CN117122564B - Fluorouracil injection and preparation method thereof - Google Patents
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Abstract
The invention relates to the technical field of medical preparations, and in particular discloses a fluorouracil injection and a preparation method thereof. The fluorouracil injection comprises a carrier for loading fluorouracil, a pH regulator, a cosolvent and water. The preparation method of the invention is that the fluorouracil-loaded carrier, water and sodium hydroxide aqueous solution are mixed, stirred and dissolved, and then the pH value is regulated to 8.9-9.4. Compared with the prior art, the fluorouracil injection prepared by the invention has the advantages of high encapsulation efficiency, good targeting property and the like, can greatly improve the efficacy of the medicine and reduce side effects.
Description
Technical Field
The invention relates to the technical field of medical preparations, in particular to fluorouracil injection and a preparation method thereof.
Background
Fluorouracil, also known as 5-fluorouracil, has the chemical formula C 4H3FN2O2, is a pyrimidine analogue, belongs to one of antimetabolites, and is mainly used for treating tumors. Fluorouracil is a homologue of uracil, which is a component of ribonucleic acid. The drug acts as an antimetabolite, which, upon intracellular conversion to potent fluorouracil deoxynucleotides, interferes with DNA synthesis by blocking the conversion of deoxynucleotide to thymidylate by intracellular thymidylate synthase. Fluorouracil can also interfere with RNA synthesis. Fluorouracil is widely distributed in body fluids and disappears from the blood within 4 hours after intravenous administration. After being converted into nucleotides, it is preferentially taken up by actively dividing tissues and tumors, and fluorouracil easily enters cerebrospinal fluid. About 20% are excreted in prototype from urine, the remainder being largely metabolized in the liver by mechanisms that normally metabolize uracil. Fluorouracil and 6-mercaptopurine, which are the earliest anticancer drugs, are both extracted from sea cucumber. The product is not completely absorbed by oral administration and is difficult to predict, so that the product is administrated by injection and rapidly distributed into various tissues of the whole body including cerebrospinal fluid and tumor tissues after intravenous injection.
Fluorouracil causes serious adverse reactions in the digestive tract and bone marrow suppression are the greatest defects in its existence. The defects of lack of selectivity to organisms, short half-life of plasma, low bioavailability and the like are also the bottleneck of clinical application of fluorouracil. To increase the selectivity of fluorouracil and mitigate adverse effects, it may be encapsulated or loaded.
Chinese patent 201310439021.9 provides a compound fluorouracil injection and its preparation method. The encapsulation rate of the compound fluorouracil injection is 65-75%, and the average particle size is less than or equal to 0.30 mu m. The compound fluorouracil injection has higher encapsulation efficiency and good stability. The preparation method changes the mixing mode of the fat-soluble component and the water-soluble component in the prescription of the compound fluorouracil injection, thereby obtaining higher encapsulation efficiency than the product prepared by the prior art.
Chinese patent 202211427711.8 discloses a compound fluorouracil injection and a preparation method thereof, wherein the injection comprises the following components: fluorouracil, a pH regulator, a plant extract, an isotonic regulator and water for injection; fluorouracil is carried by a carrier, the fluorouracil is encapsulated by the carrier formed by crosslinking cholesterol modified starch and sodium alginate, the fluorouracil is connected in the carrier formed by crosslinking surface modified starch and sodium alginate, the solubility of the fluorouracil can be obviously improved, a compact crosslinking structure has better mechanical property, the fluorouracil can be better encapsulated, the fluorouracil is not easy to leak, the targeted slow release is realized in a human body, and the fluorouracil has stable quality and good clinical medication safety.
However, the prior art still has the problems of low encapsulation efficiency, insufficient nano-carrier load and weak targeting, and the defects can influence the efficacy and bioavailability of fluorouracil and cause side effects to influence the health of patients, so that the fluorouracil-loaded form which is more effective and safer is developed and applied to injections has very important significance.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to solve the technical problem of a fluorouracil injection and a preparation method thereof.
Typically, the extracellular pH of the tumor is about 6.5 to 7.2, whereas the normal tissue is 7.3 to 7.4; the pH value in cancer cells is 5.0-6.0, and the pH value of lysosomes is 4.0-5.0. Thus, coating a chemotherapeutic drug in a stimuli-responsive nano-delivery system with targeting function is an ideal strategy to achieve cell targeting and drug release at specific sites. In the invention, fluorouracil is loaded by an isopropyl amino methyl-polyethylene glycol-polyamide-amine compound, wherein the polyamide-amine is compounded with polyethylene glycol and then further subjected to Michael addition with 2- (diisopropylamino) ethyl methacrylate to obtain the compound, and the fluorouracil can be subjected to state change under the acidic condition of tumors after being loaded by the compound, so that the drugs are quickly released into tumor cells, and the targeting of the drugs is realized. The polyamide-amine is very suitable for serving as a drug carrier due to the dendritic structure and smaller diameter, however, the internal space is completely open, the drug is easy to release in advance before reaching tumor cells, a layer of protective layer can be formed on the outer surface of the polyethylene glycol by a long chain of the polyethylene glycol, meanwhile, the property of neutrality of the polyethylene glycol surface can be changed, so that the polyethylene glycol is not easy to endocytose and escape of lysosomes, after being compounded with the polyethylene glycol, the polyethylene glycol is further subjected to Michael addition with 2- (diisopropylamino) ethyl methacrylate, and amine groups are introduced into the surface of the carrier, so that the pH sensitivity can be converted into a hydrophilic/hydrophobic state under acidic/alkaline conditions, and the internal 'closure' is realized, therefore, the directional release of fluorouracil under the acidic tumor condition can be realized, the obtained fluorouracil carrier has high encapsulation rate and strong targeting property, the drug efficacy can be greatly improved, and the side effects are reduced.
In order to achieve the above purpose, the invention provides a preparation method of fluorouracil injection, comprising the following steps:
adding 0.1 to 0.5 weight part of sodium hydroxide into 1 to 5 weight parts of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.3-1.2 parts by weight of fluorouracil-loaded carrier into 8-15 parts by weight of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding the sodium hydroxide aqueous solution to adjust the pH to 8.9-9.4, stirring and mixing uniformly, sterilizing and filtering the obtained mixed solution, and filling to obtain fluorouracil injection.
Preferably, the fluorouracil-loaded carrier is obtained after loading fluorouracil and isopropyl aminomethyl-polyethylene glycol-polyamide-amine complex.
Further preferably, the preparation method of the fluorouracil-loaded carrier comprises the following steps:
s1, mixing polyethylene glycol, 4-dimethylaminopyrimidine, glyoxylic acid and dicyclohexylcarbodiimide in a solvent, stirring, and performing aftertreatment to obtain a polyethylene glycol derivative;
S2, adding polyamide-amine and anhydrous sodium sulfate into a solvent, mixing, adding a polyethylene glycol derivative under inert atmosphere, heating and stirring, and performing post-treatment to obtain a polyethylene glycol-polyamide-amine compound;
S3, mixing polyethylene glycol-polyamide-amine compound, polyethylene glycol, rhodium trichloride trihydrate and 2- (diisopropylamino) ethyl methacrylate in a solvent, heating, stirring, cooling, dialyzing, and performing aftertreatment to obtain isopropylaminomethyl-polyethylene glycol-polyamide-amine compound;
S4, adding fluorouracil into an acidic PBS buffer solution, adding an isopropyl aminomethyl-polyethylene glycol-polyamide-amine compound, regulating the pH value to be weak alkaline by using an aqueous solution of NaOH, stirring for encapsulation, regulating the pH value to be neutral by using an aqueous solution of HCl, filtering, and freeze-drying to obtain the fluorouracil-loaded carrier.
Most preferably, the preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 4-15 parts by weight of polyethylene glycol into 25-50 parts by weight of dichloromethane, adding 0.1-0.5 part by weight of 4-dimethylaminopyridine, 0.5-2.5 parts by weight of glyoxylic acid and 3.5-6.5 parts by weight of dicyclohexylcarbodiimide, stirring at room temperature for 16-28 hours, filtering, concentrating filtrate under reduced pressure, adding water into the filtrate, adding dichloromethane for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0-5 ℃ to obtain a precipitate, filtering, and drying residues to obtain polyethylene glycol derivatives;
S2, adding 2.5-5.5 parts by weight of polyamide-amine and 1-4 parts by weight of anhydrous sodium sulfate into 35-65 parts by weight of methanol, adding 0.01-0.1 part by weight of polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 70-80 ℃, stirring for 10-20 hours, cooling to room temperature, filtering, washing residues with diethyl ether, and drying to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 1.5 to 3.5 parts by weight of polyethylene glycol-polyamide-amine compound into 15 to 30 parts by weight of water, adding 2 to 4 parts by weight of polyethylene glycol and 0.01 to 0.05 part by weight of rhodium trichloride trihydrate and 1 to 3 parts by weight of 2- (diisopropylamino) ethyl methacrylate, heating to 45 to 55 ℃, stirring for 6 to 10 hours, cooling to room temperature, dialyzing (less than 7500 Da) for 3 days, washing residues with methanol, and freeze-drying to obtain an isopropylaminomethyl polyethylene glycol-polyamide-amine compound;
S4, adding 0.5-1.0 part by weight of fluorouracil into 500-1000 parts by weight of PBS buffer solution with pH=4-5, adding 0.25-0.6 part by weight of isopropyl amino methyl-polyethylene glycol-polyamide-amine compound, adjusting pH=8-8.5 by using 0.1mol/LNaOH aqueous solution, stirring for 1-3 hours for encapsulation, adjusting pH to be neutral by using 0.1mol/L HCl aqueous solution, precipitating, filtering, and freeze-drying residues to obtain the fluorouracil-loaded carrier.
Further, the filter membrane has a diameter of 0.2 μm.
Further, the temperature of the degerming filtration is less than or equal to 35 ℃, the filtration pressure difference is less than or equal to 0.3MPa, and the filtration flow rate is less than or equal to 69.8L/h.
The invention has the beneficial effects that:
1. In the invention, fluorouracil is loaded by an isopropyl amino methyl-polyethylene glycol-polyamide-amine compound, wherein the polyamide-amine is compounded with polyethylene glycol and then further subjected to Michael addition with 2- (diisopropylamino) ethyl methacrylate to obtain the compound, and the fluorouracil can be subjected to state change under the acidic condition of tumors after being loaded by the compound, so that the drugs are quickly released into tumor cells, and the targeting of the drugs is realized.
2. Compared with the prior art, the fluorouracil carrier obtained by the invention has high encapsulation efficiency and strong targeting property, can greatly improve the efficacy of the drug and reduce side effects.
Detailed Description
Polyamide-amine, PAMAM-G4.0, CAS number: 163442-67-9 from the biological technology of the seashore.
Example 1
A preparation method of fluorouracil injection comprises the following steps:
adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.8g of fluorouracil-loaded carrier into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 9.5g of polyethylene glycol (PEG-2000) into 30mL of methylene dichloride, adding 0.25g of 4-dimethylaminopyridine, 1.25g of glyoxylic acid and 5.5g of dicyclohexylcarbodiimide, stirring at room temperature for 18h, filtering, concentrating filtrate under reduced pressure of-0.9 MPa, adding water into the filtrate to disperse, adding methylene dichloride for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0 ℃ to precipitate, filtering, and drying the residue at 40 ℃ for 8h to obtain a polyethylene glycol derivative;
S2, adding 3.5g of polyamide-amine and 3g of anhydrous sodium sulfate into 50mL of methanol, adding 0.05g of the polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 75 ℃, stirring for 14h, cooling to room temperature, filtering, washing residues with diethyl ether, and drying at 40 ℃ for 8h to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 2.5g of polyethylene glycol-polyamide-amine compound into 25mL of water, adding 3g of polyethylene glycol (PEG-2000), 0.02g of rhodium trichloride trihydrate and 2g of 2- (diisopropylamino) ethyl methacrylate, heating to 53 ℃, stirring for 8h, cooling to room temperature, dialyzing (less than 7500 Da) for 3d, washing the residue with methanol, and freeze-drying at-30 ℃ under 10Pa for 48h to obtain an isopropylaminomethyl-polyethylene glycol-polyamide-amine compound;
S4, 1g of fluorouracil is added into 1LpH =4 PBS buffer, 0.6g of isopropyl amino methyl-polyethylene glycol-polyamide-amine complex is added, the pH value is regulated to be 8 by 0.1mol/LNaOH aqueous solution, the mixture is stirred for 3 hours for encapsulation, the pH value is regulated to be neutral by 0.1mol/L HCl aqueous solution, precipitation occurs, filtration is carried out, and the residue is frozen and dried for 48 hours at the temperature of minus 30 ℃ under the pressure of 10Pa, so that the fluorouracil-loaded carrier is obtained.
Comparative example 1
A preparation method of fluorouracil injection comprises the following steps:
Adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.5g of fluorouracil into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolution, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
Example 2
A preparation method of fluorouracil injection comprises the following steps:
adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.8g of fluorouracil-loaded carrier into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
1g of fluorouracil is added into 1L of PBS buffer solution with pH=4, 0.6g of polyamide-amine is added, the pH=8 is regulated by 0.1mol/LNaOH aqueous solution, the mixture is stirred for 3 hours for encapsulation, the pH is regulated to be neutral by 0.1mol/L HCl aqueous solution, precipitation occurs, the mixture is filtered, and the residue is frozen and dried for 48 hours at-30 ℃ under 10Pa to obtain the fluorouracil-loaded carrier.
Example 3
A preparation method of fluorouracil injection comprises the following steps:
adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.8g of fluorouracil-loaded carrier into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 9.5g of polyethylene glycol (PEG-2000) into 30mL of methylene dichloride, adding 0.25g of 4-dimethylaminopyridine, 1.25g of glyoxylic acid and 5.5g of dicyclohexylcarbodiimide, stirring at room temperature for 18h, filtering, concentrating filtrate under reduced pressure of-0.9 MPa, adding water into the filtrate to disperse, adding methylene dichloride for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0 ℃ to precipitate, filtering, and drying the residue at 40 ℃ for 8h to obtain a polyethylene glycol derivative;
S2, adding 3.5g of polyamide-amine and 3g of anhydrous sodium sulfate into 50mL of methanol, adding 0.05g of the polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 75 ℃, stirring for 14h, cooling to room temperature, filtering, washing residues with diethyl ether, and drying at 40 ℃ for 8h to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 1g of fluorouracil into 1LpH =4 PBS buffer, adding 0.6g of polyethylene glycol-polyamide-amine complex, adjusting pH to 8 by using 0.1mol/LNaOH aqueous solution, stirring for 3h for encapsulation, adjusting pH to be neutral by using 0.1mol/L HCl aqueous solution, precipitating, filtering, and freeze-drying the residue at-30 ℃ under 10Pa for 48h to obtain the fluorouracil-loaded carrier.
Example 4
A preparation method of fluorouracil injection comprises the following steps:
adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.8g of fluorouracil-loaded carrier into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 9.5g of polyethylene glycol (PEG-2000) into 30mL of methylene dichloride, adding 0.25g of 4-dimethylaminopyridine, 1.25g of glyoxylic acid and 5.5g of dicyclohexylcarbodiimide, stirring at room temperature for 18h, filtering, concentrating filtrate under reduced pressure of-0.9 MPa, adding water into the filtrate to disperse, adding methylene dichloride for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0 ℃ to precipitate, filtering, and drying the residue at 40 ℃ for 8h to obtain a polyethylene glycol derivative;
S2, adding 3.5g of polyamide-amine and 3g of anhydrous sodium sulfate into 50mL of methanol, adding 0.05g of the polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 75 ℃, stirring for 14h, cooling to room temperature, filtering, washing residues with diethyl ether, and drying at 40 ℃ for 8h to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 2.5g of polyethylene glycol-polyamide-amine compound into 25mL of water, adding 3g of polyethylene glycol (PEG-2000), 0.02g of rhodium trichloride trihydrate and 2g of 2- (diisopropylamino) ethyl methacrylate, heating to 53 ℃, stirring for 8h, cooling to room temperature, dialyzing (less than 7500 Da) for 3d, washing the residue with methanol, and freeze-drying at-30 ℃ under 10Pa for 48h to obtain an isopropylaminomethyl-polyethylene glycol-polyamide-amine compound;
S4 fluorouracil 0.6g was added to 1LpH =4 PBS buffer, and then 0.6g isopropyl aminomethyl-polyethylene glycol-polyamide-amine complex was added, pH=8 was adjusted with 0.1mol/LNaOH aqueous solution, and the mixture was stirred for 3 hours for encapsulation, pH was adjusted to neutrality with 0.1mol/L HCl aqueous solution, precipitation occurred, filtration was performed, and the residue was lyophilized at-30℃under 10Pa for 48 hours to give fluorouracil-loaded carrier.
Example 5
A preparation method of fluorouracil injection comprises the following steps:
adding 0.15g of sodium hydroxide into 1.5mL of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.8g of fluorouracil-loaded carrier into 10mL of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adding 0.09g of 3mol/L sodium hydroxide aqueous solution for regulating the pH to 9.0, stirring for uniform mixing, sterilizing and filtering the obtained mixed solution by a filter membrane with the thickness of 0.2 mu m, and filling at 30 ℃ at the temperature of 0.3MPa and 69.5L/h to obtain the fluorouracil injection.
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 9.5g of polyethylene glycol (PEG-2000) into 30mL of methylene dichloride, adding 0.25g of 4-dimethylaminopyridine, 1.25g of glyoxylic acid and 5.5g of dicyclohexylcarbodiimide, stirring at room temperature for 18h, filtering, concentrating filtrate under reduced pressure of-0.9 MPa, adding water into the filtrate to disperse, adding methylene dichloride for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0 ℃ to precipitate, filtering, and drying the residue at 40 ℃ for 8h to obtain a polyethylene glycol derivative;
S2, adding 3.5g of polyamide-amine and 3g of anhydrous sodium sulfate into 50mL of methanol, adding 0.05g of the polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 75 ℃, stirring for 14h, cooling to room temperature, filtering, washing residues with diethyl ether, and drying at 40 ℃ for 8h to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 2.5g of polyethylene glycol-polyamide-amine compound into 25mL of water, adding 3g of polyethylene glycol (PEG-2000), 0.02g of rhodium trichloride trihydrate and 2g of 2- (diisopropylamino) ethyl methacrylate, heating to 53 ℃, stirring for 8h, cooling to room temperature, dialyzing (less than 7500 Da) for 3d, washing the residue with methanol, and freeze-drying at-30 ℃ under 10Pa for 48h to obtain an isopropylaminomethyl-polyethylene glycol-polyamide-amine compound;
S4 fluorouracil 1.5g was added to 1LpH =4 PBS buffer, and then 0.6g isopropyl aminomethyl-polyethylene glycol-polyamide-amine complex was added, pH=8 was adjusted with 0.1mol/LNaOH aqueous solution, and the mixture was stirred for 3 hours for encapsulation, pH was adjusted to neutrality with 0.1mol/L HCl aqueous solution, precipitation occurred, filtration was performed, and the residue was lyophilized at-30℃under 10Pa for 48 hours to obtain fluorouracil-loaded carrier.
Test example 1
70 Healthy mice (18-20 g) with the same body type are injected with 0.2mL of 2×10 7/mL H22 cancer cell suspension, 7 groups of 10 mice are divided after 7d feeding, fluorouracil injection is injected into each group at the dosage of 20mg/kg/d, the blank control group is injected with the same amount of physiological saline, the mice are killed after continuous 7d injection, tumors are dissected, taken out and weighed, and each group is averaged, so that the tumor inhibition rate is calculated.
TABLE 1 anticancer effects of fluorouracil injection
| Test protocol | Tumor weight/g | Tumor inhibition/% |
| Blank control | 2.10±0.11 | - |
| Comparative example 1 | 0.75±0.05 | 64.28 |
| Example 1 | 0.21±0.03 | 90.0 |
| Example 2 | 0.55±0.04 | 73.81 |
| Example 3 | 0.46±0.06 | 78.10 |
| Example 4 | 0.31±0.07 | 85.24 |
| Example 5 | 0.33±0.04 | 84.28 |
Comparison of comparative example 1 and example 1 shows that the fluorouracil is encapsulated in the carrier to greatly inhibit the growth of tumor, which is probably due to the fact that the carrier of example 1 not only can achieve higher encapsulation rate, but also can realize directional release of fluorouracil through polyethylene glycol chains and surface response groups, and has high targeting property, thus having good curative effect, and not only being capable of improving bioavailability of fluorouracil, but also reducing side effects. In example 2, after the fluorouracil is encapsulated by the polyamide-amine, the channels inside the polyamide-amine are completely open, so that the fluorouracil cannot realize targeting to tumors, and the drug is likely to be released in advance, while the polyamide-amine-polyethylene glycol complex in example 3 can form a protective layer on the surface of the nano-carrier after the fluorouracil is encapsulated by the polyethylene glycol chain, but the oriented release in different environments cannot be realized, so that the tumor inhibiting effect of examples 2-3 is not as good as that of example 1. In examples 4 to 5, fluorouracil and isopropylaminomethyl-polyethylene glycol-polyamide-amine were used in different proportions from example 1, which resulted in a difference in encapsulation effect and thus in a difference in the final release amount, and thus, a difference in tumor suppression rate was exhibited.
Test example 2
The fluorouracil injection prepared in the comparative examples and examples was added to a dialysis bag (molecular weight cut-off < 7500 Da) and then immersed in a 15mLPBS (pH 6.5 or 7.4) solution, and shaken at 37℃at a rotational speed of 100rpm. The release solution outside the 3mL dialysis bag was taken at different time points and an equal amount of PBS solution was replenished. Fluorouracil released at each time point was measured and the cumulative release rate was calculated.
Table 2 fluorouracil in vitro Release test
As can be seen from a comparison of comparative example 1 with example 1, fluorouracil is not encapsulated in comparative example 1, and thus is rapidly released under different pH conditions, so that directional release of tumor cells is not achieved. Whereas the release of fluorouracil under acidic conditions in example 1 was more rapid, probably because the prepared carrier had pH sensitivity and could undergo a hydrophilic/hydrophobic state transition under acidic/alkaline conditions, thus achieving an internal "closure", thus enabling the targeted release of fluorouracil under acidic conditions of the tumor, which was also demonstrated by the release of fluorouracil from different pH environments, the release rate of fluorouracil under alkaline environments was significantly less, whereas the carriers in examples 2-3 were likewise incapable of achieving a change of state under different acidic and alkaline conditions, due to the lack of a responsive group, and thus could not achieve a targeted release under different pH conditions. The difference in the release amounts of examples 4 to 5 from example 1 is mainly related to the mass ratio of fluorouracil, isopropylaminomethyl-polyethylene glycol-polyamide-amine at the time of encapsulation.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (4)
1. The preparation method of the fluorouracil injection is characterized by comprising the following steps: adding 0.1 to 0.5 weight part of sodium hydroxide into 1 to 5 weight parts of water, stirring and dissolving to obtain sodium hydroxide aqueous solution; adding 0.3-1.2 parts by weight of fluorouracil-loaded carrier into 8-15 parts by weight of water, adding the sodium hydroxide aqueous solution, stirring for dissolving, adjusting pH to 8.9-9.4 with the sodium hydroxide aqueous solution, stirring and mixing uniformly, sterilizing and filtering the obtained mixed solution, and filling to obtain fluorouracil injection;
The preparation method of the fluorouracil-loaded carrier comprises the following steps:
S1, adding 4-15 parts by weight of polyethylene glycol into 25-50 parts by weight of dichloromethane, adding 0.1-0.5 part by weight of 4-dimethylaminopyridine, 0.5-2.5 parts by weight of glyoxylic acid and 3.5-6.5 parts by weight of dicyclohexylcarbodiimide, stirring at room temperature for 16-28 hours, filtering, concentrating filtrate under reduced pressure, adding water into the filtrate, adding dichloromethane for extraction, mixing organic phases, pouring into diethyl ether, cooling to 0-5 ℃ to obtain a precipitate, filtering, and drying residues to obtain polyethylene glycol derivatives;
S2, adding 2.5-5.5 parts by weight of polyamide-amine and 1-4 parts by weight of anhydrous sodium sulfate into 35-65 parts by weight of methanol, adding 0.01-0.1 part by weight of polyethylene glycol derivative in the step S1 under inert atmosphere, heating to 70-80 ℃, stirring for 10-20 hours, cooling to room temperature, filtering, washing residues with diethyl ether, and drying to obtain a polyethylene glycol-polyamide-amine compound;
S3, adding 1.5 to 3.5 parts by weight of polyethylene glycol-polyamide-amine compound into 15 to 30 parts by weight of water, adding 2 to 4 parts by weight of polyethylene glycol and 0.01 to 0.05 part by weight of rhodium trichloride trihydrate and 1 to 3 parts by weight of 2- (diisopropylamino) ethyl methacrylate, heating to 45 to 55 ℃, stirring for 6 to 10 hours, cooling to room temperature, dialyzing (less than 7500 Da) for 3 days, washing residues with methanol, and freeze-drying to obtain an isopropylaminomethyl polyethylene glycol-polyamide-amine compound;
S4, adding 0.5-1.0 part by weight of fluorouracil into 500-1000 parts by weight of PBS buffer solution with pH=4-5, adding 0.25-0.6 part by weight of isopropyl amino methyl-polyethylene glycol-polyamide-amine compound, adjusting pH=8-8.5 by using 0.1mol/LNaOH aqueous solution, stirring for 1-3 hours for encapsulation, adjusting pH to be neutral by using 0.1mol/L HCl aqueous solution, precipitating, filtering, and freeze-drying residues to obtain the fluorouracil-loaded carrier.
2. The method for preparing fluorouracil injection according to claim 1, characterized in that: the filter membrane diameter was 0.2 μm.
3. The method for preparing fluorouracil injection according to claim 1, characterized in that: the temperature of the degerming filtration is less than or equal to 35 ℃, the filtration pressure difference and the filtration flow rate are less than or equal to 69.8L/h.
4. A fluorouracil injection, which is characterized in that: a method according to any one of claims 1 to 3.
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