CN108478804B - A kind of polyacrylic acid-S-S-drug copolymer and preparation method thereof - Google Patents

Abstract

The invention discloses a polyacrylic acid-S-S-drug copolymer and a preparation method thereof. It belongs to the field of polymer chemistry and pharmaceutical preparations. The drug and cystamine dihydrochloride are prepared into drug derivatives; then polyacrylic acid and drug derivatives are condensed into polyacrylic acid-S-S-drug copolymer, which can spontaneously form amphiphilic polymer micelles in an aqueous solution , the connecting bond is a disulfide bond, which can be cleaved responsively at the lesion site to release the drug. In addition, polyacrylic acid can improve the water solubility of the drug, which can be used to prepare redox-sensitive polymers that improve the solubility of poorly soluble drugs. medicine. The invention also discloses a preparation method of the PAA‑S‑S‑GA copolymer and its use as an anticancer drug carrier.

Description

A kind of polyacrylic acid-S-S-drug copolymer and preparation method thereof

technical field

The invention relates to the field of pharmaceutical preparations and polymer chemistry, in particular to a redox type, water-soluble pharmaceutical preparation that can effectively improve insoluble drugs and a preparation method thereof.

Background technique

The idea of covalently combining water-soluble polymers with chemotherapeutic drugs was first proposed in the 1970s, and since then, it has become a rapidly growing field with the development of synthesis and polymers. In the 1990s, it began to enter the clinic, such as poly(L-glutamic acid)-paclitaxel copolymer. Other conjugates are also under development, such as polyethylene glycol-camptothecin with polyethylene glycol as a carrier. Polyethylene glycol is an FDA-approved hydrophilic polymer with low toxicity and immunogenicity, but the fact is that even at the tumor site, the polyethylene glycol linker may be difficult to break to release the drug, resulting in a significant anti-cancer effect decline. The rapidly developing tumor microenvironment-sensitive drug delivery system, which can release drugs responsively, provides a new strategy for overcoming the barriers of drug solubility and site-specific delivery of chemotherapeutic drugs.

Gambogic Acid (GA, C ₃₈ H ₄₄ O ₈ ) is one of the main active compounds with anti-tumor effect. As an extract of Chinese medicine Garcinia cambogia, it has been used for thousands of years. GA has been proven to have anti-tumor effects in many tumor types, and has become a hot spot in natural product anti-tumor research in recent years. Due to its large toxic and side effects, poor water solubility, and low selectivity, its current anti-tumor clinical research is limited.

Glutathione (glutathione, GSH) is a naturally occurring tripeptide in the human body. The content of glutathione in tumor tissues and cells is high, but the content of glutathione in normal cells of cancer patients is lower than that of healthy people. High levels of GSH in tumor cells are often resistant to chemotherapy, and some researchers have tried to reduce GSH levels in cancer cells by using GSH-depleting drugs such as butthionine imide sulfate (BSO). However, the effect of using BSO is limited and untargeted, and the drug also reduces the content of GSH in normal cells, which further aggravates the side effects caused by radiotherapy and chemotherapy. The high concentration of glutathione in the tumor site can reduce the disulfide bond, while the concentration of glutathione in normal tissues and blood vessels is low, the disulfide bond can exist stably, and the high concentration of glutathione itself can also reduce the disulfide bond. will be oxidized and thus consumed.

Polyacrylic acid has good biocompatibility, non-toxic and harmless, and can be modified. Small molecule drug polyacrylic acid polymer selectively enters tumor cells through high permeability, lymphatic reflux barrier and endocytosis of solid tumor tissue, reducing drug Toxic and side effects, prolong the residence time of the drug at the tumor site.

Therefore, a linkage sensitive to pH conditions and enzyme systems of tumor tissue was developed to connect chemotherapeutic drugs with water-soluble polymers, so as to ensure the solubility of the drugs in water and release them from the conjugates at the tumor site in time. Pharmaceutical formulations of drugs have practical significance.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a high molecular polymer prodrug for intelligently responsive drug release, which connects polyacrylic acid and insoluble drugs to disulfide bonds through covalent bonds, so as to improve the water solubility of the insoluble drugs, and at the same time, due to tumor The content of reduced glutathione in tissues and tumor cells is high, which can hydrolyze and break disulfide bonds, which can not only target tumor tissues, but also reduce the toxic and side effects to normal cells.

The technical scheme adopted in the present invention is: a polyacrylic acid-S-S-drug copolymer having the structural formula shown in (I):

Wherein, x=5-10 mol%, y=90-95 mol%, and R is a drug compound with a carboxyl group. Preferably, the drug compound with carboxyl group is selected from gambogic acid, rhein, valsartan, methotrexate, exenatide acetate, IDN-6556, AGI-1067, azoserine, chlorobenzene Alanine, N-Acetyl-L-Phenylalanine and N-Acetyl-L-Valine. More preferably, the drug compound with carboxyl group is gambogic acid.

Preferably, in the above-mentioned polyacrylic acid-S-S-drug copolymer, the weight-average molecular weight of the polyacrylic acid segment is 50 kDa.

The above-mentioned preparation method of a polyacrylic acid-S-S-drug copolymer comprises the following steps: 1) preparing a drug and cystamine dihydrochloride into a drug derivative; 2) condensing the polyacrylic acid and the drug derivative into a polyacrylate Acrylic-S-S-Drug Copolymer.

A preparation method of polyacrylic acid-S-S-gambogic acid copolymer, comprising the steps:

1) Dissolve gambogic acid in dichloromethane, stir until fully dissolved, add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and HOBT under ice bath (1-Hydroxybenzotriazole), stirred overnight in the dark at room temperature, added cystamine dihydrochloride to the reaction solution, and added methanol as a solubilizer, adjusted the pH to 7.4 with triethylamine, stirred for 24h, and the obtained product was treated with NaHCO ₃ Wash with aqueous solution, add anhydrous sodium sulfate to the organic layer, dry, filter, remove dichloromethane by rotary evaporation under reduced pressure, and dry in vacuum to obtain gambogic acid derivative;

2) Dissolve polyacrylic acid (PAA) in DMF, stir until completely dissolved, add EDC and HOBT under ice bath, and stir overnight in the dark to obtain mixed solution A; dissolve gambogic acid derivative in DMF, under ice bath It was added dropwise to the mixed solution A, stirred for 24 hours, the post-reaction solution was added dropwise to ice water, the precipitate was collected, dissolved in water, dialyzed for two days, and freeze-dried to obtain polyacrylic acid-S-S-gambogic acid copolymer. Preferably, by weight ratio, gambogic acid:polyacrylic acid=(1.2-1.3):1. In the dialysis, the molecular weight of the dialysis bag is 50kDa, and the dialysis medium is distilled water.

The polyacrylic acid-S-S-gambogic acid copolymer has the structural formula shown in (II):

Among them, x=5～10mol%, y=90～95mol%.

Compared with the prior art, the present invention has the following beneficial effects:

The polymer drug copolymer of the invention improves the water solubility of the insoluble gambogic acid, is linked by disulfide bonds, has good release response performance, enhances the targeting of the copolymerized drug, and greatly prolongs the stay of the anticancer drug in the tumor. The time and critical micelle concentration test showed that the polymer drug copolymer was easy to form micelles, and cell experiments showed that it had a good inhibitory effect on liver cancer. The polymer drug copolymer has the function of targeted and intelligent drug release. The particle size is about 160 nm, which is helpful for the accumulation of nanoparticles at the tumor site. After the responsive fracture, the drug is released, which is helpful for the penetration of the drug. The present invention designs and develops a polyacrylic acid-S-S-gambogic acid copolymer drug delivery system by adopting the polyacrylic acid polymer targeted drug delivery technology and the high concentration of glutathione at the tumor site as the target, thereby increasing the gambogic acid targeting Therapeutic effect, reducing toxic and side effects, thereby improving bioavailability.

The polymer-drug copolymer of the present invention has redox response performance, good water solubility, less toxic and side effects, and the hydrophilic section is polyacrylic acid. In aqueous solution, amphiphilic polymer micelles are spontaneously formed due to the hydrophilic and hydrophobic interaction, and the drug can be released responsively at the tumor site. The application of the polymer-drug copolymer of the present invention as an anticancer drug carrier can effectively improve the water solubility of poorly soluble drugs.

Description of drawings

Figure 1 shows the particle size and potential changes of the prepared PAA-S-S-GA copolymers under different hydration volumes.

Figure 2a shows the particle size distribution of the prepared PAA-S-S-GA under the optimal hydration volume.

Figure 2b is the zeta potential diagram of the prepared PAA-S-S-GA under the optimal hydration volume.

Figure 3 is a CMC graph of the prepared PAA-S-S-GA.

Figure 4 is the DSC detection chart of the prepared PAA-S-S-GA.

Figure 5 is a graph showing the change of drug release of the prepared PAA-S-S-GA under different concentrations of glutathione.

Detailed ways

The technical solutions of the present invention are further described below through specific embodiments. It should be understood by those skilled in the art that the embodiments are only for helping the understanding of the present invention, and should not be regarded as a specific limitation of the present invention.

Example 1 Polyacrylic acid-S-S-gambogic acid copolymer (PAA-S-S-GA)

(1) Preparation method

1. Dissolve 1.57g of gambogic acid in 50mL of dichloromethane, stir until completely dissolved, add 620mg of EDC and 438mg of HOBT under ice bath, and stir overnight in the dark at room temperature to obtain a bright yellow reaction solution, which is added to the reaction solution 1.69 g of cystamine dihydrochloride was added, and 10 mL of methanol was added to help dissolve, and the pH was adjusted to 7.4 with triethylamine, and stirred for 24 h. After the obtained reaction solution was washed three times with NaHCO ₃ aqueous solution, the organic layer was dried by adding anhydrous sodium sulfate, and filtered. The dichloromethane was removed by rotary evaporation under reduced pressure, and dried in vacuo to obtain a yellow oily compound, which is a gambogic acid derivative, and the next reaction was carried out directly.

m/z: 763.3 [M+H] ⁺ ; 1H NMR (CDCl ₃ ) δ 8.64 (s, 1H), 6.71 (d, 1H), 6.58 (d, 1H), 6.14 (t, 1H), 5.60 ( d, 1H), 5.3(m, 1H), 5.13(m, 1H), 4.92(s, 2H), 3.48(q, 1H), 3.4(m, 1H), 3.22(m, 1H), 2.95(m ,1H), 2.86(m,1H), 2.64(m,1H), 2.35(m,1H), 2.15(br,2H), 2.1(m,1H), 2.01(m,3H), 1.87(s, 3H), 1.73(s, 6H), 1.61(d, 6H), 1.47(s, 3H), 1.35(s, 3H), 1.3(s, 3H).

2. Dissolve 1.25g of polyacrylic acid (M50kDa) in 20mL of DMF, stir until completely dissolved, add 620mg of EDC and 438mg of HOBT under ice bath, and stir overnight in the dark to obtain a colorless and transparent mixed solution A. The gambogic acid derivative was dissolved in 20 mL of DMF, added dropwise to the mixture A under an ice bath, stirred for 24 h, the reacted solution was added dropwise to a large amount of ice water, the precipitate was collected, dissolved in water, dialyzed (dialysis bag molecular weight) 50kDa, the dialysis medium is distilled water) for two days, remove the small molecule drugs and impurities, freeze-dried to obtain a yellow fluffy solid, namely polyacrylic acid-S-S-gambogic acid copolymer, denoted as PAA-S-S-GA.

IR(KBr,cm ^-1 ): 3442, 2974, 2926, 2845, 2574, 1716, 1640, 1263, 1172, 1101, 1039, 947, 813; other chemical shifts appear in the one-dimensional H spectrum compared to polyacrylic acid alone There are ¹ H NMR (DMSO) δ7.46-7.60 (m, 2H), 6.55 (m, 1H), 5.57-5.63 (m, 1H), 5.33 (m, 1H), 5.06 (br, 2H), which can be proved The synthesis of polymer drug conjugates was successful.

(2) Investigation on the hydration volume of PAA-S-S-GA

Take 10mg PAA-S-S-GA, disperse in 15mL, 20mL, 30mL, 40mL, 60mL of PBS buffer solution with a concentration of 10mmol/L, respectively, and stir at room temperature for ten minutes to form a nanomicelle solution, and obtain the PAA-S-S-GA concentration respectively. 0.67mg/mL, 0.50mg/mL, 0.33mg/mL, 0.25mg/mL, 0.17mg/mL, and the particle size and zeta potential were compared. The results are shown in Figure 1. When the concentration of PAA-S-S-GA is 0.33 mg/mL, it has a good particle size distribution, the particle size is about 160 nm, and the absolute value of zeta potential is high, indicating the stability and dispersion of micelles. Well, the particle size distribution and zeta potential at the optimal concentration (0.33 mg/mL) are shown in Figure 2a and Figure 2b.

(3) Determination of critical micelle concentration (CMC) of PAA-S-S-GA

The critical micelle concentration of the polymer was detected by the pyrene fluorescent probe method. The acetone solution of pyrene was prepared at a concentration of 1×10 ^-4 mg/mL. Take 10 mg of PAA-SS-GA and transfer it to a 10 mL volumetric flask, and make constant volume to obtain a micelle solution with a concentration of 1.00 mg/mL. 100 μL of the prepared pyrene solution was added to a centrifuge tube and dried with nitrogen. Then, 5 mL of different concentrations of PAA-SS-GA polymer solutions were added to each centrifuge tube, and the final concentration of pyrene in each centrifuge tube was 2×10 ^-6 mg/mL. The prepared solution was equilibrated at room temperature for 24h for detection. The detection conditions were as follows: the excitation wavelength was 334 nm, the excitation slit was 5 nm, the emission wavelength range was 350 nm to 500 nm, the emission slit was 5 nm, and the scanning speed was 240 nm/min. The PAA-SS-GA polymer concentrations are 0.0005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 mg/mL, respectively, and the ratio of the peak heights at 373 nm and 384 nm is the ordinate Y, the polymer The logarithm of the solution concentration is plotted on the abscissa X. The results are shown in Figure 3. The concentration at the intersection of the two straight lines in the figure is the CMC value of the polymer. It is inferred that the CMC of the polymer is about 0.01 mg/mL, with a lower The critical micelle concentration can still maintain a certain stability after a certain dilution. It shows that the PAA-SS-GA copolymer has good water solubility and stability.

(4) DSC detection of PAA-S-S-GA

Take GA, PAA, PAA-S-S-GA and GA, PAA physical mixture respectively for DSC detection, the temperature range is 25 ℃ -230 ℃, the results are shown in Figure 4, the curves are GA, PAA, GA and PAA physical from top to bottom Mixture, PAA-S-S-GA. It can be seen from Figure 4 that the GA curve has a larger peak at 80 °C. Similar to the physical mixing curves of PAA and GA. However, the DSC curves of PAA and PAA-S-S-GA did not appear the large peak of GA, and the deviation from the straight line was small. This indicates that GA is oxidized in the amorphous or molecular state in PAA-S-S-GA, and there is almost no free crystalline GA.

(5) Investigation of glutathione-sensitive release degree of PAA-S-S-GA

Take 1 mL of PAA-S-S-GA, transfer it to a dialysis bag (MWCO, 50 kDa), and use 10 mL of PBS (pH 7.4), 0.1% (w/v) SDS and GSH (0 mM, 2 mM, 10 mM, 40 mM) as the release medium. , the release time was 48h, and the temperature was 37°C. At the same time, the dialysate group without GSH was used as a control, and 1mL of dialysate was taken at different time intervals for high performance liquid chromatography analysis, and 1mL of the corresponding fresh buffer was added to restore the volume. . The released drug concentration was detected by high performance liquid chromatography, and the results are shown in Figure 5. It can be seen from Figure 5 that at the concentrations of 0 mM and 2 mM glutathione, the copolymer hardly breaks, and the release amount is small, indicating that it can maintain a certain stability at low concentrations of glutathione, while at 10 mM and 40 mM. At the same concentration of glutathione, the release is rapid, and the release amount can reach about 70% in 10h, indicating that it has excellent response performance.

(6) PAA-S-S-GA pharmacodynamics test

Taking the prepared PAA-S-S-GA as the test sample, the excellent antitumor effect shown in the following pharmacodynamic test was shown:

Determination of growth inhibitory activity (GI50) on HepG-2 cells: After tumor cells were trypsinized, they were dispersed into single cells and suspended in DMEM containing penicillin (25U/mL) and streptomycin (25μg/mL). in the culture medium. The cells were inoculated in a 96-well culture plate and incubated at 37°C in an air containing 5% CO ₂ and a relative humidity of 100% for 24 h. xanthic acid) culture medium, parallel wells were set for each concentration, after culturing for 24 hours, the culture medium containing the test samples was discarded, and the conventional culture medium was added to culture for 48 hours. , U.S. Sigma company product) culture medium, the final concentration of MTT is 0.5mg/mL, add DMSO to dissolve after 4h of incubation, 1h purple crystals are completely dissolved, detect 570nm/630nm on SK601 microplate reader (Japan Seikagaku company product) Optical Density (OD). Calculate the half growth inhibition rate of the test sample to tumor cells according to the following formula:

Inhibition rate=(TT ₀ )/(CT ₀ )×100%

T represents the OD value of the cells in the test sample group

T ₀ represents the OD value of the control plate cells when the test sample is added

Table 1

Table 1 shows the inhibitory effect of PAA-S-S-GA copolymer and gambogic acid on HepG-2 hepatoma cells. It can be seen from Table 1 that the compound of the present invention (polyacrylic acid-S-S-gambogic acid copolymer) shows excellent targeting Antitumor action, as an antitumor agent, is effective for the prevention and treatment of diseases, especially the treatment of liver cancer.

Claims (7)

1. The polyacrylic acid-S-S-drug copolymer is characterized in that the polyacrylic acid-S-S-drug copolymer is a polyacrylic acid-S-S-gambogic acid copolymer and has a structural formula shown as (II):

wherein x is 5 to 10 mol% and y is 90 to 95 mol%.

2. The polyacrylic acid-S-drug copolymer of claim 1, wherein the polyacrylic acid segment has a weight average molecular weight of 50 kDa.

3. The method of claim 1, comprising the steps of: 1) preparing a drug derivative from the drug and cystamine dihydrochloride; 2) then the polyacrylic acid and the drug derivative are condensed into polyacrylic acid-S-S-drug copolymer.

4. The method for preparing polyacrylic acid-S-S-drug copolymer according to claim 3, comprising the steps of:

1) dissolving gambogic acid in dichloromethane, stirring to dissolve completely, adding EDC and HOBT under ice bath, stirring overnight at room temperature in a dark place, adding cystamine dihydrochloride into the reaction solution, adding methanol for assisting dissolution, regulating p H to 7.4 with triethylamine, stirring for 24h, and adding NaHCO into the obtained product₃Washing with water solution, drying the organic layer with anhydrous sodium sulfate, filtering, removing dichloromethane by rotary evaporation under reduced pressure, and vacuum drying to obtain gambogic acid derivative;

2) dissolving polyacrylic acid in DMF, stirring to be completely dissolved, adding EDC and HOBT in ice bath, and stirring overnight in a dark place to obtain a mixed solution A; dissolving the gambogic acid derivative in DMF, dropwise adding the solution into the mixed solution A in ice bath, stirring for 24h, dropwise adding the reacted solution into ice water, collecting the precipitate, dissolving the precipitate with water, dialyzing for two days, and freeze-drying to obtain the polyacrylic acid-S-S-gambogic acid copolymer.

5. The method for preparing a polyacrylic acid-S-S-drug copolymer according to claim 4, wherein the ratio of gambogic acid to polyacrylic acid (1.2-1.3): 1 is calculated by mass.

6. The method of claim 4, wherein in step 2), the molecular weight of the dialysis bag is 50kDa, and the dialysis medium is distilled water.

7. The use of a polyacrylic acid-S-gambogic acid copolymer of claim 1 in the preparation of an anti-tumor pharmaceutical preparation.

Images