size (M _w = 441.4), high chemical stability and biological stability, high tumor permeability, easy to combine with drugs, and organic and aqueous solvents The advantages of compatibility and low cost have enabled the rapid development of folic acid-mediated tumor targeting research.

In recent years, folic acid-targeted polymer drug delivery systems have been extensively studied at home and abroad. J.R.Baker Jr.'s research group at the University of Michigan modified the targeting group folic acid on the surface of polyamide-amine (PAMAM) dendrimers. There are specific interactions, which can effectively improve the therapeutic effect of anticancer drugs (Choi, Y. Chemistry & Biology 2005, 12, 35), however, the clinical application of dendrimers largely depends on the controllable preparation, function With the development of chemical and other related research, there are still problems such as difficulty and high cost in the controllable preparation of highly regular monodisperse peptide dendrimers.

Contents of the invention

The purpose of the present invention is to provide a polymer with a novel chemical structure, which has the function of drug targeting carrier.

The polymer drug carrier containing folic acid targeting of the present invention contains folic acid targeting group, polyoxyethylene-oxypropylene-oxyethylene (PEO ₆₁ -PPO ₄₀ -PEO ₆₁ , Pluronic F87) as a hydrophilic segment, polypropylene Lactide [poly(lactic acid), PLA] is an amphiphilic block copolymer folic acid-polyoxyethylene-polyoxypropylene-polyoxyethylene-polylactic acid (FA-F87-PLA) as a hydrophobic segment. Its chemical structure is as follows :

Its synthetic route is as Fig. 1, and concrete preparation method is:

(1) According to the weight ratio, first put F87 (20g, 2.60mmol) into a dry round bottom flask, and add 100ml of dry CH ₂ Cl ₂ to dissolve it. Then dissolve the mixture of 4-dimethylaminopyridine (DMAP) (0.19 mg, 1.56 mmol) and folic acid (folic acid, FA) (0.83 g, 1.89 mmol) in 10 ml of anhydrous dimethyl sulfoxide (DMSO), and add to the reaction flask inside. Then, a solution of 1,3-dicyclohexylcarbodiimide (DCC) (0.36g, 1.74mmol) in CH ₂ Cl ₂ (25ml) was added to the reaction flask (in an ice bath environment) with a dropping funnel, and the addition was completed Then continue to react for 48h. After the reaction, 8% NaHCO ₃ solution was used to extract the reaction solution to remove impurities such as unreacted FA. Then the extract was spin-dried, dissolved in CH ₂ Cl ₂ , precipitated into frozen ether and purified twice, and the precipitate was dried to obtain FA-F87-OH;

(2) Use FA-F87-OH as a macroinitiator and stannous octoate as a catalyst to initiate ring-opening polymerization of the cyclic monomer lactide (LA) under anhydrous and oxygen-free conditions. Finally the desired copolymer is obtained. The specific synthesis method is: after the reaction bottle is vacuumed and dehumidified by argon gas, FA-F87-OH, lactide and stannous octoate are added under argon gas, and the amount of lactide is FA-F87-OH 50-100% of the weight, the amount of stannous octoate is 0.1-0.15% of the lactide weight, the reactant is heated to 140-160 ° C, under stirring, the reaction continues for 7-9 hours; the reactant is dissolved in dichloromethane , then sink into methanol, a white substance precipitates out, filter; then dissolve the polymer with dichloromethane, sink into methanol, filter, and dry to obtain FA-F87-PLA copolymer.

The invention also relates to the application of the FA-F87-PLA copolymer in embedding the anticancer drug paclitaxel.

The method of entrapping paclitaxel is:

1. Preparation of FA-F87-PLA nanoparticles embedded with paclitaxel

Weigh 3 mg of FA-F87-PLA polymer and 0.3 mg of paclitaxel (Paclitaxel, PTX) into a stoppered test tube, add 3.3 ml of dimethylsulfoxide (DMSO) to dissolve. The DMSO solution was then dispersed in 10 g of ultrapure water, poured into a dialysis bag and dialyzed for 24 hours to remove unencapsulated paclitaxel.

2. Determination of paclitaxel embedding rate and drug loading

Get 4ml of the nanoparticle aqueous solution in step 1 to freeze-dry, then add 4ml of acetonitrile-water (7: 3v/v) mixed solution to dissolve, then test with high performance liquid chromatography (HPLC), the test conditions are as follows: C18 column, with 1.0 Acetonitrile-water (7:3v/v) at a flow rate of ml·min ^-1 was used as the mobile phase. The peak area was detected at 227nm, and the paclitaxel concentration in the sample was calculated by comparing it with the main peak area of the standard. After testing, the retention time of paclitaxel was 3.89min.

The formula for calculating the embedding rate is as follows:

The FA-F87-PLA amphiphilic block copolymer involved in the present invention is a polymer with a novel chemical structure. And compared with the existing FA-F127-PLA copolymer, it has a higher drug embedding rate and increases the anti-tumor effect of the drug.

Description of drawings

Fig. 1 is the synthetic route of polymer FA-F87-PLA of the present invention.

detailed description

Pluronic F87 (PEO ₆₁ -PPO ₄₀ -PEO ₆₁ ,) was purchased from Sigma.

Example 1

The preparation method of FA-F87-PLA copolymer of the present invention is as follows:

1. First put Pluronic F87 (PEO ₆₁ -PPO ₄₀ -PEO ₆₁ ,) (20g, 2.60mmol) into a dry round bottom flask, add 100ml of dry CH ₂ Cl ₂ to dissolve. Then dissolve the mixture of 4-dimethylaminopyridine (DMAP) (0.19 mg, 1.56 mmol) and folic acid (folic acid, FA) (0.83 g, 1.89 mmol) in 10 ml of anhydrous dimethyl sulfoxide (DMSO), and add to the reaction flask inside. Then, a solution of 1,3-dicyclohexylcarbodiimide (DCC) (0.36g, 1.74mmol) in CH ₂ Cl ₂ (25ml) was added to the reaction flask (in an ice bath environment) with a dropping funnel, and the addition was completed Then continue to react for 48h. After the reaction, 8% NaHCO ₃ solution was used to extract the reaction solution to remove impurities such as unreacted FA. Then the extract was spin-dried, dissolved in CH ₂ Cl ₂ and precipitated into frozen ether for purification twice. The precipitate was taken and dried to obtain FA-F87-OH. The weight of the obtained copolymer was 11.61 g, and the yield was 55.7%.

2. After the reaction bottle is vacuumed and dehumidified by argon, add 2.5g of FA-F87-OH, 2.5g of lactide and 3mg of stannous octoate under the condition of argon, heat the reactant to 120°C, and stir Next, the reaction continued for 6 hours; the reactant was dissolved in dichloromethane, then sank into methanol, and a white substance was precipitated, filtered; then the polymer was dissolved with dichloromethane, and sank into methanol, filtered, dried, and finally 0.86 g of FA-F87-PLA copolymer (carrier) was obtained, and the yield was 17.2%.

Structural characterization ¹ H NMR (400MHz, CDCl ₃ , ppm): 1.1-1.2(m, CH ₃ of PPO block in F87), 1.5-1.7(m, CH ₃ of PLA block), 3.3-3.7(m, OCH ₂ CH ₂ of PEO block and OCH ₂ CH of PPO block in F87), 5.1-5.3 (m, CH of PLA block).

The molecular weight of the prepared FA-F87-PLA copolymer and the segment content of PLA are calculated by the nuclear magnetic spectrum of FA-F87-PLA, and the result is that the molecular weight (Mn) of FA-F87-PLA is 24800, and the content of the PLA segment The content is 67.1 wt%, that is, the structural formula of the corresponding polymer is FA-PEO ₆₁ -PPO ₄₀ -PEO ₆₁ -PLA ₂₃₁ . The content of FA in FA-F87-PLA copolymer was measured by ultraviolet spectrophotometer. According to the ultraviolet absorption peak of FA at 288nm, FA has been successfully connected to the terminal group of F87-PLA copolymer.

Embodiment 2 FA-F87-PLA copolymer of the present invention embeds paclitaxel

1. Preparation of FA-F87-PLA nanoparticles embedded with paclitaxel

Weigh 3 mg of FA-F87-PLA polymer and 0.3 mg of paclitaxel (Paclitaxel, PTX) into a stoppered test tube, add 3.3 ml of dimethylsulfoxide (DMSO) to dissolve. The DMSO solution was then dispersed in 10 g of ultrapure water, poured into a dialysis bag and dialyzed for 24 hours to remove unencapsulated paclitaxel.

2. Determination of paclitaxel embedding rate and drug loading

Get 4ml of the nanoparticle aqueous solution in step 1 to freeze-dry, then add 4ml of acetonitrile-water (7:3v/v) mixed solution to dissolve, then use high-performance liquid chromatography (HPLC) to test, the test conditions are as follows: C18 column, at 1.0 Acetonitrile-water (7:3v/v) at a flow rate of ml·min ^-1 was used as the mobile phase. The peak area was detected at 227nm, and the paclitaxel concentration in the sample was calculated by comparing it with the main peak area of the standard. After testing, the retention time of paclitaxel was 3.89min.

The formula for calculating the embedding rate is as follows:

It can be seen from the calculation results that the paclitaxel embedding rate of FA-F87-PLA copolymer is 45.4%.

3. Synthesis of FA-F127-PLA copolymer

1. Synthesis of FA-F127-OH: first add 0.42g, 0.95mmol FA and 0.18g 1,3-dicyclohexylcarbodiimide (DCC) to 35ml anhydrous dimethyl sulfoxide (DMSO), at room temperature After stirring for 12 hours, 10 g, 0.79 mmol of Pluronic F127 and 0.097 g of 4-dimethylaminopyridine (DMAP) were added thereto, and stirring was continued at room temperature for 24 hours. The reaction was then centrifuged for 5 minutes. The supernatant was dialyzed with DMSO for 3 hours, the molecular weight cut-off of the dialysis bag was 3500, and then dialyzed with twice distilled water for 24 hours. The reaction solution in the dialysis bag was spin-dried and dissolved in 5 ml of dichloromethane, dropped into anhydrous ether, filtered, and vacuum-dried to obtain 5 g of FA-F127-OH with one end modified with FA.

2. Synthesis of FA-F127-PLA: After the reaction bottle is vacuumed and dehumidified by argon, 5g of FA-Pluronic-OH, 2.5g of lactide and 2.5mg of stannous octoate are added under argon. The reactant was heated to 150°C, and the reaction continued for 6 hours under stirring; the reactant was sunk in methanol, and a white substance precipitated out, filtered; then the polymer was dissolved with dichloromethane, sunk in methanol, filtered, and dried , 3.8 g of FA-F127-PLA copolymer (carrier) was finally obtained, and the yield was 50.7%.

Structural characterization ¹ H NMR (400MHz, DMSO-d ⁶ , ppm): 1.04-1.05(m, CH ₃ of PPO block inPluronic), 1.28-1.30(m, CH ₂ CH ₂ CO of folate), 1.47-1.49(m ,CH ₃ of PLA block),3.33-3.52(m,OCH ₂ CH ₂ of PEO block and OCH ₂ CH of PPO block in Pluronic),5.21-5.22(m,CHof PLA block),6.5-7.7(d,benzene -H of folate).

FA-F127-PLA copolymer was used to embed paclitaxel in the same way, and the embedding rate was tested. The embedding rate of paclitaxel was calculated to be 24.6%.

From the comparison of the results, it can be seen that the paclitaxel entrapment rate of FA-F87-PLA copolymer is nearly 2 times higher than that of FA-F127-PLA copolymer.

Claims (4)

1. a kind of polymer, its molecular structural formula is as follows：

2. polymer according to claim 1, its preparation method is comprised the following steps：

(1) according to weight ratio, first by 2.60mmol F87 (PEO₆₁-PPO₄₀-PEO₆₁) it is put into dry round-bottomed flask, add 100ml is dried CH₂Cl₂Middle dissolving；Then 0.19mg 1.56mmol 4- diformazan ammonia is dissolved with 10ml anhydrous dimethyl sulfoxides (DMSO) The mixture of yl pyridines (DMAP) and 0.83g, 1.89mmol Folic Acid (FA), in adding reaction bulb；Then by 0.36g, The CH of 1.74mmol 1,3- dicyclohexylcarbodiimides (DCC)₂Cl₂Solution 25ml Dropping funnels are added in ice bath environment and reacted In bottle, continue to react 48h after dripping；8% NaHCO after having reacted₃Solution extractive reaction liquid is removing unreacted FA etc. Impurity；Then CH is used after extract is spin-dried for₂Cl₂Dissolving postprecipitation enters to freeze purification among ether and twice, takes and be obtained after precipitation drying FA-F87-OH；

(2) it is catalyst to do macromole evocating agent and stannous octoate with FA-F87-OH, under conditions of anhydrous, anaerobic, causes ring Shape monomers lactide (LA) carries out ring-opening polymerization, finally gives required copolymer；Specifically synthetic method is：Reaction bulb leads to After crossing evacuation-logical argon deoxygenation dehumidifying, FA-F87-OH, lactide and stannous octoate are added under the conditions of argon, lactide Measure as the 50-100% of FA-F87-OH weight, the amount of stannous octoate is the 0.1-0.15% of lactide weight, and reactant is heated To 140-160 DEG C, under stirring, reaction continues 7-9 hours；Then the dissolving of reactant dichloromethane is sunk in methanol, there is white Material settles out, and filters；Then with dichloromethane polymer is dissolved again, and is sunk in methanol, filtered, be dried, obtain FA-F87- PLA copolymer.

3. application of the polymer according to claim 1 in the carrier for preparing treating cancer medicine.

4. application according to claim 3, it is characterised in that the treating cancer medicine is paclitaxel.