CN108392636B - Preparation method of graphene drug carrier with folic acid and WGA as targeting factors - Google Patents
Preparation method of graphene drug carrier with folic acid and WGA as targeting factors Download PDFInfo
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Abstract
The invention relates to a graphene nano-drug carrier with a folic acid and wheat germ agglutinin dual targeting factor, which is prepared by taking graphene oxide, 6-carboxymethyl-gamma-cyclodextrin, folic acid and Wheat Germ Agglutinin (WGA) as main raw materials, and a preparation method thereof. According to the method, graphene oxide and paratoluensulfonyl chloride are used as raw materials, graphene oxide hydroxyl is activated, L-lysine is grafted, and hydrazine hydrate is used for reducing the graphene oxide grafted with the lysine; then reacting the graphene nano-drug carrier with 6-carboxymethyl-gamma-cyclodextrin and folic acid under the common catalysis of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to generate nano-graphene grafted with gamma-CD and folic acid simultaneously, and then assembling the wheat germ agglutinin and the gamma-CD to prepare the graphene nano-drug carrier with the folic acid and wheat germ agglutinin dual targeting factors.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of a graphene nano-drug carrier with folic acid and wheat germ agglutinin dual-targeting factors, which is prepared by taking graphene oxide, 6-carboxymethyl-gamma-cyclodextrin, folic acid and Wheat Germ Agglutinin (WGA) as main raw materials.
Background
In recent decades, various nanomaterials of different sizes, shapes and chemical compositions have been studied as nanocarriers for therapeutic drugs, including nanoparticles of metals and metal oxides, polymeric micelles, liposomes, dendrimers and carbon nanotubes. Among them, due to the advantages in properties, as a new and competitive drug delivery system, graphene and graphene oxide have great application potential in systemic, targeted, and local drug delivery systems. The graphene has large specific surface area and high drug loading capacity. Due to the special conjugated structure, the conjugate has superior performance in loading drugs, particularly anticancer drugs. However, graphene is too rigid, and may cause some damage to normal cells. In addition, graphene is highly hydrophobic and has limited dispersibility in aqueous solutions. The graphene surface is subjected to grafting modification of a drug-loading factor and a targeting factor, so that the existing problems can be overcome, and the graphene surface has drug-loading and multiple active targeting capabilities.
The invention takes graphene oxide, 6-carboxymethyl-gamma-cyclodextrin, folic acid and wheat germ agglutinin as main raw materials to prepare a graphene nano-drug carrier with folic acid and wheat germ agglutinin dual targeting factors.
Disclosure of Invention
According to the method, graphene oxide and paratoluensulfonyl chloride are used as raw materials, graphene oxide hydroxyl is activated, L-lysine is grafted, and hydrazine hydrate is used for reducing the graphene oxide grafted with the lysine; then reacting the graphene nano-drug carrier with 6-carboxymethyl-gamma-cyclodextrin and folic acid under the common catalysis of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to generate nano-graphene grafted with gamma-CD and folic acid simultaneously, and then assembling the wheat germ agglutinin and the gamma-CD to prepare the graphene nano-drug carrier with the folic acid and wheat germ agglutinin dual targeting factors.
Detailed Description
Example 1
(1) Acylchlorinated graphene oxide
Dispersing 10.00g of graphene oxide (the purity is 99%, the thickness is less than or equal to 5nm, and the size is 2-8 um) in 100ml of chloroform, heating to 55 ℃, respectively and slowly dripping 5.50g of thionyl chloride and 2.00g of N-methylpyrrolidone under stirring, heating to boiling, carrying out reflux reaction for 3 hours, cooling to 25 ℃, centrifuging for 10 minutes at 3000 revolutions per minute, pouring out supernatant, scraping out precipitate, freezing and drying to obtain brown solid powder, and storing for later use;
(2) L-lysine grafted graphene oxide
Adding 5.00g of L-lysine into 50ml of distilled water, stirring and dissolving at 25 ℃, cooling to 10 ℃, slowly adding the product obtained in the step (1), stirring and reacting for 5 hours, heating to 30 ℃, continuously stirring and reacting for 2 hours, cooling to 25 ℃, filtering, washing for 3 times with absolute ethyl alcohol, and freeze-drying to obtain white solid powder for later use, namely wheat germ agglutinin;
(3) Reduction of L-lysine grafted graphene oxide
At 25 deg.C, 100ml of distilled water is taken, and N is introduced 2 10 minutes, 20.00g of L-ascorbic acid are added in N 2 Stirring and dissolving under flowing, adding 5.00g of the sample in the step (2), keeping the temperature at 25 ℃,stirring for reaction for 24h, filtering, washing with distilled water for 6 times, and vacuum drying to obtain black solid powder for later use;
(4) Grafting of 6-carboxymethyl-gamma-cyclodextrin and folic acid
Adding 2.50g of 6-carboxyl-gamma-cyclodextrin and 0.50g of folic acid into 50ml of distilled water, stirring at room temperature to dissolve, adding the sample obtained in the step (3), and adding the mixture in N 2 Stirring and dispersing under flowing down, cooling to 0 ℃, slowly adding 3.00g30% EDC aqueous solution and 2.00g10% NHS aqueous solution, reacting for 2h at 0 ℃, then reacting for 48h at 25 ℃, filtering, washing with distilled water for 3 times, and drying under vacuum to obtain black solid powder for later use;
(5) Assembly of WGA
And (3) dispersing the product obtained in the step (4) in 500ml of water for injection, adjusting the freezing point of a NaCl solution to be-0.52 ℃, adding 0.2gWGA and 50Hz, performing ultrasonic oscillation for 1h to obtain a black suspension, and sealing and storing.
Example 2:
(1) Particle size measurement of Fine particles
Samples (2 ml,50w and 2 Hz) obtained in example 1 (5) were taken at 36.7 ℃ and subjected to ultrasonic oscillation at 20ml,50w and 20Hz respectively for different times, and then dropped on a copper net with a supporting membrane, excess liquid was removed by filter paper, heavy metal complex dye was dropped again, excess liquid was removed by filter paper, and the mixture was naturally dried for 45min, and the particle size of the fine particles was measured by a scanning electron microscope. See table 1.
TABLE 1 average particle size of microparticles at rest for various periods of time
Time of sonication, min | 5 | 10 | 15 | 20 | 25 | 30 |
Average particle diameter, um | 0.17 | 0.23 | 0.25 | 0.28 | 0.27 | 0.27 |
(2) Zeta potential measurement
The Zeta potential of microparticles in a solution diluted by different factors was measured at 36.7 ℃ after 20ml,50w, and 20Hz of samples obtained in example 1 (5) were sonicated for different times. See table 2.
TABLE 2 Zeta potential for samples diluted at different times
Time of sonication, min | 5 | 10 | 15 | 20 | 25 | 30 |
Zeta potential, mV | 11.20 | 11.22 | 11.21 | 11.19 | 11.18 | 11.19 |
Example 3:
(1) Particle size determination after conjugation with N-acetylneuraminic acid
20ml of each sample was taken at 36.7 ℃ and added to an aqueous solution containing 0.5% NaCl and 2% N-acetylneuraminic acid, the samples were allowed to stand for various periods of time, dropped on a copper mesh having a support film, excess liquid was removed by filter paper, heavy metal complex dye was dropped again, excess liquid was removed by filter paper, dried naturally for 45min, and the particle size of the fine particles was measured by scanning electron microscopy. See table 3.
TABLE 3 average particle size of microparticles bound to N-acetylneuraminic acid and then allowed to stand for various periods of time
Rest time, h | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Average particle diameter, um | 0.22 | 0.27 | 0.31 | 0.34 | 0.35 | 0.35 | 0.35 |
(2) Zeta potential determination after binding to N-acetylneuraminic acid
20ml of the sample solution was taken at 36.7 ℃ and added to an aqueous solution containing 0.5% of NaCl and 2% of N-acetylneuraminic acid, and the Zeta potential of the sample was measured at different times. See table 4.
TABLE 4 Zeta potential of stationary samples at different times after binding to N-acetylneuraminic acid
Rest time, h | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Zeta potential, mV | -1.28 | -1.75 | -2.04 | -2.10 | -2.14 | -2.14 | -2.15 |
Claims (1)
1. A preparation method of a graphene nano-drug carrier with dual targeting factors of folic acid and wheat germ agglutinin, which is prepared by taking graphene oxide, 6-carboxymethyl-gamma-cyclodextrin, folic acid and wheat germ agglutinin as main raw materials, comprises the following steps:
(1) Acylchlorinated graphene oxide
Dispersing 10.00g of graphene oxide with the purity of 99%, the thickness of less than or equal to 5nm and the size of 2-8um in 100ml of chloroform, heating to 55 ℃, respectively and slowly dripping 5.50g of thionyl chloride and 2.00g of N-methyl pyrrolidone under stirring, heating to boiling, carrying out reflux reaction for 3 hours, cooling to 25 ℃, centrifuging for 10 minutes, removing supernatant, scraping out precipitate, freezing and drying to obtain brown solid powder, and storing for later use;
(2) L-lysine grafted graphene oxide
Adding 5.00g of L-lysine into 50ml of distilled water, stirring and dissolving at 25 ℃, cooling to 10 ℃, slowly adding the product obtained in the step (1), stirring and reacting for 5 hours, then heating to 30 ℃, continuing stirring and reacting for 2 hours, cooling to 25 ℃, filtering, washing for 3 times with absolute ethyl alcohol, and freeze-drying to obtain white solid powder for later use;
(3) Reduction of L-lysine grafted graphene oxide
At a temperature of 25 ℃, taking100ml of distilled water was added with N 2 10 minutes, 20.00g of L-ascorbic acid are added in N 2 Stirring and dissolving under flowing, adding 5.00g of the sample obtained in the step (2), keeping the temperature at 25 ℃, stirring and reacting for 24h, filtering, washing with distilled water for 6 times, and drying in vacuum to obtain black solid powder for later use;
(4) Grafting of 6-carboxymethyl-gamma-cyclodextrin and folic acid
Adding 2.50g of 6-carboxyl-gamma-cyclodextrin and 0.50g of folic acid into 50ml of distilled water, stirring at room temperature to dissolve, adding the sample obtained in the step (3), and adding the mixture in N 2 Stirring and dispersing under the flowing-down condition, cooling to 0 ℃, slowly adding 3.00g30% EDC aqueous solution and 2.00g10% NHS aqueous solution, reacting at 0 ℃ for 2h, then reacting at 25 ℃ for 48h, filtering, washing with distilled water for 3 times, and drying in vacuum to obtain black solid powder for later use;
(5) Assembly of WGA
And (3) dispersing the product obtained in the step (4) in 500ml of water for injection, adjusting the freezing point of a NaCl solution to be-0.52 ℃, adding 0.2gWGA and 50Hz, performing ultrasonic oscillation for 1h to obtain a black suspension, and sealing and storing.
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CN104940142A (en) * | 2015-07-13 | 2015-09-30 | 河南中医学院 | Preparing method of matrine nanoparticles modified by wheat germ agglutinin and application of matrine nanoparticles |
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