CN113248700A

CN113248700A - Synthesis and application of fullerol grafted polymer carrier

Info

Publication number: CN113248700A
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: 2021-08-13
Anticipated expiration: 2041-04-13
Also published as: CN113248700B

Abstract

The invention relates to synthesis of a fullerol grafted high molecular compound, a preparation method of a drug delivery system and application thereof. The synthesis of the fullerol grafted high molecular compound is that the fullerol and the high molecular compound with single amino end are subjected to addition reaction in aqueous solution (or mixed solution of water and organic solvent) to obtain a product. The synthesis condition is mild, no by-product and pollution are generated, 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 fullerol grafted distearoylphosphatidylethanolamine-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 inhibition rate of the drug-loaded micelle on normal cells is reduced to a greater extent.

Description

Synthesis and application of fullerol 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 fullerol grafted polymer carrier.

Background

Fullerenes are a series of caged spherical nano-molecules formed from carbon atoms. In recent years, fullerene has been found to have excellent biological activity, such as antioxidant action, antiviral action, antitumor and immunoregulatory action. However, fullerene cannot be dissolved in water, which hinders further application of fullerene, researchers attach polar functional groups to the structure of fullerene to increase its solubility in water, wherein the introduction of hydroxyl groups results in a fullerene alcohol (i.e., a hydroxylated fullerene) with hydrophilic properties, which has the following advantages: 1. the double bond in the structure can participate in the reactions of oxidation reduction, addition, nucleophilic, electrophilic substitution and the like, so that a series of derivatives can be obtained, and various groups can be grafted according to the requirement; 2. fullerol has protective effect on heart, liver, kidney, lung, and brain of animals, such as Fullerol C₆₀(OH)₂₄Has effects of preventing and relieving cardiotoxicity and hepatotoxicity induced by adriamycin. Fullerol C₆₀(OH)₂₄The antioxidant property of (A) can prevent the oxidative stress reaction of rat kidney, testis and lung caused by adriamycin. The fullerol can reduce mucositis during chemotherapy and restore irinotecan-induced leukopenia; 3. fullerol C₆₀(OH) X has the advantages of inhibiting tumor growth and tumor metastasis, small dosage and low toxicity. C₆₀(OH)₂₂Cancer metastasis can also be inhibited by inhibiting angiogenesis.

The drug delivery system prepared by the high molecular material is more and more valued by people, can realize the functions of slow release, targeted delivery, solubilization, attenuation and the like of the drug, and has obtained FDA approval for a plurality of high molecular anti-tumor drug delivery systems to be put on the market. The nano-scale drug delivery system comprises nanoparticles, micelles, liposomes, nano-vesicles, polymer drug composites and the like; the micron-sized drug delivery system comprises microspheres, micro-vesicles, microgels and the like; macroscopic drug delivery systems include polymeric hydrogels, implantable materials, and the like. These new dosage forms alter the traditional mode of administration; the medicine property is improved; changes the in vivo pharmacokinetic properties of the drug.

The micelle is a colloid solution which is thermodynamically stable and is formed by self-assembly of amphiphilic high molecular polymers in water. The traditional preparation method of the micelle drug delivery system comprises a direct dissolution method, a dialysis method and a self-assembly solvent evaporation method. Micellar solutions are not kinetically stable systems and are typically stored after lyophilization. The problems existing at present include unstable redissolution after freeze-drying, increase of complexity of preparation process due to addition of a stabilizer, adverse effect of drug stability due to addition of the stabilizer and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide synthesis and application of a fullerol grafted polymer carrier. According to the invention, the fullerol is grafted to the high polymer material through a mild chemical reaction, so that the drug-loading characteristic of the high polymer material is retained, the excellent physical property and physiological activity of the fullerol are increased, and the fullerol has a synergistic effect especially as an anti-tumor drug carrier. The preparation method of the fullerol grafted distearoylphosphatidylethanolamine-polyethylene glycol doxorubicin-loaded micelle is simple and mild in conditions.

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

1) dissolving fullerol in water at a concentration of 1-1000 mg/mL;

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

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

4) directly freeze-drying the reaction solution prepared in the step 3), or freeze-drying the dialyzate through dialysis to obtain the fullerol 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, C₆₀(OH) n, wherein n is an integer from 16 to 58.

In the synthesis of the fullerol grafted polymer carrier, the organic solvent is any one or combination of more of dimethyl sulfoxide, dimethyl formamide, methanol, ethanol, acetone and the like.

In the synthesis of the fullerol grafted polymer carrier, the polymer material is a natural or synthetic compound with a single amino end in the structure and comprises distearoyl phosphatidyl ethanolamine-polyethylene glycol-amino DSPE-PEG-NH₂Poly (lactide, glycolide) polyethylene glycol-amino PLGA-PEG-NH₂Polyethylene glycol-amino mPEG-NH₂Stearic acid-polyethylene glycol-amino SA-PEG-NH₂Cholesterol-polyethylene glycol-amino CLS-PEG-NH₂Aminocyclodextrin beta-CD-NH₂And the like.

In the synthesis of the fullerol grafted polymer carrier provided by the invention, functional groups such as acid-base groups including carboxyl, sulfonic group, sulfydryl, amino and the like can be introduced into the fullerol in the structure of the fullerol grafted polymer carrier; the targeting group comprises 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 fullerol grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol drug-loaded micelle synthesized based on the method, which comprises the following steps:

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

2) dissolving doxorubicin hydrochloride in water to prepare a drug stock solution, wherein the concentration of the doxorubicin hydrochloride is 2 mg/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 prepare the micelle solution.

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

1) the synthesis condition of the fullerol grafted polymer is mild, no by-product and pollution are generated, the operation is simple, and the method can be used for industrial production;

2) fullerol grafted distearoylphosphatidylethanolamine-polyethylene glycol/doxorubicin hydrochloride micelle (C)₆₀The preparation conditions of (OH) -DSPE-PEG/DOX & HCl) are mild, the operation is simple, and the shaking table of a conventional instrument can be used for realizing clinical on-site preparation. A series of problems that the micelle solution is inconvenient to store and the redissolution is difficult to prepare the freeze-drying agent are avoided;

3) preparation of C₆₀The carrier of the (OH) -DSPE-PEG/DOX & HCl micelle has a polyethylene glycol structure, so that the blood circulation time of the micelle can be prolonged, and the micelle is prevented from being cleared by reticuloendothelial systems, macrophages and the like in vivo.

4) Preparation of C₆₀The (OH) -DSPE-PEG/DOX HCl micelle has the particle size of about 300 nanometers, and can retain tumor tissues through an EPR effect;

5) preparation of C₆₀The (OH) -DSPE-PEG/DOX & HCl micelle has carrier with Fullerol structure to reduce the toxicity of the micelle medicine administrating system to normal cells.

Drawings

FIGS. 1-7 are schematic diagrams of the synthesis of fullerene alcohol grafted polymers;

FIGS. 8-14 are infrared spectra of the fullerol grafted polymer;

FIGS. 15-21 show the fullerene alcohol grafted polymers¹An H-NMR spectrum;

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

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

fig. 25 is an in vitro cytostatic profile of drug-loaded micelles.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise indicated, the reagents used in the following examples are analytical grade reagents and are commercially available from a regular channel.

Fullerols (C) used in the following examples unless otherwise specified₆₀(OH)_22-24) Purchased from Suzhou constant ball technology, Inc.;

distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH) used in the examples below, unless otherwise specified₂) (MW 2800), distearoylphosphatidylethanolamine-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) is from Yuqian medicine science and technology Limited;

distearoylphosphatidylethanolamine-polyethylene glycol-aldehyde (DSPE-PEG-CHO) (MW 5800) used in the following examples was purchased from west asian chemical technology (shandong) limited, unless otherwise specified.

Example 1: fullerol grafted distearoylphosphatidylethanolamine-polyethylene glycol (C)₆₀(OH) -DSPE-PEG) and the synthetic scheme is shown in figure 1.

Fullerol 115mg (0.1mmol), distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH)₂) (MW 2800)280mg (0.1mmol) of this compound was put into 10mL of water, heated to 50 ℃, and the reaction was stirred for 8 hours to terminate the reaction. At this time, if it is to be reversedThe reaction solution was completely dissolved by dropping the solution into ethanol, and the solution was brown. (the aqueous solution of Fullerol was dropped into ethanol, and Fullerol was completely precipitated as particles and could not be dissolved.) the reaction solution was lyophilized to obtain brown solid (3). IR (cm)^-1) 3421,2917,2884,2851,1738,1624,1467,1344,1281,1249,1110,954,843.1H-NMR: δ 3.58ppm (m),3.26ppm (d), δ 3.05ppm(s), δ 02.98ppm (m), δ 12.90ppm (d), δ 2.75ppm (m), δ 2.67ppm(s), δ 2.42ppm (d), δ 2.33ppm (m), δ 1.79ppm(s), δ 1.47ppm(s), δ 1.17ppm(s), δ 0.77 ppm(s). With the raw material DSPE-PEG-NH₂(1) And C₆₀IR spectrum of (OH) (2) and¹comparison of H-NMR spectra, C₆₀1624 in the infrared spectrum of (OH) -DSPE-PEG (3) reflects the characteristic structure of fullerol, which¹Delta 1.79ppm(s) in the H-NMR spectrum is a characteristic peak of the fullerol, and the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 8,¹the H-NMR is shown in FIG. 15.

Example 2: carboxylic-fullerol grafted distearoylphosphatidylethanolamine-polyethylene glycol ((C)₆₀(OH) (COOH) -DSPE-PEG) synthesis, scheme of synthesis is shown in FIG. 2.

C from example 1₆₀(OH) -DSPE-PEG (MW 4000)100mg (0.025mmol), and glycine 9mg (0.12mmol) were put into 10mL of water, heated to 50 ℃, and stirred for 6 hours to complete the reaction. The reaction was dialyzed 3 times (MW 500) against ultrapure water, and the dialysate was lyophilized to give brown solid (5). IR (cm)^-1) 3424,2917,2880,2850,1739,1606,1467,1381,1346,1282,1250,1108,951,843.1H-NMR: δ 3.59ppm (m), δ 3.43ppm(s),3.27ppm (d), δ 03.06ppm (m), δ 12.94ppm (m), δ 2.77ppm(s), δ 2.67ppm(s), δ 2.42ppm(s), δ 1.91ppm(s), δ 1.79ppm(s), δ 1.48ppm(s), δ 1.18ppm(s), δ 0.77 ppm(s). With the raw material (C)₆₀IR spectra and of (OH) -DSPE-PEG (3) and Glycine (4)¹Comparison of H-NMR spectra, C₆₀1606 in the infrared spectrum of (OH) (COOH) -DSPE-PEG (5) reflects the characteristic structure of the fullerol,¹δ 1.79ppm(s) in an H-NMR spectrum is a characteristic peak of the fullerol, δ 1.91ppm(s) is a characteristic peak of CH obtained by adding C (C) bond in a fullerol structure to NH2 of glycine, and δ 3.43ppm(s) is a characteristic peak of the glycine, and an infrared spectrum result is proved, wherein an infrared spectrum is shown in a figure 9，¹The H-NMR is shown in FIG. 16.

Example 3: mannose-Fullerol grafted Distearoylphosphatidylethanolamine-polyethylene glycol (Manno-C)₆₀(OH) -DSPE-PEG) and the synthetic scheme is shown in FIG. 3.

C from example 1₆₀(OH) -DSPE-PEG (MW 4000)100mg (0.025mmol) and p-aminophenyl-D-mannopyranose 20mg (0.075mmol) were put into 10mL of water, heated to 50 ℃ and stirred for 6 hours to terminate the reaction. The reaction was dialyzed 3 times (MW 500) against ultrapure water, and the dialysate was lyophilized to give brown solid (7). IR (cm)^-1):3405,2917,2851,1739,1608,1592,1515,1494,1467,1455,1345,1296,1250,1108,1003,954,850.¹H-NMR: δ 8.14ppm (d), δ 7.95ppm (d),7.17ppm (d), δ 06.90ppm (d), δ 16.70ppm (d), δ 26.41ppm (d), δ 35.64ppm(s), δ 44.07ppm(s), δ 53.93ppm (d), δ 63.58ppm (m),3.26ppm (d), δ 72.98ppm (m), δ 82.76ppm(s), δ 2.70ppm (m), δ 2.42ppm(s), δ 2.33ppm (m), δ 2.23ppm(s), δ 1.79ppm(s), δ 1.44ppm(s), δ 1.16ppm(s), δ 0.76 ppm(s). IR spectrum sum of the product with raw materials (C60(OH) -DSPE-PEG (3) and p-aminobenzo-D-mannopyranose (6))¹H-NMR spectrum comparison, 1608 in the Manno-C60(OH) -DSPE-PEG (7) infrared spectrum reflects the characteristic structure of fullerol, 1592,1515,1494 reflects the characteristic structure of p-aminobenzene-D-mannopyranose,¹delta 1.79ppm(s) in an H-NMR spectrum is a characteristic peak of the fullerol, delta 8.14ppm (D),7.17ppm (D), delta 6.70ppm (D), delta 6.41ppm (D), delta 5.64ppm(s) are characteristic peaks of the p-aminobenzene-D-mannopyranose, and the result of an infrared spectrum is proved, and an infrared spectrogram is shown in figure 10,¹the H-NMR is shown in FIG. 17.

Example 4: fullerol grafted (amide bond connected) distearoylphosphatidylethanolamine-polyethylene glycol ((C)₆₀(OH) - (CONH) -DSPE-PEG) synthesis, scheme of synthesis is shown in FIG. 4.

100mg (0.035mmol) of distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl group (DSPE-PEG-COOH) (MW 2850), 19mg (0.1mmol) of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), and 11mg (0.1mmol) of N-hydroxysuccinimide (NHS) were put into 4ml of a total of DMMSO, dissolved by ultrasound, and reacted for 3 hours with stirring at normal temperature. Adding into the reaction solutionEthylenediamine 4mg (0.07mmol)20uL was stirred at room temperature overnight. Dialyzing the reaction solution in ultrapure water for 3 times (MW 500), and lyophilizing the dialyzate to obtain a white solid which is distearoylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-CONH-C)₂H₄NH₂)(9)。IR(cm^-1):3382,2918,2889,2851,2741,2695,1741,1544,1467,1360,1343,1281,1242,1149,1111,1061,963,843.¹H-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)。

Fullerol 40mg (0.035mmol), DSPE-PEG-CONH-C₂H₄NH₂(MW 2900) (0.035mmol), 100mg (0.035mmol) and 10mL of water were put into the flask, and the mixture was heated to 50 ℃ and stirred for 8 hours to terminate the reaction. At this time, when the reaction solution was dropped into ethanol, the reaction solution was completely dissolved and the solution was brown. The reaction was lyophilized to give a brown solid (10). IR (cm)^-1):3420,2917,2888,2851,1738,1594,1467,1360,1343,1280,1242,1147,1112,1062,963,843.¹H-NMR: δ 5.16ppm(s),4.08ppm(s), δ 3.58ppm (m),3.47ppm(s),3.24ppm(s), δ 2.60ppm(s), δ 2.52ppm(s), δ 2.21ppm (m), δ 1.79ppm(s), δ 1.48ppm(s), δ 1.18ppm(s), δ 0.78 ppm(s). With the raw material (C)₆₀(OH)(2)、DSPE-PEG-COOH(8)、DSPE-PEG-CONH-C₂H₄NH₂(9) IR spectrum of) and¹comparison of H-NMR spectra, C₆₀1594,1360 in the infrared spectrum of (OH) - (CONH) -DSPE-PEG (10) reflects the characteristic structure of the fullerol,¹delta 1.79ppm(s) in the H-NMR spectrum is a characteristic peak of the fullerol, and the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 11,¹the H-NMR is shown in FIG. 18.

Example 5: fullerol grafted (hydrazone bond linked) distearoylphosphatidylethanolamine-polyethylene glycol ((C)₆₀(OH) - (CONHN ═ C) -DSPE-PEG) synthesis, scheme for synthesis is shown in fig. 5.

Adding 100mg (0.017mmol) of distearoylphosphatidylethanolamine-polyethylene glycol-aldehyde (DSPE-PEG-CHO) (MW 5800) into 2mL of water, adding 20mg (0.17mmol) of oxalyl dihydrazide into 2mL of MSO, ultrasonic dissolving, mixing, and mixing1 drop of glacial acetic acid is added into the solution, and the reaction is stirred at room temperature for 48 hours. Adding 4mL of water into the reaction solution, filtering with a filter membrane (0.45um), dialyzing the filtrate in ultrapure water for 3 times (MW 500), and lyophilizing the dialysate to obtain 92mg of white solid which is distearoylphosphatidylethanolamine-polyethylene glycol-hydrazino (DSPE-PEG-C NNH-COCONHNH)₂)(12)。IR:3421,3287,2887,2741,2695,1688,1657,1540,1467,1413,1360,1343,1281,1242,1149,1112,1061,963,843.¹H-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)。

Fullerol 20mg (0.017mmol), DSPE-PEG-C ═ NNH-COCONHNH₂(MW 5900)100mg (0.017mmol) was poured into 10mL of water, heated to 50 ℃ and stirred for 8 hours to terminate the reaction. At this time, when the reaction solution was dropped into ethanol, the reaction solution was completely dissolved and the solution was brown. The reaction was lyophilized to give a brown solid (13). IR (cm)^-1):3432,2886,1637,1602,1467,1415,1360,1343,1281,1242,1148,1113,1061,963,843.¹H-NMR: δ 7.81ppm (d),7.76ppm (d), δ 3.97ppm (m), δ 3.58ppm (m),3.20ppm(s), δ 2.89ppm(s), δ 1.79ppm(s), δ 1.15ppm (m), δ 0.76 ppm(s). And the raw material (C60(OH) (2), DSPE-PEG-CHO (11), DSPE-PEG-C ═ NNH-COCONHNH₂(12) IR spectrum of) and¹H-NMR spectrum comparison, 1602,1360 in the infrared spectrum of C60(OH) - (CONHN ═ C) -DSPE-PEG (13) reflects the characteristic structure of fullerol, which is¹Delta 1.79ppm(s) in the H-NMR spectrum is a characteristic peak of the fullerol, and the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 12,¹the H-NMR is shown in FIG. 19.

Example 6: fullerol grafted polyethylene glycol (C)₆₀(OH) -PEG) Synthesis, scheme for the synthesis is shown in FIG. 6.

Fullerol 115mg (0.1mmol), methoxy-polyethylene glycol-amino (mPEG-NH)₂) (MW 2000)200mg (0.1mmol) of water was poured into 10mL of water, heated to 50 ℃, and the reaction was stirred for 8 hours to terminate the reaction. At this time, when the reaction solution was dropped into ethanol, the reaction solution was completely dissolved and the solution was brown. The reaction was lyophilized to give a brown solid (15). IR (cm)^-1):3421,2887,2741,2695,1716,1625,1467,1360,1343,1280,1242,1147,1113,1061,964,843.¹H-NMR: δ 3.79ppm (t), δ 3.62ppm (m),3.41ppm(s), δ 1.79ppm(s), δ 1.09ppm (d). With the raw material (C)₆₀(OH)(2)、mPEG-NH₂(14) IR spectrum of) and¹comparison of H-NMR spectra, C₆₀1625 in the infrared spectrum of (OH) -PEG (15) reflects the characteristic structure of fullerol, which¹Delta 1.79ppm(s) in the H-NMR spectrum is a characteristic peak of the fullerol, and the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 13,¹the H-NMR is shown in FIG. 20.

Example 7: fullerol grafted poly (lactide, glycolide) -polyethylene glycol ((C)₆₀(OH) -PLGA-PEG) synthesis, scheme for synthesis is shown in FIG. 7.

Fullerol 11.5mg (0.01mmol), poly (lactide, glycolide) -polyethylene glycol-amino (PLGA-PEG-NH)₂) (MW 20000)200mg (0.01mmol) was put into 10mL of water, heated to 50 ℃ and stirred for 8 hours to terminate the reaction. At this time, when the reaction solution was dropped into ethanol, the reaction solution was completely dissolved and the solution was brown. The reaction was lyophilized to give a brown solid (17). IR (cm)^-1):3421,2995,2945,2883,1759,1613,1455,1424,1383,1360,1345,1278,1188,1097,953,843.¹H-NMR: δ 5.98ppm (m),5.20ppm (m), δ 4.91ppm(s), δ 04.52ppm (m), δ 14.14ppm (dd), δ 24.06ppm (m), δ 33.79ppm(s), δ 3.51ppm (m),3.35ppm (m), δ 1.79ppm(s), δ 1.46ppm(s), δ 1.33ppm (m), δ 1.27ppm (m), δ 1.18ppm (m), δ 0.83ppm (t), δ 0.77ppm (t). With the raw material (C)₆₀(OH)(2)、PLGA-PEG-NH₂(16) IR spectrum of) and¹comparison of H-NMR spectra, C₆₀1613,1345 in the infrared spectrum of (OH) -PLGA-PEG (17) reflects the characteristic structure of fullerol, which¹Delta 1.79ppm(s) in the H-NMR spectrum is a characteristic peak of the fullerol, and the result of the infrared spectrum is proved, the infrared spectrum is shown in figure 14,¹the H-NMR is shown in FIG. 21.

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

Preparation C₆₀(OH) -DSPE-PEG stock solution 5mL (10mg/mL), C was weighed₆₀(OH) -DSPE-PEG 50mg, 5mLDissolving the mixture in ultrapure water by ultrasonic wave for later use.

5mL (10mg/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.

Preparing 5mL (2mg/mL) of DOX & HCl stock solution, weighing 10mg of DOX & HCl, putting into 5mL of ultrapure water, and ultrasonically dissolving for later use.

Get C₆₀(OH) -DSPE-PEG stock solution and DOX & HCl stock solution were each 2mL, mixed, heated to 60 ℃ and stirred slowly for 30 minutes. Cooling, filtering with filter membrane (0.22um), and storing the filtrate at 4 deg.C.

Taking 2mL of DSPE-PEG stock solution and DOX & HCl stock solution respectively, mixing, heating to 60 ℃, and slowly stirring for 30 minutes. Cooling, filtering with filter membrane (0.22um), and storing the filtrate at 4 deg.C.

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

Preparation C₆₀(OH) -DSPE-PEG stock solution 5mL (20mg/mL), C was weighed₆₀(OH) -DSPE-PEG 100mg, put into 5mL of ultrapure water, and ultrasonically dissolved for later use.

5mL (20mg/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 suitable amount of the micelle solution (example 9) was taken out of the copper mesh, left to stand for 5min, blotted dry with filter paper, negatively stained with a 2% phosphotungstic acid solution dropwise for 5min, and after air-dried naturally, the morphology of the micelles on the copper mesh was observed by a transmission electron microscope (H-7650, Hitachi, Japan).

C₆₀TEM images of (OH) -DSPE-PEG/DOX & HCl micelles and 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 subjecting to C with a laser particle size analyzer (Zetasizer Nano ZS-90, Marvin, UK)₆₀(OH) -DSPE-PEG/DOX & HCl micelles, average particle size, Zeta potential and PDI of the DSPE-PEG/DOX & HCl micelles were measured.

Preparation of a standard curve: reverse phase chromatographic column: diamonsil TM C18 column (150 mm. times.4.6 mm,5 μm); mobile phase: methanol-water 65: 35; sample introduction amount: 20 mu L of the solution; flow rate: 1.0m L/min column temperature: 37 ℃; detection wavelength: 233 nm.

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

Drug loading and encapsulation efficiency: 0.5mL of the micelle solution was put into an ultrafiltration tube (MWCO 30,000, Millipore), centrifuged at high speed (12000r/min) for 10 minutes, and the filtrate was taken to measure the concentration of DOX & HCl therein by HPLC, whereby the content of free DOX & HCl in the micelle solution (W1) was obtained. And adding methanol into the micelle solution, destroying the micelle structure, adding a mobile phase to a constant volume (diluted by 20 times), and measuring the concentration of DOX & HCl by an HPLC method to obtain the total content (W2) of DOX & HCl in the micelle solution, wherein the mass of the carrier in the solution is W3. The Entrapment Efficiency (EE) and drug Loading (LC) were calculated according to the following formulas. EE ═ W2-W1)/W2 × 100%, LC ═ W2-W1)/(W2 + W3) × 100%.

The results of the average particle diameter, Zeta potential, PDI, Encapsulation Efficiency (EE) and drug Loading (LC) are as follows

Example 11: in vitro drug Release study, C₆₀The 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: an accurately weighed amount of DOX & HCl (5.0mg) was put into a 50mL volumetric flask, to which ultrapure water was added to dissolve and dilute to a set volume. DOX & HCl series solutions were prepared at concentrations of 2, 5, 10, 20, 40 mg/L: stock solutions of 0.2, 0.5, 1.0, 2.0, 4.0mL were extracted and placed into 10mL volumetric flasks, diluted to constant volume with ultra pure water and then UV spectrophotometer ((SHIMADZU UV-2550) readings are recorded with a regression equation of y-0.02125 x-0.00258 (r-0.9997) indicating that the peak area of DOX-HCl has a good linearity at concentrations of 0-40 mg/L.

Taking C prepared in example 9₆₀1mL of (OH) -DSPE-PEG/DOX & HCl micelles or DSPE-PEG/DOX & HCl micelles was placed in a dialysis bag (MW 1000) which was placed in a release medium of pH 7.4 phosphate buffer (30 mL). Incubate in 37 ℃ water bath. The concentration of released DOX · HCl was measured by a UV-Vis spectrophotometer at a predetermined time point. The release rate of the drug is determined by DOX & HCl (from C) in a certain time₆₀(OH) -DSPE-PEG/DOX & HCl micelle Release or DSPE-PEG/DOX & HCl micelle Release) divided by C₆₀(OH) -initial DOX & HCl content of DSPE-PEG/DOX & HCl micelles or DSPE-PEG/DOX & HCl micelles is determined.

After 48 hours of incubation, C₆₀The 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: the cytostatic rate study and the in vitro cytostatic rate map are shown in FIG. 25.

Cells were plated in 96-well plates (3X 10)³Cells/well, 90 μ Ι/well) for 24h, and then drug-containing medium (10 μ Ι) is added. In free DOX & HCl and the same concentration of C as prepared in example 9₆₀(OH) -DSPE-PEG/DOX & HCl micelles orCells were incubated at 37 ℃ and 5% CO in the presence of DSPE-PEG/DOX & HCl micelles₂Incubation was continued for 72 hours with 3 replicates per concentration.

Apoptosis was determined by 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 at λ 450nm by a microplate reader (MK-3, Thermo Fisher Scientific). Detection of DOX & HCl, DSPE-PEG/DOX & HCl and C₆₀(OH) -DSPE-PEG/DOX & HCl has inhibitory effect on tumor cells and normal cells. The inhibition rate of tumor cell growth was calculated according to the following formula: inhibition rate ═ [ (OD450 control well-OD 450 administration well)/(OD 450 control well-OD 450 blank well)]×100％。

Compared with cells treated by free DOX & HCl, the inhibition rate difference of the DSPE-PEG/DOX & HCl micelle and DOX & HCl of equivalent dose is small, and the DSPE-PEG/DOX & HCl micelle has no advantage in-vitro tumor inhibition. The inhibition rate of the C60(OH) -DSPE-PEG/DOX & HCl micelle on tumor cells (HepG2, BEL-7402, SGC-7901 and OE19) is slightly reduced, but the inhibition rate on normal cells (L02 and GES-1) is obviously reduced: the L02 cells were significantly reduced below 500nM concentration, with about a 30% reduction in inhibition at 500nM for C60(OH) -DSPE-PEG/DOX HCl micelles compared to C60(OH) -DSPE-PEG/DOX HCl micelles and DOX; GES-1 cells show a significant decrease below 250nM concentration. The carrier grafted by the fullerol is relatively weak in toxicity to normal cells, and plays a role in protecting the normal cells to a certain extent.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

The fullerol has other potential medicinal values in the field of medicine, for example, the fullerol can effectively relieve inflammatory reaction of mouse graft rejection (GVHD), including liver injury and intestinal injury and reduce the accumulation of white blood cells; the fullerol can reduce inflammation and oxidative stress of myocardial cells caused by myocardial ischemia/reperfusion injury; fullerol can prevent intervertebral disc degeneration, and treat intervertebral disc degeneration by inhibiting inflammatory reaction of vertebra bone marrow stromal cells and lipogenesis; the fullerol nanoparticles can be used for treating neuroinflammation of lumbago and backache; through the combination with the polymer carrier, the stability of the fullerol in the body is improved, the half-life period in the body is prolonged, and the fullerol grafted polymer synthesized by the method has the potential of being applied to the field of pharmacy.

Fullerols are used for preventing skin aging and have been widely used in the field of cosmetics; can activate atrophic hair follicle for caring hair. The grafting polymer greatly improves the lipophilicity of the fullerol, improves the skin absorption capacity, and the synthesized fullerol grafting polymer can also be applied to the field of cosmetics. The above are all included in the scope of the present invention.

Claims

1. The synthesis of the fullerol grafted polymer carrier is characterized by comprising the following steps:

1) dissolving fullerol in water at a concentration of 1-1000 mg/mL;

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

2. The synthesis of a fullerol grafted polymer carrier as claimed in claim 1, wherein in step 1) the fullerol is a hydroxylated derivative of fullerene, C₆₀(OH) n, wherein n is an integer from 16 to 58.

3. The synthesis of a fullerol grafted polymer carrier according to claim 1, wherein the organic solvent is any one or more of dimethyl sulfoxide, dimethyl formamide, methanol, ethanol, acetone.

4. The synthesis of a fullerol grafted polymeric carrier as claimed in claim 1, wherein said polymeric material is a natural or synthetic compound having a single amino terminus in its structure.

5. The synthesis of a fullerol grafted polymer carrier according to claim 1, wherein the fullerol in the structure of the fullerol grafted polymer carrier is fullerol or fullerol introduced with a functional group, the functional group is an acidic group or a targeting group, and the acidic group comprises any one of carboxyl, sulfonic acid, sulfhydryl and amino; the targeting group comprises any one of folic acid, biotin, asialo, lactose, galactose, mannose, glucose, glycyrrhetinic acid and nano magnetic beads.

6. A drug delivery system prepared based on the fullerol grafted polymeric carrier of any one of claims 1-5, comprising micelles, liposomes, nanoparticles, nanovesicles or microspheres.

7. A preparation method of a drug-loaded micelle is characterized in that the method is a preparation method of a fullerol grafted distearoyl phosphatidyl ethanolamine-polyethylene glycol drug-loaded micelle, and the drug-loaded micelle is a water-soluble drug.

8. The preparation method of the drug-loaded micelle of claim 7, wherein the preparation method of the fullerol grafted distearoylphosphatidylethanolamine-polyethylene glycol drug-loaded micelle 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 0.1-100 mg/mL;

9. The application of the fullerene alcohol grafted polymer carrier drug delivery system in tumor treatment and normal cell toxicity reduction.

10. The application of fullerol grafted polymer in cosmetics and eliminating inflammation of body is provided.