CN108578705B - Folic acid grafted fullerol adriamycin-loaded prodrug and preparation method and application thereof - Google Patents
Folic acid grafted fullerol adriamycin-loaded prodrug and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a folic acid grafted fullerol doxorubicin-loaded prodrug, which is formed by combining 1 molecule of doxorubicin with 1 molecule of fullerol introduced with a hydrazide group through a hydrazone bond and 1 molecule of folic acid through an amide bond. The invention also provides a preparation method and application of the folic acid grafted fullerol doxorubicin-loaded prodrug. The folic acid grafted fullerol doxorubicin-loaded prodrug takes water-soluble fullerol as a drug carrier, introduces a small molecular hydrazide group into the structure, is connected with folic acid through an amido bond and is connected with doxorubicin through a hydrazone bond to form the folic acid grafted fullerol doxorubicin-loaded prodrug, is mediated and phagocytized by a folic acid receptor of a tumor cell membrane after reaching a tumor tissue, is dissociated by the hydrazone bond through a weakly acidic environment of a cell endosome, releases a drug and the carrier, has small toxic and side effects, can realize targeted therapy and has good curative effect.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the field of pharmaceutical chemicals, and particularly relates to a folic acid grafted fullerol doxorubicin-loaded prodrug, and a preparation method and application thereof.
[ background of the invention ]
Doxorubicin (DOX), also called Doxorubicin, hydroxyrubicin or hydroxydaunorubicin. Adriamycin is an antitumor antibiotic, can inhibit the synthesis of RNA and DNA, has the action mechanism that DOX is embedded into DNA/RNA to inhibit the synthesis of nucleic acid, has stronger inhibition effect on RNA, has wider antitumor spectrum, belongs to a periodic nonspecific medicament, and has killing effect on tumor cells in various growth periods. The traditional Chinese medicine composition is clinically used for treating acute lymphocytic leukemia, acute myelocytic leukemia, malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing's sarcoma, wilms' tumor, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous cell carcinoma, testicular cancer, gastric cancer, liver cancer and the like. Doxorubicin is a broad-spectrum antitumor agent, can produce a wide range of biochemical effects on the body, has a strong cytotoxic effect, and also has high cytotoxicity on normal tissue cells, so doxorubicin has the following side effects and disadvantages for treating cancer:
1) common alopecia, bone marrow depression, oral ulcers, anorexia, nausea or even vomiting;
2) after a few patients inject the product, the original radiation field can show skin redness or pigmentation;
3) when the product is applied to patients with leukemia and malignant lymphoma, especially the patients who use the product for the first time can cause hyperuricemia due to massive destruction of tumor cells, so that joint pain or renal function damage can be caused;
4) the product has cardiotoxicity, and can cause delayed severe heart failure.
Fullerene is a series of cage-shaped and spherical nano molecules formed by carbon atoms, can be widely applied to various fields, and currently, fullerene and derivatives thereof are more and more concerned in the field of pharmacy because of the fact that the fullerene and the derivatives have the effects of resisting oxidation and viruses and also have the effects of resisting tumors and regulating immunity. For example, some hydroxylated C60 and C60-malonic acid react very readily with free radicals in the body, which, by being likened to a sponge that absorbs free radicals, protects cells from damage caused by oxidative stress. Under high concentration and ultraviolet irradiation, the C60 water-soluble derivative can generate singlet active oxygen to cause tumor cell death without obvious damage to normal cells. C60 has been proved to have anti-tumor activity and immunoregulation property under non-lighting condition, for example, the discovery of Fuller C (60) O (x) H (y) (y is more than 10 and less than 50) and carboxylic fullerene has the advantages of inhibiting tumor growth and tumor metastasis, and has small dosage and low toxicity. In addition, the C60 fullerol and the derivatives thereof have anti-hepatoma cell activity in vivo and in vitro, and the fullerol C60(OH) X can obviously activate macrophages and inhibit the growth of tumors. In addition, it was found that the fullerol C60(OH)20 can reduce the expression of angiogenesis factor in tumor tissue, and has the function of resisting tumor metastasis. In addition, the C60 molecule has excellent chemical modifiability, and because the double bond in the structure can generate Diels-Alder reaction (Diels-Alder reaction), Bingel (Bingel) reaction and the like, a series of derivatives can be conveniently prepared, and the C60 molecule is an ideal matrix for drug design and can be connected with various groups as required.
The folate receptor is a membrane glycoprotein linked with glycosylated phosphatidyl alcohol, and is highly expressed in most malignant tumors, such as ovarian cancer, breast cancer, brain cancer, lung cancer, and the like. The folate receptor is 100-fold higher than normal tissues by 300-fold, and can specifically bind folate and analogs thereof, and take it into the cytosol by mediating endocytosis. The folic acid is introduced into the structure of the prodrug, the folic acid receptor can be used as an action target, and the prodrug can be actively targeted and taken into tumor cells, so that the distribution of the drug in the tumor cells is improved.
[ summary of the invention ]
In order to solve the defect that the existing adriamycin as a broad-spectrum antitumor drug has large toxic and side effects, the invention provides a preparation method and application of a folic acid grafted fullerol adriamycin-loaded prodrug and a folic acid grafted fullerol-loaded adriamycin prodrug which have small toxic and side effects, can be used for targeted therapy and have good curative effect.
The invention provides a folic acid grafted fullerol doxorubicin-loaded prodrug, which is formed by combining 1 molecule of doxorubicin, which is obtained by introducing 1 molecule of fullerol into a hydrazide group, with 1 molecule of doxorubicin through a hydrazone bond and 1 molecule of folic acid through an amide bond, and has the following structural formula:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6.
The invention also provides a preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug, which comprises the following steps:
(1) in solvent water, carrying out addition reaction on fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(2) reacting folic acid with N-hydroxysuccinimide NHS to generate folic acid succinimide ester by using carbodiimide EDC as a catalyst;
(3) carrying out acylation reaction on the hydrazino fullerol generated in the step (1) and the folic acid succinimidyl ester generated in the step (2) to generate folic acid grafted hydrazino fullerol, wherein the structural formula of the folic acid grafted hydrazino fullerol is as follows:
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(4) and (4) carrying out condensation reaction on the adriamycin and the folic acid grafted hydrazino fullerol generated in the step (3) to generate the folic acid grafted fullerol doxorubicin-loaded prodrug.
In a preferred embodiment of the preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention, the step (1) is specifically as follows: dissolving fullerol in solvent water, adding oxalyl dihydrazide, stirring and reacting for 3-7 days at 25-50 ℃, dialyzing by taking water as a solvent, and freeze-drying to obtain a dark brown chromogenic solid, namely the hydrazino fullerol.
In a preferred embodiment of the preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention, the step (2) is specifically as follows: dissolving folic acid FA in dimethyl sulfoxide DMSO (dimethyl sulfoxide), adding N-hydroxysuccinimide NHS and carbodiimide EDC, and reacting at room temperature in a dark place under the protection of inert gas to obtain folic acid succinimide ester.
In a preferred embodiment of the preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention, the step (3) is specifically as follows: dissolving the hydrazino fullerol prepared in the step (1) in a mixed solvent consisting of water and dimethyl sulfoxide (DMSO), adding the folic acid succinimide ester prepared in the step (2), stirring and reacting for 4-8 hours at room temperature in a dark condition to prepare the folic acid grafted hydrazino fullerol, wherein the mass ratio of the hydrazino fullerol to the folic acid succinimide ester is 1: 1.
in a preferred embodiment of the preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention, the step (4) is specifically as follows: dissolving adriamycin in a dimethyl sulfoxide (DMSO) solvent, adding the dissolved adriamycin into the folic acid grafted hydrazino fullerol prepared in the step (3), stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating, and carrying out suction filtration to obtain a reddish brown solid, namely the folic acid grafted fullerol supported adriamycin prodrug, wherein the mass ratio of the adriamycin to the folic acid grafted hydrazino fullerol is (1-1.3): 1.
in a preferred embodiment of the preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention, the step (4) is specifically as follows: adding doxorubicin hydrochloride into the folic acid grafted hydrazino fullerol prepared in the step (3), stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating and filtering to obtain a reddish brown solid, namely the folic acid grafted fullerol supported doxorubicin prodrug, wherein the mass ratio of the doxorubicin to the folic acid grafted hydrazino fullerol is (1-1.3): 1.
the invention also discloses a preparation method of the folic acid grafted fullerol doxorubicin-loaded prodrug, which comprises the following steps:
(1) in solvent water, carrying out addition reaction on fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(2) condensing doxorubicin and hydrazino fullerol generated in the step (1) to obtain a prodrug of the fullerol carried with the doxorubicin;
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(3) reacting folic acid with N-hydroxysuccinimide NHS to generate folic acid succinimide ester by using carbodiimide EDC as a catalyst;
(4) and (3) carrying out acylation reaction on the fullerene alcohol-loaded adriamycin prodrug generated in the step (2) and the folic acid succinimide ester generated in the step (3) to generate the folic acid grafted fullerene alcohol-loaded adriamycin prodrug.
The invention also provides application of the folic acid grafted fullerol doxorubicin-loaded prodrug in preparation of antitumor drugs.
Compared with the prior art, the folic acid grafted fullerol doxorubicin-loaded prodrug and the preparation method and application thereof provided by the invention have the following beneficial effects:
the folic acid grafted fullerol doxorubicin-loaded prodrug takes water-soluble fullerol as a drug carrier, a small molecule hydrazide group is introduced into the structure, the folic acid is connected with the fullerene through an amido bond and is connected with the doxorubicin through a hydrazone bond to form the prodrug, after the prodrug reaches tumor tissues, the folate receptor mediated phagocytosis of tumor cell membranes is utilized, the hydrazone bond is dissociated by utilizing the weak acid environment of a cell endosome, and the drug and the carrier are released.
Secondly, the folic acid grafted fullerol doxorubicin-loaded prodrug has the advantages of simple molecular structure, easy synthesis, mild reaction conditions and simple and convenient operation method.
And thirdly, grafting folic acid to enable the adriamycin prodrug to have tumor targeting, and combining with folic acid receptors on the surfaces of tumor cells, the adriamycin prodrug can be actively mediated to enter the tumor cells rich in folic acid receptors.
The hydrazone bond mediated medicine in tumor cells has a quick release function, and can trigger the hydrolysis of the acid sensitive hydrazone bond under the condition of the pH of an endosome, so that the medicine can be quickly released in the tumor cells, and the curative effect is good.
And fifthly, the carrier fullerol has good water solubility, can improve the solubility of the prodrug molecules, has an anti-tumor effect and can play a synergistic effect with adriamycin.
[ description of the drawings ]
Fig. 1 is a flowchart illustrating steps of a method for preparing a folic acid grafted fullerene alcohol doxorubicin-loaded prodrug according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a synthesis reaction route of a folic acid grafted fullerol doxorubicin-loaded prodrug according to an embodiment of the present invention;
FIG. 3 is a flowchart illustrating the steps of a method for preparing a folic acid grafted fullerol doxorubicin-loaded prodrug according to example two of the present invention;
FIG. 4 is a schematic diagram of a synthesis reaction route of a folic acid grafted fullerol doxorubicin-loaded prodrug provided in example two of the present invention;
FIG. 5 is an infrared spectrum obtained by detecting raw materials of fullerol, folic acid and doxorubicin and the product folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example III by Fourier infrared spectroscopy;
FIG. 6 is a 1H-NMR spectrum obtained by detecting raw materials of fullerol, folic acid and doxorubicin and the product folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example III by using a 1H nuclear magnetic resonance method;
FIG. 7 is a DSC (differential scanning calorimetry) spectrum obtained by detecting raw materials of fullerol, folic acid and doxorubicin and a product of folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example three;
FIG. 8 is a graph of in vitro dissolution curves of folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example three of the present invention in PBS solution under different pH conditions.
[ detailed description ] embodiments
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.
The invention provides a folic acid grafted fullerol doxorubicin-loaded prodrug, which is formed by combining 1 molecule of doxorubicin introduced with 1 molecule of fullerol with hydrazide groups through hydrazone bonds and 1 molecule of folic acid through amide bonds, and has the following structural formula:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6.
The folic acid grafted fullerol doxorubicin-loaded prodrug is formed by taking water-soluble fullerol as a drug carrier, introducing a small molecular hydrazide group into the structure, connecting the small molecular hydrazide group with folic acid through an amide bond, and connecting the small molecular hydrazide group with doxorubicin through a hydrazone bond, wherein the hydrazone bond is formed by condensation of a hydrazine group and a carbonyl group in doxorubicin, and the amide bond is formed by reaction of the hydrazine group and an activated carboxyl group in a folic acid structure. The adriamycin prodrug has tumor targeting property by grafting folic acid, can actively mediate the prodrug to enter tumor cells rich in the folic acid receptors by combining with the folic acid receptors on the surfaces of the tumor cells, has a quick release function on the drug in the tumor cells mediated by hydrazone bonds, can trigger the hydrolysis of the acid sensitive hydrazone bonds under the condition of the pH of an endosome, and can be quickly released in the tumor cells, so that the curative effect is good.
Example one
Referring to fig. 1 and 2, the present invention provides a method for preparing a folic acid grafted fullerol doxorubicin-loaded prodrug, which comprises the following steps:
s11, in solvent water, carrying out addition reaction on the fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
specifically, the method comprises the following steps: dissolving fullerol in solvent water, adding oxalyl dihydrazide, stirring and reacting for 3-7 days at 25-50 ℃, dialyzing by taking water as a solvent, and freeze-drying to obtain a dark brown chromogenic solid, namely the hydrazino fullerol.
S12, reacting folic acid with N-hydroxysuccinimide NHS and carbodiimide EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide for short) to generate folic acid succinimide ester; wherein, the carbodiimide EDC is used as a catalyst;
specifically, the method comprises the following steps: dissolving folic acid FA in dimethyl sulfoxide DMSO (dimethyl sulfoxide), adding N-hydroxysuccinimide NHS and carbodiimide EDC, and reacting at room temperature in a dark place under the protection of inert gas to obtain folic acid succinimide ester.
S13, carrying out acylation reaction on the hydrazino fullerol generated in the step S11 and the folic acid succinimide ester generated in the step S12 to generate folic acid grafted hydrazino fullerol, wherein the structural formula of the folic acid grafted hydrazino fullerol is as follows:
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
specifically, the method comprises the following steps: dissolving the hydrazino fullerol prepared in the step S11 in a mixed solvent composed of water and dimethyl sulfoxide (DMSO), adding the folic acid succinimide ester prepared in the step S12, stirring and reacting for 4-8 hours at room temperature in a dark condition to prepare the folic acid grafted hydrazino fullerol, wherein the mass ratio of the hydrazino fullerol to the folic acid succinimide ester is 1: 1, the volume ratio of the mixed solvent water to the dimethyl sulfoxide DMSO is 1: 1.
it should be noted that, in the specific operation process, the end point of the reaction can also be determined by TLC analysis, specifically: the developing agent is isopropanol: ammonia water: when the reaction solution of step S13 was observed by TLC method using 9:1:2 (or another developing solvent) as water and the folic acid succinimide ester produced in step S12 as a control, the reaction was terminated when the TLC plate layer showed that the free folic acid succinimide ester in the reaction solution was substantially disappeared.
S14, condensing the adriamycin or the adriamycin hydrochloride with the folic acid grafted hydrazino fullerol generated in the step S13 to generate the folic acid grafted fullerol adriamycin-carried prodrug.
Specifically, the method comprises the following steps: when adriamycin is used as a raw material, dissolving adriamycin in a dimethyl sulfoxide (DMSO) solvent, adding the dissolved adriamycin into the folic acid grafted hydrazino fullerol prepared in the step S13, stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating and filtering to obtain a reddish brown solid, namely, the folic acid grafted fullerol supported adriamycin prodrug, wherein the mass ratio of the adriamycin to the folic acid grafted hydrazino fullerol is (1-1.3): 1; when doxorubicin hydrochloride is used as a raw material, directly adding doxorubicin hydrochloride into the folic acid grafted hydrazino-fullerene alcohol prepared in the step S13, stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating, and carrying out suction filtration to obtain a reddish brown solid, namely the folic acid grafted fullerene alcohol-loaded doxorubicin prodrug, wherein the mass ratio of the doxorubicin to the folic acid grafted hydrazino-fullerene alcohol is (1-1.3): 1.
it should be noted that steps S11 and S12 are not in sequence in the present application, and may be performed simultaneously in the specific preparation process, and the preparation of hydrazino fullerol and the preparation of folic acid succinimidyl ester are not related to each other.
Example two
Referring to fig. 3 and 4, the present invention further provides a method for preparing a folic acid grafted fullerene alcohol doxorubicin-loaded prodrug, which is different from the first embodiment in that after a small-molecule hydrazide group is introduced into fullerene, the fullerene alcohol is firstly subjected to a condensation reaction with doxorubicin to obtain a fullerene alcohol-loaded doxorubicin prodrug, and then the fullerene alcohol-loaded doxorubicin prodrug is subjected to an acylation reaction with folic acid succinimide ester to generate a folic acid grafted fullerene alcohol doxorubicin-loaded prodrug, that is, when the fullerene alcohol introduced with the small-molecule hydrazide group is connected with folic acid and doxorubicin, reaction sequences are different.
The method comprises the following steps:
s21, in solvent water, carrying out addition reaction on the fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
specifically, the method comprises the following steps: dissolving fullerol in solvent water, adding oxalyl dihydrazide, stirring and reacting for 3-7 days at 25-50 ℃, dialyzing by taking water as a solvent, and freeze-drying to obtain a dark brown chromogenic solid, namely the hydrazino fullerol.
S22, condensing doxorubicin or doxorubicin hydrochloride with the hydrazino-fullerol generated in the step S21 to obtain a prodrug of the doxorubicin-loaded fullerol;
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
specifically, the method comprises the following steps: when adriamycin is used as a raw material, firstly, adriamycin is dissolved in dimethyl sulfoxide (DMSO) serving as a solvent to prepare an adriamycin solution, then, the hydrazino-fullerene alcohol prepared in the step S21 is dissolved in a mixed solvent of water and DMSO to obtain a hydrazino-fullerene alcohol solution, the adriamycin solution is put into the hydrazino-fullerene alcohol solution, glacial acetic acid serves as a catalyst, the mixture is stirred and reacts for 3-4 days at room temperature in a dark condition, and the adriamycin prodrug carried by the fullerol is prepared, wherein the mass ratio of the hydrazino-fullerene alcohol to the adriamycin is 1: (1-1.3); when doxorubicin hydrochloride is used as a raw material, firstly, the hydrazino-fullerol prepared in the step S21 is dissolved in a mixed solvent of water and DMSO to obtain a hydrazino-fullerol solution, then doxorubicin hydrochloride is directly added into the hydrazino-fullerol solution, and the mixture is stirred and reacted for 3-4 days at room temperature in a dark place by using glacial acetic acid as a catalyst to prepare the doxorubicin prodrug loaded on the fullerol, wherein the mass ratio of the hydrazino-fullerol to the doxorubicin is 1: (1-1.3).
S23, reacting folic acid, N-hydroxysuccinimide NHS and carbodiimide EDC under a dark reaction condition to generate folic acid succinimide ester; wherein, the carbodiimide EDC is used as a catalyst;
specifically, the method comprises the following steps: dissolving folic acid FA in dimethyl sulfoxide DMSO (dimethyl sulfoxide), adding N-hydroxysuccinimide NHS and carbodiimide EDC, and reacting at room temperature in a dark place under the protection of inert gas to obtain folic acid succinimide ester.
S24, the fullerene alcohol-loaded adriamycin prodrug generated in the step S22 and the folic acid succinimide ester generated in the step S23 are subjected to acylation reaction to generate the folic acid grafted fullerene alcohol-loaded adriamycin prodrug.
Specifically, the method comprises the following steps: and (2) adding a proper amount of the folic acid succinimide ester solution prepared in the step (S23) into the fullerol doxorubicin-loaded prodrug solution prepared in the step (S22), stirring and reacting for 4-8 hours at room temperature in a dark place, adding methanol or ethanol into the reaction solution, precipitating, and performing suction filtration to obtain a reddish brown solid, namely the folic acid grafted fullerol doxorubicin-loaded prodrug, wherein the ratio of the amount of the fullerol doxorubicin-loaded prodrug to the amount of the folic acid succinimide ester is 1: 1.
EXAMPLE III
Preparation of folic acid grafted fullerol doxorubicin-loaded prodrug
Preparing hydrazino fullerol: dissolving 0.1g of fullerol in 10mL of purified water, adding 0.05g of oxalyl dihydrazide, stirring, reacting at room temperature for 7 days, placing the reaction solution in a dialysis bag (MW1000) for dialysis three times, and freeze-drying the dialysate to obtain a dark brown solid; preparation of folate succinimide ester solution: putting 1.0g (2.23mmol) of folic acid into a 50mL round-bottom flask, adding 40mL of dimethyl sulfoxide (DMSO), performing ultrasonic treatment until the folic acid is completely dissolved, adding 0.28g (2.48mmol) of NHS and 0.56g (2.48mmol) of EDC, introducing N2 for protection, keeping out of the sun, stirring at room temperature for reaction overnight, and performing suction filtration to remove insoluble substances to obtain an orange-red solution, namely a folic acid succinimide ester solution; weighing 100mg of prepared dark brown solid hydrazino fullerol, dissolving in 5mL of water and 5mL of mixed solution of 5mLDMSO, adding 1.6mL of prepared folic acid succinimide ester solution, and stirring for reaction for 24 hours at room temperature in a dark place to obtain folic acid grafted hydrazino fullerol reaction liquid; adding 80mg of doxorubicin hydrochloride and 1 drop of glacial acetic acid into the prepared folic acid grafted hydrazino fullerol reaction solution, stirring and reacting for 4 days under the condition of room temperature and light shielding, finally adding 50mL of anhydrous methanol, separating out a precipitate, performing suction filtration, and washing with methanol for three times to obtain a reddish brown solid, namely the folic acid grafted fullerol doxorubicin-loaded prodrug.
Example four
Referring to fig. 5, fourier infrared spectroscopy is used to detect the raw materials of fullerol, folic acid and doxorubicin, and the product of folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example three.
Taking appropriate amount of fullerol, folic acid, adriamycin and folic acid grafted fullerol adriamycin-loaded prodrug, respectively mixing with KBr, milling, tabletting, and performing infrared spectrum scanning within the scanning range of 400-4000cm-1。
EXAMPLE five
Please refer to fig. 6, adopt1And detecting the raw materials of the fullerol, the folic acid and the adriamycin by an H nuclear magnetic resonance method and the product prepared in the third embodiment, wherein the folic acid grafted fullerol carries the adriamycin prodrug.
1HNM spectrum detection is carried out on a 500MHz nuclear magnetic resonance instrument, Tetramethylsilane (TMS) is used as an internal reference, the fullerene alcohol uses D2O as a solvent, folic acid, adriamycin and folic acid grafted fullerene alcohol-carried adriamycin prodrug use deuterated DMSO as a solvent, 1H-NMR analysis is respectively carried out,
1: the fullerol has weak proton peaks at delta 3.26 and 1.07; 2: folic acid has proton peaks at delta 8.65,8.15,7.64,6.95,6.66,4.50,4.33,2.32,2.04, 1.91; 3: doxorubicin has proton peaks at δ 7.90, 7.67,5.47,5.30, 4.93,4.88,4.60,4.37,4.20,3.99,3.60,3.02,2.98,2.91,2.86,2.16,2.09,1.89,1.70,1.17, 1.06; 4: the folic acid grafted fullerol loaded doxorubicin prodrug has proton peaks at delta 8.65, 8.09,7.93,7.65, 6.94,6.65,4.50,4.34, 4.00,3.00,2.68,2.33,1.91,1.24,1.15, 0.98. Wherein 8.65, 7.65, 6.94,6.65,4.50,4.34 and 1.91 belong to characteristic peaks of folic acid; 7.93,4.00,3.00,1.15, belongs to the characteristic peak of adriamycin, judges that the structure of the product is connected with the structures of folic acid and adriamycin, and proves the result of infrared spectrum.
EXAMPLE six
Referring to fig. 7, the raw materials of fullerol, folic acid and doxorubicin and the product of folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example three were examined by differential scanning calorimetry.
The detection conditions are 30-300 ℃, the heating rate is 15 ℃/min, the N2 flows 100mL/min, and the alumina crucible is used.
The product folic acid grafted fullerol adriamycin-carried prodrug 4 is obviously different from the product folic acid grafted fullerol 1, folic acid 2 and adriamycin 3: the fullerol has two obvious endothermic peaks at 73 ℃ and 217 ℃; folic acid has a large endothermic peak at 148 deg.C, and a weak endothermic peak at 201 deg.C and 269 deg.C; doxorubicin has two endothermic peaks at 210 ℃ and 237 ℃. The folic acid grafted fullerol adriamycin-loaded prodrug has a larger endothermic peak at 67 ℃, which is different from the three raw materials. The DSC pattern indicated that the product was not the same substance as the starting material, nor a mixture of starting materials, but rather formed covalent bonds.
EXAMPLE seven
Referring to fig. 8, the product folic acid grafted fullerol doxorubicin-loaded prodrug prepared in example three was tested for dissolution rate in vitro.
The in vitro dissolution rate detection method comprises the following steps: fully dissolving the product folic acid grafted fullerols-loaded adriamycin prodrug by DMSO, filtering the solution by a membrane, detecting the adriamycin concentration in the filtrate by a fluorescence spectrophotometer, and calculating the adriamycin content according to a standard curve, wherein the concentration of the adriamycin in the filtrate is about 500 mg/L. Putting 2mL into dialysis bag (with a relative molecular weight of 1000), adding 2mL of phosphate buffer solution (PBS, pH7.4, pH 5.8, pH 4.9), tightening the bag, putting into a conical flask containing 26mL of PBS solution with corresponding pH value, placing in a constant temperature water bath, and maintaining at 37 deg.C. At set time intervals, 0.5mL of PBS solution was removed, the doxorubicin concentration was measured using a spectrofluorometer, and the same volume of fresh PBS solution was replenished.
In vitro dissolution results of the product: the release rate of the folic acid grafted fullerol doxorubicin-loaded prodrug under physiological conditions at pH7.4 is significantly lower than the release rate under acidic conditions at pH 5.8 and pH 4.9. At pH7.4, the cumulative release was only 18% for 8h, 22% for 24h and 27% for 72 h; under the condition of pH 5.8, the cumulative release amount of 8h is 30%, the cumulative release amount of 24h is 39% and the cumulative release amount of 72h is 45%; at pH 4.9, the cumulative release was 36% for 8h, 52% for 24h and 78% for 72 h. This result corresponds to the degradation behavior of hydrazone bonds. The product folic acid grafted fullerol adriamycin-carried prodrug has targeting ligand folic acid, so that the folic acid grafted fullerol adriamycin-carried prodrug has active targeting on tumor tissues with higher expression of folic acid receptors. The product is slow in release speed in normal tissues, and once phagocytized by tumor cells and enters intracellular endosomes, the adriamycin can be rapidly released in the acidic environment of the endosomes (pH is approximately equal to 5.0).
The folic acid grafted fullerol doxorubicin-loaded prodrug provided by the invention and the preparation method and application thereof have the following beneficial effects:
the folic acid grafted fullerol doxorubicin-loaded prodrug takes water-soluble fullerol as a drug carrier, a small molecule hydrazide group is introduced into the structure, the folic acid is connected with the fullerene through an amido bond and is connected with the doxorubicin through a hydrazone bond to form the prodrug, after the prodrug reaches tumor tissues, the folate receptor mediated phagocytosis of tumor cell membranes is utilized, the hydrazone bond is dissociated by utilizing the weak acid environment of a cell endosome, and the drug and the carrier are released.
Secondly, the folic acid grafted fullerol doxorubicin-loaded prodrug has the advantages of simple molecular structure, easy synthesis, mild reaction conditions and simple and convenient operation method.
And thirdly, grafting folic acid to enable the adriamycin prodrug to have tumor targeting, and combining with folic acid receptors on the surfaces of tumor cells, the adriamycin prodrug can be actively mediated to enter the tumor cells rich in folic acid receptors.
The hydrazone bond mediated medicine in tumor cells has a quick release function, and can trigger the hydrolysis of the acid sensitive hydrazone bond under the condition of the pH of an endosome, so that the medicine can be quickly released in the tumor cells, and the curative effect is good.
And fifthly, the carrier fullerol has good water solubility, can improve the solubility of the prodrug molecules, has an anti-tumor effect and can play a synergistic effect with adriamycin.
While the foregoing is directed to embodiments of the present invention, it will be understood by those skilled in the art that various changes may be made without departing from the spirit and scope of the invention.
Claims (9)
1. The folic acid grafted fullerol doxorubicin-loaded prodrug is formed by combining 1 molecule of fullerol, which is introduced with a hydrazide group, with 1 molecule of doxorubicin through a hydrazone bond and 1 molecule of folic acid through an amide bond, and has the following structural formula:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6.
2. The method for preparing the folic acid grafted fullerol doxorubicin prodrug of claim 1, comprising the steps of:
(1) in solvent water, carrying out addition reaction on fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(2) reacting folic acid with N-hydroxysuccinimide NHS to generate folic acid succinimide ester by using carbodiimide EDC as a catalyst;
(3) carrying out acylation reaction on the hydrazino fullerol generated in the step (1) and the folic acid succinimidyl ester generated in the step (2) to generate folic acid grafted hydrazino fullerol, wherein the structural formula of the folic acid grafted hydrazino fullerol is as follows:
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(4) and (3) carrying out condensation reaction on the adriamycin or the adriamycin hydrochloride and the folic acid grafted hydrazino fullerol generated in the step (3) to generate the folic acid grafted fullerol adriamycin-carried prodrug.
3. The method for preparing a folic acid grafted fullerol doxorubicin prodrug according to claim 2, wherein the step (1) is specifically as follows: dissolving fullerol in solvent water, adding oxalyl dihydrazide, stirring and reacting for 3-7 days at 25-50 ℃, dialyzing by taking water as a solvent, and freeze-drying to obtain a dark brown solid, namely the hydrazino fullerol.
4. The method for preparing a folic acid grafted fullerol doxorubicin prodrug according to claim 2, wherein the step (2) is specifically as follows: dissolving folic acid FA in dimethyl sulfoxide DMSO (dimethyl sulfoxide), adding N-hydroxysuccinimide NHS and carbodiimide EDC, and reacting at room temperature in a dark place under the protection of inert gas to obtain folic acid succinimide ester.
5. The method for preparing a folic acid grafted fullerol doxorubicin prodrug according to claim 2, wherein the step (3) is specifically as follows: dissolving the hydrazino fullerol prepared in the step (1) in a mixed solvent consisting of water and dimethyl sulfoxide (DMSO), adding the folic acid succinimide ester prepared in the step (2), stirring and reacting for 4-8 hours at room temperature in a dark condition to prepare the folic acid grafted hydrazino fullerol, wherein the mass ratio of the hydrazino fullerol to the folic acid succinimide ester is 1: 1.
6. the method for preparing a folic acid grafted fullerol doxorubicin prodrug according to claim 2, wherein the step (4) is specifically as follows: dissolving adriamycin in a dimethyl sulfoxide (DMSO) solvent, adding the dissolved adriamycin into the folic acid grafted hydrazino fullerol prepared in the step (3), stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating, and carrying out suction filtration to obtain a reddish brown solid, namely the folic acid grafted fullerol supported adriamycin prodrug, wherein the mass ratio of the adriamycin to the folic acid grafted hydrazino fullerol is (1-1.3): 1.
7. the method for preparing a folic acid grafted fullerol doxorubicin prodrug according to claim 2, wherein the step (4) is specifically as follows: adding doxorubicin hydrochloride into the folic acid grafted hydrazino fullerol prepared in the step (3), stirring and reacting for 3-4 days at room temperature in a dark place, then adding methanol or ethanol, precipitating and filtering to obtain a reddish brown solid, namely the folic acid grafted fullerol supported doxorubicin prodrug, wherein the mass ratio of the doxorubicin to the folic acid grafted hydrazino fullerol is (1-1.3): 1.
8. the method for preparing the folic acid grafted fullerol doxorubicin prodrug of claim 1, comprising the steps of:
(1) in solvent water, carrying out addition reaction on fullerol and oxalyl dihydrazide to generate hydrazino fullerol, wherein the structural formula of the hydrazino fullerol is as follows:
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(2) condensing doxorubicin and hydrazino fullerol generated in the step (1) to obtain a prodrug of the fullerol carried with the doxorubicin;
wherein the content of the first and second substances,
n is an integer, and n is 24-28;
m is an integer, and m is 2-6;
(3) reacting folic acid with N-hydroxysuccinimide NHS to generate folic acid succinimide ester by using carbodiimide EDC as a catalyst;
(4) and (3) carrying out acylation reaction on the fullerene alcohol-loaded adriamycin prodrug generated in the step (2) and the folic acid succinimide ester generated in the step (3) to generate the folic acid grafted fullerene alcohol-loaded adriamycin prodrug.
9. Use of the folic acid grafted fullerol doxorubicin-loaded prodrug according to any one of claims 1-8 in the preparation of an anti-tumor medicament.
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