CN113248700B

CN113248700B - Synthesis and application of fullerene alcohol grafted polymer carrier

Info

Publication number: CN113248700B
Application number: CN202110394445.2A
Authority: CN
Inventors: 徐蓓华; 覃江江
Original assignee: Institute Of Oncology And Basic Medicine Chinese Academy Of Sciences Preparatory; Zhejiang Chinese Medicine University ZCMU
Current assignee: Institute Of Oncology And Basic Medicine Chinese Academy Of Sciences Preparatory; Zhejiang Chinese Medicine University ZCMU
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2023-06-06
Anticipated expiration: 2041-04-13
Also published as: CN113248700A

Abstract

The invention relates to synthesis of a fullerene alcohol grafted polymer compound, a preparation method of a drug delivery system and application thereof. The synthesis of the fullerene alcohol grafted polymer compound is that the fullerene alcohol and the polymer compound with a single amino end are subjected to an addition reaction in an aqueous solution (or a mixed solution of water and an organic solvent) to obtain a product. The synthesis condition is mild, no by-product is produced, no pollution is produced, the operation is simple, and the method can be used for industrial production. The invention also provides a preparation method of the drug-loaded micelle, namely a preparation method of the fullerene alcohol grafted distearoyl phosphatidylethanolamine-polyethylene glycol/doxorubicin hydrochloride micelle (C60 (OH) -DSPE-PEG/DOX. HCl), which has mild preparation conditions and simple operation, and can realize clinical on-site preparation. The invention also provides an in-vitro cytotoxicity experimental result of the drug-loaded micelle, and the drug-loaded micelle has a great degree of reduction on the inhibition rate of normal cells.

Description

Synthesis and application of fullerene alcohol grafted polymer carrier

Technical Field

The invention belongs to the technical field of polymer nano drug delivery systems, and particularly relates to synthesis and application of a fullerene alcohol grafted polymer carrier.

Background

Fullerenes are a series of caged spherical nanomolecules formed from carbon atoms. In recent years, fullerenes have been found to have good biological activities such as antioxidant effect, antiviral effect, antitumor and immunoregulatory effect, etc. However, fullerene is insoluble in water, preventing its further application, and researchers have attached polar functional groups to fullereneIn the structure of (2), the solubility in water is increased, wherein after hydroxyl is introduced, fullerol with hydrophilic property (namely, hydroxylated fullerene) is obtained, and the fullerol has the following advantages: 1. the double bond in the structure can participate in oxidation-reduction, addition, nucleophilic, electrophilic substitution and other reactions, so that a series of derivatives are obtained, and various groups can be grafted according to the needs; 2. fullerol has protective effect on animal heart, liver, kidney, lung, brain, etc., such as Fullerol C ₆₀ (OH) ₂₄ Has preventive and alleviating effects on cardiotoxicity and hepatotoxicity induced by doxorubicin. Fulleritol C ₆₀ (OH) ₂₄ Can be used for preventing oxidation stress of rat kidney, testis and lung caused by doxorubicin. Fulleritol can reduce mucositis during chemotherapy and restore irinotecan-induced leukopenia; 3. fulleritol C ₆₀ (OH) X has the advantages of inhibiting tumor growth and tumor metastasis, and has small dosage and low toxicity. C (C) ₆₀ (OH) ₂₂ Cancer metastasis can also be inhibited by inhibiting angiogenesis.

The drug delivery system prepared by the polymer material is more and more paid attention to, and can realize the effects of drug slow release, targeted delivery, solubilization, attenuation and the like, and a plurality of polymer anti-tumor drug delivery systems are approved by FDA and marketed. The polymer material is used as a carrier, and the nanoscale drug delivery system comprises nanoparticles, micelles, liposomes, nanovesicles, polymer drug complexes and the like; the micron-sized drug delivery system comprises microspheres, microvesicles, microgels and the like; macroscopic drug delivery systems include polymeric hydrogels, implantable materials, and the like. These new dosage forms change the traditional mode of administration; improving the drug properties; changes the in vivo pharmacokinetic properties of the drug.

The micelle is a thermodynamically stable colloid solution formed by self-assembling amphiphilic high molecular polymer in water. The traditional micelle administration system preparation method comprises a direct dissolution method, a dialysis method and a self-assembly solvent evaporation method. Micellar solutions are non-kinetically stable systems, typically stored after lyophilization. The existing problems are unstable re-dissolution after freeze-drying, the complexity of the preparation process is increased by adding the stabilizer, and the addition of the stabilizer is not beneficial to the stabilization of the medicine.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention aims to provide synthesis and application of a fullerene alcohol grafted polymer carrier. According to the invention, the fullerene alcohol is grafted to the high polymer material through a mild chemical reaction, so that not only is the drug carrying characteristic of the high polymer material maintained, but also the excellent physical characteristic and the physiological activity of the fullerene alcohol are increased, and the fullerene alcohol is especially used as an antitumor drug carrier to have a synergistic effect. The provided fullerene alcohol grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol doxorubicin-loaded micelle has the advantages of simple preparation method and mild condition.

The invention provides synthesis of a fullerene alcohol grafted polymer carrier, which comprises the following steps:

1) Fulleritol is dissolved in water with the concentration of 1-1000mg/mL;

2) The polymer material is dissolved in water or in an organic solvent which can be mixed with water or in a mixed solvent of water and the organic solvent, and the concentration is 1-2000mg/mL;

3) The solution prepared in the step 1) and the solution prepared in the step 2) are prepared according to the molar ratio of the fullerene alcohol to the polymer of 1:1-1:5, mixing, stirring and reacting at normal temperature or under heating condition to obtain a reaction solution;

4) And 3) directly freeze-drying the reaction solution prepared in the step 3), or freeze-drying the dialyzate through dialysis to obtain the fullerene alcohol grafted polymer.

In a preferred embodiment of the synthesis of the fullerene grafted polymer carrier provided by the present invention, in the step 1), the fullerene is a hydroxylated derivative of fullerene, C ₆₀ (OH) n, wherein n is an integer from 16 to 58.

In the synthesis of the fullerene alcohol grafted polymer carrier provided by the invention, the organic solvent is any one or a combination of more of dimethyl sulfoxide, dimethylformamide, methanol, ethanol, acetone and the like.

In the synthesis of the fullerene alcohol grafted polymer carrier provided by the invention, the polymer material is a natural or synthetic compound with a single amino end in the structure, and distearoyl phosphatidylethanolamine-polyethylene glycol-amino DSPE-P is adoptedEG-NH ₂ Poly (lactide, glycolide) polyethylene glycol-amino PLGA-PEG-NH ₂ Polyethylene glycol-amino mPEG-NH ₂ Stearic acid-polyethylene glycol-amino SA-PEG-NH ₂ Cholesterol-polyethylene glycol-amino CLS-PEG-NH ₂ Amino cyclodextrin beta-CD-NH ₂ Etc.

In the synthesis of the fullerene grafted polymer carrier provided by the invention, functional groups such as acid-base groups including carboxyl, sulfonic acid groups, mercapto, amino and the like can be introduced into the fullerene in the fullerene grafted polymer carrier structure; targeting groups including folic acid, biotin, asialo, lactose, galactose, mannose, glucose, glycyrrhetinic acid, nano magnetic beads, and the like.

The invention also provides a preparation method of the fullerene alcohol grafted distearoyl phosphatidylethanolamine-polyethylene glycol drug-loaded micelle synthesized based on the method, which comprises the following steps:

1) Dissolving fullerene grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol in water to prepare a carrier stock solution, wherein the concentration of the fullerene grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol carrier is 1-100mg/mL;

2) Dissolving doxorubicin hydrochloride in water to prepare a pharmaceutical stock solution, wherein the concentration of the doxorubicin hydrochloride is 2mg/mL;

3) Adding the drug stock solution prepared in the step 2) into the carrier stock solution prepared in the step 1) under magnetic stirring, wherein the molar ratio of the drug to the carrier is 1:10-1:0.5, heating to 50-60 ℃, and slowly stirring for 5-60 minutes to obtain micelle solution.

Compared with the prior art, the synthesis and application of the fullerene alcohol grafted polymer carrier provided by the invention have the following beneficial effects:

1) The synthesis condition of the fullerene alcohol grafted polymer is mild, no by-product or pollution is caused, the operation is simple, and the method can be used for industrial production;

2) Fullerol grafted distearoyl phosphatidylethanolamine-polyethylene glycol/doxorubicin hydrochloride micelle (C) ₆₀ The preparation condition of (OH) -DSPE-PEG/DOX. HCl is mild, the operation is simple, and the conventional instrument cradle is utilizedCan realize the clinical on-site preparation. Avoiding a series of problems of difficult re-dissolution and the like of the freeze-dried preparation caused by inconvenient storage of micelle solution;

3) Prepared C ₆₀ The carrier of the (OH) -DSPE-PEG/DOX & HCl micelle has a polyethylene glycol structure, so that the micelle blood circulation time can be prolonged, and the micelle is prevented from being cleared by reticuloendothelial systems, macrophages and the like in vivo.

4) Prepared C ₆₀ (OH) -DSPE-PEG/DOX & HCl micelle, particle diameter is about 300 nanometers, can retain tumor tissue through EPR effect;

5) Prepared C ₆₀ The carrier of the (OH) -DSPE-PEG/DOX HCl micelle has a fullerene alcohol structure, so that the toxicity of the micelle drug delivery system to normal cells can be reduced.

Drawings

FIGS. 1-7 are synthetic route patterns of fullerene alcohol grafted polymer;

FIGS. 8-14 are IR spectra of Fullerol grafted polymers;

FIGS. 15-21 are Fullerol grafted polymers ¹ H-NMR spectrum;

FIGS. 22-23 are TEM images of drug-loaded micelles;

FIG. 24 is an in vitro release rate profile of drug-loaded micelles;

FIG. 25 is an in vitro cell inhibition profile of drug-loaded micelles.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Unless otherwise indicated, the reagents used in the following examples were all analytical grade reagents and were commercially available from regular sources.

Fulleritol (C) used in the following examples unless otherwise specified ₆₀ (OH) _22-24 ) Purchased from su zhou HengqiuTechnology limited;

distearoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH) used in the examples below, unless otherwise specified ₂ ) (mw=2800), distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH) (mw=2850), methoxy-polyethylene glycol-amino (mPEG-NH) ₂ ) (mw=2000), poly (lactide, glycolide) -polyethylene glycol-amino (PLGA-PEG-NH) ₂ ) (mw=20000) from medical science and technology limited;

distearylphospholipid ethanolamine-polyethylene glycol-aldehyde (DSPE-PEG-CHO) (mw=5800) used in the following examples was purchased from siella chemical technologies (shandong) limited unless otherwise indicated.

Example 1: fullerol grafted distearoyl phosphatidylethanolamine-polyethylene glycol (C) ₆₀ Synthesis of (OH) -DSPE-PEG, synthetic route is shown in FIG. 1.

Fullerol 115mg (0.1 mmol), distearoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH) ₂ ) (mw=2800) 280mg (0.1 mmol) was added to 10mL of water, and the mixture was heated to 50 ℃ and stirred for 8 hours to complete the reaction. At this time, if the reaction liquid is dropped into ethanol, the reaction liquid may be completely dissolved, and the solution may be brown. (dropping the aqueous solution of fullerol into ethanol, whereby fullerol is completely precipitated as particles and is not dissolved.) the reaction solution was freeze-dried to obtain brown solid (3). IR (cm) ^-1 ) 3421,2917,2884,2851,1738,1624,1467,1344,1281,1249,1110,954,843.1H-NMR: delta 3.58ppm (m), 3.26ppm (d), delta 3.05 ppm(s), delta 02.98ppm (m), delta 12.90ppm (d), delta 2.75ppm (m), delta 2.67 ppm(s), delta 2.42ppm (d), delta 2.33ppm (m), delta 1.79 ppm(s), delta 1.47 ppm(s), delta 1.17 ppm(s), delta 0.77 ppm(s). With the raw material DSPE-PEG-NH ₂ (1) And C ₆₀ IR spectrum of (OH) (2) and ¹ comparison of H-NMR spectra, C ₆₀ 1624 in the (OH) -DSPE-PEG (3) IR spectrum reflects the characteristic structure of fullerols, which ¹ Delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerol, which confirms the result of the infrared spectrum, the infrared spectrum is shown in figure 8, ¹ H-NMR is shown in FIG. 15.

Example 2: carboxyl-fullerene grafted distearoyl phosphatidylethanolamine-polyEthylene glycol ((C) ₆₀ Synthesis of (OH) (COOH) -DSPE-PEG, synthetic route is shown in FIG. 2.

C prepared in example 1 ₆₀ 100mg (0.025 mmol) of (OH) -DSPE-PEG (MW=4000), 9mg (0.12 mmol) of glycine were added to 10mL of water, heated to 50℃and stirred for 6 hours to complete the reaction. The reaction solution was dialyzed 3 times against ultrapure water (mw=500), and after lyophilization of the dialysate, brown solid (5) was obtained. IR (cm) ^-1 ) 3424,2917,2880,2850,1739,1606,1467,1381,1346,1282,1250,1108,951,843.1H-NMR: delta 3.59ppm (m), delta 3.43 ppm(s), 3.27ppm (d), delta 03.06ppm (m), delta 12.94ppm (m), delta 2.77 ppm(s), delta 2.67 ppm(s), delta 2.42 ppm(s), delta 1.91 ppm(s), delta 1.79 ppm(s), delta 1.48 ppm(s), delta 1.18 ppm(s), delta 0.77 ppm(s). With the raw material (C) ₆₀ IR spectra and of (OH) -DSPE-PEG (3) and glycine (4) ¹ Comparison of H-NMR spectra, C ₆₀ 1606 in the (OH) (COOH) -DSPE-PEG (5) infrared spectrum reflects the characteristic structure of fullerols, ¹ delta 1.79 ppm(s) in the H-NMR spectrum is a characteristic peak of fullerol, delta 1.91 ppm(s) is a C=C bond in the NH2 addition fullerol structure of glycine, the obtained CH characteristic peak, delta 3.43 ppm(s) is a characteristic peak of glycine, the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 9, ¹ H-NMR is shown in FIG. 16.

Example 3: mannose-Fullerol grafted distearoyl phosphatidylethanolamine-polyethylene glycol (Manno-C) ₆₀ Synthesis of (OH) -DSPE-PEG, synthetic route is shown in FIG. 3.

C prepared in example 1 ₆₀ 100mg (0.025 mmol) of (OH) -DSPE-PEG (MW=4000), 20mg (0.075 mmol) of p-aminobenzene-D-mannopyranose, and 10mL of water were added, and the mixture was heated to 50℃and stirred for 6 hours to complete the reaction. The reaction solution was dialyzed 3 times against ultrapure water (mw=500), and after lyophilization of the dialysate, brown solid (7) was obtained. IR (cm) ^-1 ):3405,2917,2851,1739,1608,1592,1515,1494,1467,1455,1345,1296,1250,1108,1003,954,850. ¹ H-NMR：δ8.14ppm(d),δ7.95ppm(d),7.17ppm(d),δ6.90ppm(d),δ6.70ppm(d),δ6.41ppm(d),δ5.64ppm(s),δ4.07ppm(s),δ3.93ppm(d),δ3.58ppm(m),3.26ppm(d),δ2.98ppm(m),δ2.76ppm(s),δ2.70ppm(m),δ2.42ppm(s),δ2.33ppm(m),δ2.23ppm(s),δ1.79ppm(s),δ1.44ppm(s),δ1.16ppm(s),δ0.76 ppm(s). IR spectrum and IR spectrum with starting material (C60 (OH) -DSPE-PEG (3) and p-aminobenzene-D-mannopyranose (6)) ¹ Comparing H-NMR spectrum, 1608 in Manno-C60 (OH) -DSPE-PEG (7) infrared spectrum reflects characteristic structure of fullerol, 1592,1515,1494 reflects characteristic structure of p-aminobenzene-D-mannopyranose, ¹ delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerene alcohol, delta 8.14ppm (D), 7.17ppm (D), delta 6.70ppm (D), delta 6.41ppm (D), delta 5.64 ppm(s) are characteristic peak of p-aminobenzene-D-mannopyranose, and the infrared spectrum is shown in figure 10, ¹ H-NMR is shown in FIG. 17.

Example 4: fullerol grafted (amide bond linked) distearoyl phosphatidylethanolamine-polyethylene glycol ((C) ₆₀ Synthesis of (OH) - (CONH) -DSPE-PEG, synthetic route is shown in FIG. 4.

Distearoyl phosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH) (mw=2850) 100mg (0.035 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (edc·hcl) 19mg (0.1 mmol), N-hydroxysuccinimide (NHS) 11mg (0.1 mmol) were added to 4ml dmso as to be ultrasonically dissolved, and the mixture was stirred at room temperature for 3 hours. To the reaction solution was added 4mg (0.07 mmol) of ethylenediamine and 20uL, and the reaction was stirred at room temperature overnight. Dialyzing the reaction solution in ultrapure water for 3 times (MW=500), lyophilizing the dialysate to obtain white solid which is distearoyl phosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-CONH-C) ₂ H ₄ NH ₂ )(9)。IR(cm ^-1 ):3382,2918,2889,2851,2741,2695,1741,1544,1467,1360,1343,1281,1242,1149,1111,1061,963,843. ¹ H-NMR：δ5.16ppm(s),4.08ppm(s),δ3.59ppm(m),3.46ppm(s),3.24ppm(s),δ3.08ppm(t),δ2.57ppm(dd),δ2.51ppm(m),δ2.21ppm(m),δ1.48ppm(s),δ1.18ppm(s),δ0.78ppm(s)。

Fulleritol 40mg (0.035 mmol), DSPE-PEG-CONH-C ₂ H ₄ NH ₂ (mw=2900) (0.035 mmol), 100mg (0.035 mmol) was put into 10mL of water, heated to 50 ℃ and stirred for 8 hours to finish the reaction. At this time, if the reaction liquid is dropped into ethanol, the reaction liquid may be completely dissolved, and the solution may be brown. After the reaction solution was lyophilized, brown solid (10) was obtained. IR (cm) ^-1 ):3420,2917,2888,2851,1738,1594,1467,1360,1343,1280,1242,1147,1112,1062,963,843. ¹ H-NMR: delta 5.16 ppm(s), 4.08 ppm(s), delta 3.58 ppm(s), 3.47 ppm(s), 3.24 ppm(s), delta 2.60 ppm(s), delta 2.52 ppm(s), delta 2.21 ppm(s), delta 1.79 ppm(s), delta 1.48 ppm(s), delta 1.18 ppm(s), delta 0.78 ppm(s). With the raw material (C) ₆₀ (OH)(2)、DSPE-PEG-COOH(8)、DSPE-PEG-CONH-C ₂ H ₄ NH ₂ (9) IR spectrum and ¹ comparison of H-NMR spectra, C ₆₀ 1594,1360 in the (OH) - (CONH) -DSPE-PEG (10) infrared spectrum reflects the characteristic structure of fullerols, ¹ delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerol, which confirms the result of the infrared spectrum, the infrared spectrum is shown in figure 11, ¹ H-NMR is shown in FIG. 18.

Example 5: fullerol grafted (hydrazone bond linked) distearoyl phosphatidylethanolamine-polyethylene glycol ((C) ₆₀ Synthesis of (OH) - (conhn=c) -DSPE-PEG, synthetic route pattern is shown in fig. 5.

Distearoyl phosphatidylethanolamine-polyethylene glycol-aldehyde (DSPE-PEG-CHO) (mw=5800) 100mg (0.017 mmol) was added to 2mL of water, 20mg (0.17 mmol) of oxalyl dihydrazide was added to 2mL of ldmso, and the two were dissolved by ultrasonic, mixed, 1 drop of glacial acetic acid was added to the mixed solution, and the mixture was stirred at room temperature for 48 hours. Adding 4mL of water into the reaction solution, filtering with a filter membrane (0.45 um), dialyzing the filtrate in ultrapure water for 3 times (MW=500), and lyophilizing the dialysate to obtain 92mg of white solid which is distearoyl phosphatidylethanolamine-polyethylene glycol-hydrazino (DSPE-PEG-C=NNH-COCONHNH) ₂ )(12)。IR:3421,3287,2887,2741,2695,1688,1657,1540,1467,1413,1360,1343,1281,1242,1149,1112,1061,963,843. ¹ H-NMR：δ7.94ppm(d),7.85ppm(d),δ3.97ppm(m),δ3.58ppm(m),3.20ppm(s),δ2.94ppm(m),δ1.28ppm(t),δ1.15ppm(m),δ0.95ppm(t)。

20mg (0.017 mmol) of fullerol, DSPE-PEG-C=NNH-COCONHNH ₂ (mw=5900) 100mg (0.017 mmol) was added to 10mL of water, heated to 50 ℃ and stirred for 8 hours to complete the reaction. At this time, if the reaction liquid is dropped into ethanol, the reaction liquid may be completely dissolved, and the solution may be brown. After the reaction solution was lyophilized, brown solid (13) was obtained. IR (cm) ^-1 ):3432,2886,1637,1602,1467,1415,1360,1343,1281,1242,1148,1113,1061,963,843. ¹ H-NMR: delta 7.81ppm (d), 7.76ppm (d), delta 3.97ppm (m), delta 3.58ppm (m), 3.20 ppm(s), delta 2.89 ppm(s), delta 1.79 ppm(s), delta 1.15ppm (m), delta 0.76 ppm(s). With raw materials (C60 (OH) (2), DSPE-PEG-CHO (11), DSPE-PEG-C=NNH-COCONHNH ₂ (12) IR spectrum and ¹ comparison of H-NMR spectra 1602,1360 in the C60 (OH) - (CONHN=C) -DSPE-PEG (13) infrared spectra reflects the characteristic structure of fullerols, which ¹ Delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerol, which confirms the result of the infrared spectrum, the infrared spectrum is shown in figure 12, ¹ H-NMR is shown in FIG. 19.

Example 6: fullerol grafted polyethylene glycol (C) ₆₀ Synthesis of (OH) -PEG, synthetic route is shown in FIG. 6.

Fulleritol 115mg (0.1 mmol), methoxy-polyethylene glycol-amino (mPEG-NH) ₂ ) (mw=2000) 200mg (0.1 mmol) was added to 10mL of water, heated to 50 ℃, and reacted for 8 hours with stirring to terminate the reaction. At this time, if the reaction liquid is dropped into ethanol, the reaction liquid may be completely dissolved, and the solution may be brown. After the reaction solution was lyophilized, brown solid (15) was obtained. IR (cm) ^-1 ):3421,2887,2741,2695,1716,1625,1467,1360,1343,1280,1242,1147,1113,1061,964,843. ¹ H-NMR: delta 3.79ppm (t), delta 3.62ppm (m), 3.41 ppm(s), delta 1.79 ppm(s), delta 1.09ppm (d). With the raw material (C) ₆₀ (OH)(2)、mPEG-NH ₂ (14) IR spectrum and ¹ comparison of H-NMR spectra, C ₆₀ 1625 in the (OH) -PEG (15) IR spectrum reflects the characteristic structure of fullerols, which ¹ Delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerol, which confirms the result of the infrared spectrum, the infrared spectrum is shown in figure 13, ¹ H-NMR is shown in FIG. 20.

Example 7: fullerol grafted poly (lactide, glycolide) -polyethylene glycol ((C) ₆₀ Synthesis of (OH) -PLGA-PEG, synthetic route is shown in FIG. 7.

Fulleritol 11.5mg (0.01 mmol), poly (lactide, glycolide) -polyethylene glycol-amino (PLGA-PEG-NH) ₂ ) (mw=20000) 200mg (0.01 mmol) was put into 10mL of water, heated to 50 ℃, stirred and reacted for 8 hours to terminate the reaction. At this time, if the reaction liquid is dropped into ethanol, the reaction liquid can be completely dissolved,the solution was brown. After the reaction solution was lyophilized, brown solid (17) was obtained. IR (cm) ^-1 ):3421,2995,2945,2883,1759,1613,1455,1424,1383,1360,1345,1278,1188,1097,953,843. ¹ H-NMR: delta 5.98ppm (m), 5.20ppm (m), delta 4.91 ppm(s), delta 04.52ppm (m), delta 14.14ppm (dd), delta 24.06ppm (m), delta 33.79 ppm(s), delta 3.51ppm (m), 3.35ppm (m), delta 1.79 ppm(s), delta 1.46 ppm(s), delta 1.33ppm (m), delta 1.27ppm (m), delta 1.18ppm (m), delta 0.83ppm (t), delta 0.77ppm (t). With the raw material (C) ₆₀ (OH)(2)、PLGA-PEG-NH ₂ (16) IR spectrum and ¹ comparison of H-NMR spectra, C ₆₀ 1613,1345 in the (OH) -PLGA-PEG (17) IR spectrum reflects the characteristic structure of fullerols, which ¹ Delta 1.79 ppm(s) in the H-NMR spectrum is characteristic peak of fullerol, which confirms the result of the infrared spectrum, the infrared spectrum is shown in figure 14, ¹ H-NMR is shown in FIG. 21.

Example 8: fullerol grafted distearoyl phosphatidylethanolamine-polyethylene glycol/doxorubicin hydrochloride micelle (C) ₆₀ Preparation of (OH) -DSPE-PEG/DOX. HCl (final carrier concentration 0.5%)

Formulation C ₆₀ 5mL (10 mg/mL) of (OH) -DSPE-PEG stock solution was weighed C ₆₀ 50mg of (OH) -DSPE-PEG is added into 5mL of ultrapure water, and the mixture is dissolved by ultrasonic waves for standby.

5mL (10 mg/mL) of DSPE-PEG stock solution is prepared, 50mg of DSPE-PEG is weighed and put into 5mL of ultrapure water for ultrasonic dissolution for later use.

5mL (2 mg/mL) of DOX HCl stock solution was prepared, 10mg of DOX HCl was weighed, and 5mL of ultra-pure water was added thereto for ultrasonic dissolution for use.

Taking C ₆₀ 2mL each of the (OH) -DSPE-PEG stock solution and DOX HCl stock solution were mixed, heated to 60℃and stirred slowly for 30 minutes. After cooling, the mixture was filtered through a filter membrane (0.22 um), and the filtrate was stored at 4℃for further use.

The DSPE-PEG stock solution and DOX HCl stock solution were taken and mixed in 2mL each, heated to 60℃and stirred slowly for 30 minutes. After cooling, the mixture was filtered through a filter membrane (0.22 um), and the filtrate was stored at 4℃for further use.

Example 9: fullerol grafted distearoyl phosphatidylethanolamine-polyethylene glycol/doxorubicin hydrochloride micelle (C) ₆₀ Preparation of (OH) -DSPE-PEG/DOX. HCl (1% final support concentration)

Formulation C ₆₀ 5mL (20 mg/mL) of (OH) -DSPE-PEG stock solution was weighed C ₆₀ 100mg of (OH) -DSPE-PEG is added into 5mL of ultrapure water, and the mixture is dissolved by ultrasonic waves for standby.

5mL (20 mg/mL) of DSPE-PEG stock solution is prepared, 100mg of DSPE-PEG is weighed and put into 5mL of ultrapure water for ultrasonic dissolution for later use.

Example 10: morphology observation of micelles: a proper amount of micelle solution (example 9) is dipped with a copper mesh, kept stand for 5min, sucked by filter paper, and then is negatively dyed with 2% phosphotungstic acid solution dropwise for 5min, and after natural air drying, the morphology of the micelle on the copper mesh is observed by a transmission electron microscope (H-7650, hitachi, japan).

C ₆₀ TEM images of (OH) -DSPE-PEG/DOX. HCl micelles, DSPE-PEG/DOX. HCl micelles are shown in FIGS. 22-23.

Example 11: particle size distribution, PDI, zeta potential, encapsulation Efficiency (EE) and drug Loading (LC) measurements of micelles: placing the micelle solution into a sample cell, and performing laser particle size analysis (Zetasizer Nano ZS-90, mallotus, UK) on C ₆₀ The average particle size, zeta potential and PDI of the (OH) -DSPE-PEG/DOX. HCl micelles, DSPE-PEG/DOX. HCl micelles were measured.

Standard curve preparation: reverse-phase chromatographic column: diamond TM C18 column (150 mm. Times.4.6 mm,5 μm); mobile phase: methanol-water=65:35; sample injection amount: 20. Mu.L; flow rate: column temperature 1.0m L/min: 37 ℃; detection wavelength: 233nm.

A100 mg/L DOX HCl stock solution was prepared, and an accurately weighed DOX HCl (5.0 mg) was placed in a 50mL volumetric flask, and methanol was added thereto for dissolution and dilution to a set volume. DOX & HCl series solutions with concentrations of 1, 5, 10, 20, 50mg/L were prepared: 0.1, 0.5, 1.0, 2.0, 5.0mL of the stock solution was taken and placed in a 10mL volumetric flask, diluted to a constant volume with methanol, then injected separately, and subjected to linear regression with the concentration (x) on the abscissa and the peak area (y) on the ordinate to obtain a standard curve equation, y= 77.948x-90.872, (r=0.9994).

Drug loading and encapsulation efficiency: 0.5mL of the micelle solution was added to a ultrafilter tube (MWCO 30,000, millipore), centrifuged at high speed (12000 r/min) for 10 minutes, and the filtrate was taken and the DOX. HCl concentration therein was measured by HPLC to obtain the free DOX. HCl content (W1) in the micelle solution. And (3) taking the micelle solution, adding methanol to destroy the micelle structure, adding a mobile phase to fix the volume (diluting by 20 times), and measuring the DOX and HCl concentration by an HPLC method to obtain the total DOX and HCl content (W2) in the micelle solution, wherein the mass of the carrier in the solution is W3. Encapsulation Efficiency (EE) and drug Loading (LC) were calculated as follows. Ee= (W2-W1)/w2×100%, lc= (W2-W1)/(w2+w3) ×100%.

Average particle diameter, zeta potential, PDI, encapsulation Efficiency (EE), drug Loading (LC) results were as follows

Example 11: in vitro drug Release study, C ₆₀ The in vitro release rate profiles of (OH) -DSPE-PEG/DOX. HCl micelles and DSPE-PEG/DOX. HCl micelles are shown in FIG. 24.

Standard curve preparation, 100mg/L DOX HCl stock solution: accurately weighed DOX HCl (5.0 mg) was placed in a 50mL volumetric flask, and ultrapure water was added thereto for dissolution and dilution to a set volume. DOX & HCl series solutions with concentrations of 2, 5, 10, 20, 40mg/L were prepared: 0.2, 0.5, 1.0, 2.0, 4.0mL of stock solution was extracted and placed in a 10mL volumetric flask, diluted to constant volume with ultrapure water, then the UV spectrophotometer ((SHIMADZU UV-2550) reading was recorded regression equation y= 0.02125x-0.00258 (r=0.9997), indicating that the peak area of DOX HCl has a good linear relationship at concentrations of 0-40 mg/L.

Taking C prepared in example 9 ₆₀ 1mL of (OH) -DSPE-PEG/DOX. HCl micelle or DSPE-PEG/DOX. HCl micelle was placed in a dialysis bag (MW 1000), and the dialysis bag was placed in a release medium of phosphate buffer (30 mL) having a pH of 7.4. Incubate in a 37℃water bath. The concentration of DOX. HCl released was measured by a UV-Vis spectrophotometer at predetermined time points. The drug release rate is determined by DOX & HCl (from C ₆₀ The release amount of (OH) -DSPE-PEG/DOX. HCl micelle or DSPE-PEG/DOX. HCl micelle release) divided by C ₆₀ The initial DOX HCl content of the (OH) -DSPE-PEG/DOX HCl micelle or DSPE-PEG/DOX HCl micelle is determined.

After 48 hours of incubation, C ₆₀ The cumulative release rate of (OH) -DSPE-PEG/DOX. HCl micelles was 45.1% (pH 7.4), and the cumulative release rate of DSPE-PEG/DOX. HCl micelles was 49.5% (pH 7.4).

Example 12: cell inhibition studies, in vitro cell inhibition profiles are shown in FIG. 25.

Cells were plated in 96-well plates (3X 10) ³ Cells/well, 90. Mu.l/well) were incubated for 24h, and then medium containing the drug (10. Mu.l) was added. C prepared in example 9 at free DOX. HCl and equivalent concentration ₆₀ The cells were incubated at 37℃and 5% CO in the presence of (OH) -DSPE-PEG/DOX. HCl micelles or DSPE-PEG/DOX. HCl micelles ₂ Incubation was continued for 72 hours with 3 duplicate wells per concentration.

Apoptosis was measured using the CCK-8 reduction assay. Mu.l CCK-8 was added to each well and the cells were incubated at 37℃for about 1 hour. The absorbance was measured by a microplate reader (MK-3,Thermo Fisher Scientific) at λ=450 nm. Detection of DOX. HCl, DSPE-PEG/DOX. HCl and C ₆₀ Inhibition of tumor cells and normal cells by (OH) -DSPE-PEG/DOX. HCl. The inhibition of tumor cell growth was calculated according to the following formula: inhibition = [ (OD 450 control well-OD 450 dosing well)/(OD 450 control well-OD 450 blank well)]×100％。

Cells treated with free DOX & HCl are used as a control, the inhibition ratio difference between the equivalent dose DSPE-PEG/DOX & HCl micelle and DOX & HCl is smaller, and the DSPE-PEG/DOX & HCl micelle has no advantage in-vitro tumor inhibition. While the inhibition rate of C60 (OH) -DSPE-PEG/DOX & HCl micelle on tumor cells (HepG 2, BEL-7402, SGC-7901, OE19) is slightly reduced, the inhibition rate on normal cells (L02, GES-1) is obviously reduced: l02 cells decreased significantly below 500nM concentration, and at 500nM, about 30% decrease in inhibition occurred in C60 (OH) -DSPE-PEG/DOX. HCl micelles compared to C60 (OH) -DSPE-PEG/DOX. HCl micelles and DOX; GES-1 cells were significantly reduced below 250 nM. The carrier grafted by the fullerol has relatively weak toxicity to normal cells, and plays a role in protecting the normal cells to a certain extent.

The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Fulleritol also has other potential medicinal values in the medical field, such as the Fulleritol can effectively relieve inflammatory reaction caused by mouse graft rejection (GVHD), including liver injury and intestinal injury, and reduce accumulation of leucocytes; fulleritol can reduce myocardial cell inflammation and oxidative stress caused by myocardial ischemia/reperfusion injury; fulleritol can prevent disc degeneration and treat disc degeneration by inhibiting inflammatory reaction and adipogenesis of bone marrow stromal cells of vertebrae; the fullerene alcohol nanoparticle can treat nerve inflammation of lumbago and backache; through combination with a polymer carrier, the stability of the fullerene alcohol in vivo is improved, the half-life period in vivo is prolonged, and the fullerene alcohol grafted polymer synthesized by the invention has potential of being applied to the field of pharmacy.

Fulleritol, which can prevent skin aging, has been widely used in the cosmetic field; can activate atrophic hair follicle, and can be used for caring hair. The grafted polymer has raised lipophilic performance and skin absorption capacity, and the synthesized grafted polymer may be also used in cosmetics. All of which are included in the scope of the present invention.

Claims

1. A preparation method of drug-loaded micelle is characterized in that the method is a preparation method of fullerene alcohol grafted distearoyl phosphatidylethanolamine-polyethylene glycol drug-loaded micelle, and the drug-loaded micelle is a water-soluble drug; the preparation method of the fullerene alcohol grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol drug-loaded micelle specifically comprises the following steps:

2) Dissolving a water-soluble drug in water to prepare a drug stock solution, wherein the concentration of the drug is 0.1-100mg/mL;