CN113896941B - PH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere and preparation method thereof - Google Patents

PH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere and preparation method thereof Download PDF

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CN113896941B
CN113896941B CN202111318154.1A CN202111318154A CN113896941B CN 113896941 B CN113896941 B CN 113896941B CN 202111318154 A CN202111318154 A CN 202111318154A CN 113896941 B CN113896941 B CN 113896941B
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paclitaxel
porous cellulose
cellulose
molecularly imprinted
temperature
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CN113896941A (en
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付玉杰
曹劲松
赵春建
焦骄
顾成波
武晓丹
秦祥宇
邵冠松
王涛
常远航
秦冰洋
王子晗
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Northeast Forestry University
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Abstract

The invention belongs to the technical field of imprinted adsorption materials, and particularly relates to a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere and a preparation method thereof, wherein the modified porous cellulose hydrogel microsphere is obtained by mixing a cellulose solution, a sodium alginate solution and cage-type polysilsesquioxane, performing reversed-phase suspension polymerization, and crosslinking with a crosslinking agent; then, a temperature-sensitive substance is grafted on the surface of the modified porous cellulose hydrogel microsphere, so that the paclitaxel molecular imprinting adsorption polymer prepared by the invention has dual responses of pH and temperature. The pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the invention have higher adsorption capacity and desorption rate.

Description

PH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere and preparation method thereof
Technical Field
The invention belongs to the technical field of imprinted adsorption materials, and particularly relates to a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere and a preparation method thereof.
Background
Paclitaxel is a natural diterpenoid alkaloid with unique chemical structure, and can be widely used for treating various cancers such as ovarian cancer, breast cancer, lung cancer, etc. In recent years, the demand for paclitaxel has increased, but the production capacity is far from being sufficient. In addition, the content of paclitaxel in plants is very low, and the complexity of the composition and structure thereof adds great difficulty to the enrichment and separation of paclitaxel, so that it is necessary to prepare a material capable of selectively adsorbing and separating paclitaxel. Molecularly imprinted polymer materials are a new type of targeted adsorbent, which has received increasing attention due to recognition specificity. In particular, the development of molecularly imprinted materials for paclitaxel enrichment has made great progress.
In the prior art, paclitaxel is taken as a template molecule, and reversible addition-fragmentation chain transfer precipitation polymerization (RAFTPP) is utilized to prepare a corresponding paclitaxel molecularly imprinted polymer, however, the paclitaxel molecularly imprinted material prepared by the preparation method has the problems of low selective adsorption sensitivity on paclitaxel, and further lower adsorption capacity and desorption rate.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a preparation method of a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere, which comprises the following steps:
mixing an oily medium and an emulsifier to obtain an emulsion;
mixing the emulsion with a cellulose solution, a sodium alginate solution and cage type polysilsesquioxane, and carrying out reversed phase suspension polymerization to obtain primary modified cellulose;
adjusting the pH value of the primary modified cellulose to be neutral, carrying out a first crosslinking reaction on an obtained product and an aldehyde crosslinking agent, and soaking the obtained first crosslinked solid in hot water to obtain modified porous cellulose hydrogel microspheres;
mixing the modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium crosslinking agent, and carrying out a second crosslinking reaction to obtain a second crosslinked solid;
mixing the second crosslinking solid with water, N-isopropylacrylamide, a functional monomer, paclitaxel, an initiator and an organic amine crosslinking agent, and carrying out a third crosslinking reaction to obtain a primary paclitaxel polymer;
and removing the paclitaxel in the primary paclitaxel polymer to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres.
Preferably, the concentration of the cellulose solution is 3-5 wt%; the concentration of the sodium alginate solution is 2-4 wt%.
Preferably, the mass ratio of the cellulose solution to the sodium alginate solution to the cage-type polysilsesquioxane is 79-84: 10-15: 1 to 2.
Preferably, the temperature of the first crosslinking reaction is 20-30 ℃ and the time is 40-60 min.
Preferably, in the raw materials of the second crosslinking reaction, the mass ratio of the modified porous cellulose hydrogel microspheres to the paclitaxel is 6-10: 1.
preferably, the functional monomer comprises 4-vinylpyridine, 2-vinylpyridine, methacrylic acid or acrylamide.
Preferably, the organic amine-based crosslinking agent comprises a mixture of N, N' -tetramethylethylenediamine and N, N-methylenebisacrylamide.
The invention also provides the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the preparation method.
Preferably, the pore size of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere is 6-8 nm.
The invention also provides application of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the preparation method as a solid phase extraction small column filler in paclitaxel adsorption.
The invention provides a preparation method of a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere, which comprises the following steps:
mixing an oily medium and an emulsifier to obtain an emulsion; mixing the emulsion with a cellulose solution, a sodium alginate solution and cage type polysilsesquioxane, and carrying out reversed phase suspension polymerization to obtain primary modified cellulose; adjusting the pH value of the primary modified cellulose to be neutral, carrying out a first crosslinking reaction on an obtained product and an aldehyde crosslinking agent, and soaking the obtained first crosslinked solid in hot water to obtain modified porous cellulose hydrogel microspheres; mixing the modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium crosslinking agent, and carrying out a second crosslinking reaction to obtain a second crosslinked solid; mixing the second crosslinking solid with water, N-isopropylacrylamide, a functional monomer, paclitaxel, an initiator and an organic amine crosslinking agent, and carrying out a third crosslinking reaction to obtain a primary paclitaxel polymer; and removing the paclitaxel in the primary paclitaxel polymer to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres.
According to the invention, sodium alginate, cellulose and cage type polysilsesquioxane are organically combined together through reverse suspension polymerization, the sodium alginate is polyanion polysaccharide salt, the hydrogen bonding effect among sodium alginate molecules is easily influenced by pH value, the negative charge density on the surface of the sodium alginate molecules is increased along with the increase of the pH value, so that the electrostatic repulsion among anions of the sodium alginate is increased, the strength of the hydrogen bonding effect among the molecules is weakened, and the sensitivity to the pH value is shown. The cellulose molecules are rich in hydroxyl, and the sodium alginate and the cellulose solution are blended and modified by utilizing the characteristic, so that the obtained sodium alginate modified porous cellulose hydrogel microspheres have pH value response characteristics. Within a certain pH range, the polymer hydrogel microspheres prepared by the invention have no structure change, have a specific recognition effect on target paclitaxel, accurately adsorb the target paclitaxel, and adjust the pH value when desorbing, so that the structure of the microspheres is changed, and the paclitaxel is adsorbed by alcoholysis.
In addition, the cage-type polysilsesquioxane has a specific adsorption effect on the paclitaxel, and the introduction of the cage-type polysilsesquioxane is beneficial to the adsorption of the porous cellulose hydrogel microspheres on the paclitaxel. Meanwhile, the active functional group of the cage-type polysilsesquioxane can form hydrogen bond action with the paclitaxel, so the cellulose microsphere blended with the cage-type polysilsesquioxane also has certain specific adsorption action on the paclitaxel. The modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium cross-linking agent are mixed and cross-linked, paclitaxel is firstly combined with cage-type polysilsesquioxane having a specific adsorption effect on the paclitaxel, so that the paclitaxel is firstly distributed in the microspheres to a certain extent, and then the obtained second cross-linked solid and the paclitaxel are cross-linked again, so that the distribution of the paclitaxel in the microspheres is further improved.
And N-isopropyl acrylamide is used as an acrylamide derivative monomer, has a low critical solution temperature due to hydrophilic amide groups and hydrophobic isopropyl groups, is about 37 ℃, and can be structurally converted when the external environment is higher than the critical solution temperature, so that the temperature sensitivity is shown. According to the invention, N-isopropylacrylamide and cellulose hydrogel are utilized to form an interpenetrating network structure, and when the external environment is lower than the critical dissolving temperature of the N-isopropylacrylamide, the structure of the polymer hydrogel microspheres prepared by the invention is not changed, the polymer hydrogel microspheres have a specific recognition effect on target paclitaxel, and can be accurately adsorbed; when the external environment is higher than the critical dissolving temperature of the N-isopropylacrylamide, the structure of the polymer hydrogel microsphere prepared by the invention is transformed, and the taxus chinensis is absorbed out by alcoholysis.
Therefore, the imprinted polymer prepared by the preparation method can realize the accurate enrichment of the paclitaxel by adjusting the temperature and the pH value, further improve the adsorption capacity of the paclitaxel, and simultaneously realize the efficient elution of the paclitaxel by adjusting the temperature and the pH value.
In addition, the material has simple and easy synthesis method and low manufacturing cost, and is beneficial to large-scale industrial production.
Drawings
FIG. 1 is an infrared spectrum of modified porous cellulose hydrogel microspheres, pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres, and non-imprinted hydrogel microspheres;
FIG. 2 is a scanning electron microscope image of a pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere;
FIG. 3 is a physical diagram of a pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere molecularly imprinted material prepared in example 1;
FIG. 4 is a pH response test chart of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere molecularly imprinted material prepared in example 1;
fig. 5 and 6 are temperature condition test charts of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere molecularly imprinted material prepared in example 1.
Detailed Description
The invention provides a preparation method of a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere, which comprises the following steps.
Mixing an oily medium and an emulsifier to obtain an emulsion;
mixing the emulsion with a cellulose solution, a sodium alginate solution and cage type polysilsesquioxane, and carrying out reversed phase suspension polymerization to obtain primary modified cellulose;
adjusting the pH value of the primary modified cellulose to be neutral, carrying out a first crosslinking reaction on an obtained product and an aldehyde crosslinking agent, and soaking the obtained first crosslinked solid in hot water to obtain modified porous cellulose hydrogel microspheres;
mixing the modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium crosslinking agent, and carrying out a second crosslinking reaction to obtain a second crosslinked solid;
mixing the second crosslinking solid with water, N-isopropylacrylamide, a functional monomer, paclitaxel, an initiator and an organic amine crosslinking agent, and carrying out a third crosslinking reaction to obtain a primary paclitaxel polymer;
and removing the paclitaxel in the primary paclitaxel polymer to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres.
The invention mixes the oily medium and the emulsifier to obtain the emulsion.
In the invention, the oily medium preferably comprises liquid paraffin or No. 10 transformer oil; in the present invention, the emulsifier preferably comprises tween 80 and/or span 80; in the invention, the dosage ratio of the liquid paraffin to the emulsifier is preferably 120-200 mL: 2-3 g, and more preferably 120-130 mL: 2-3 g.
In the invention, the mixing mode is preferably mechanical stirring, and the rotating speed of the mechanical stirring is preferably 300-500 rpm, and more preferably 300-400 rpm; the mechanical stirring time is preferably 30-40 min, and more preferably 30-35 min. In the invention, the mixing temperature is preferably 50-70 ℃, and more preferably 50-60 ℃.
After the emulsion is obtained, the emulsion is mixed with a cellulose solution, a sodium alginate solution and cage type polysilsesquioxane for inverse suspension polymerization to obtain the primary modified cellulose.
In the present invention, the preparation of the cellulose solution preferably includes mixing cellulose and a sodium hydroxide/urea aqueous solution, and centrifuging and defoaming the mixture to obtain the cellulose solution. The mixing operation and parameters are preferably the same as those for preparing the emulsion and will not be described in detail herein. In the present invention, the concentration of the cellulose solution is preferably 3 to 5 wt%, and more preferably 3.5 to 4 wt%. In the present invention, the source of the cellulose includes microcrystalline cellulose, cotton, wood, cotton linters, wheat straw, reed, hemp, mulberry bark, paper mulberry bark or bagasse.
In the invention, the preparation of the sodium alginate solution preferably comprises mixing sodium alginate and a sodium hydroxide/urea aqueous solution, and centrifuging and defoaming to obtain the sodium alginate solution. The mixing operation and parameters are preferably the same as those for preparing the emulsion and will not be described in detail herein. In the invention, the concentration of the sodium alginate solution is preferably 2-4 wt%, and more preferably 3.5-4 wt%.
In the present invention, the cage-type polysilsesquioxane preferably includes a vinyl cage-type polysilsesquioxane and/or a methyl propenyl cage-type polysilsesquioxane, and more preferably a methyl propenyl cage-type polysilsesquioxane.
In the invention, the mass ratio of the cellulose solution to the sodium alginate solution to the cage-type polysilsesquioxane is preferably 79-84: 10-15: 1-2, more preferably 84: 15: 1.
in the present invention, the reverse phase suspension polymerization is preferably carried out by stirring, and the rotation speed of the stirring is preferably 300 to 500rpm, and more preferably 300 to 400 rpm; in the invention, the time of the reversed-phase suspension polymerization is preferably 2.5 to 3.5 hours, and more preferably 2.8 to 3 hours.
After the primary modified cellulose is obtained, the pH value of the primary modified cellulose is adjusted to be neutral, and the obtained product and an aldehyde crosslinking agent are subjected to a first crosslinking reaction to obtain the modified porous cellulose hydrogel microspheres.
In the invention, the preferred pore size of the modified porous cellulose hydrogel microspheres is 6-8 nm.
In the present invention, the aldehyde-based crosslinking agent preferably includes glutaraldehyde and/or glyoxal. In the present invention, the ratio of the aldehyde crosslinking agent to the cellulose solution is preferably 10 mL: 79-84 g, more preferably 10 mL: 84 g.
In the invention, the temperature of the first crosslinking reaction is preferably 20-30 ℃, more preferably 25-30 ℃, and the time is 30-60 min, more preferably 30-40 min.
After the first crosslinking reaction, the present invention preferably further comprises sieving the solid obtained by the first crosslinking reaction, and soaking in hot water. In the invention, the aperture of the sieving screen is 60 meshes. In the invention, the temperature of the hot water soaking is preferably 80-100 ℃, and more preferably 90-100 ℃. In the invention, the time for soaking in hot water is preferably 4.5-6.5 hours, and more preferably 5-6 hours.
After the modified porous cellulose hydrogel microspheres are obtained, the modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium crosslinking agent are mixed for a second crosslinking reaction to obtain a second crosslinked solid.
In the invention, the modified porous cellulose hydrogel microspheres are preferably prepared by using a methanol solution of the modified porous cellulose hydrogel microspheres. In the present invention, the paclitaxel is preferably performed using a methanol solution of paclitaxel. In the invention, the concentration of the modified porous cellulose hydrogel microspheres in the methanol solution of the modified porous cellulose hydrogel microspheres is preferably 15-25 mg/mL, and more preferably 18-22 mg/mL. In the invention, during the second crosslinking reaction, the mass ratio of the modified porous cellulose hydrogel microspheres to the paclitaxel is 6-10: 1, and more preferably 8-10: 1.
In the present invention, the inorganic calcium-based crosslinking agent preferably includes a saturated aqueous solution of calcium sulfate or a saturated aqueous solution of calcium carbonate.
In the invention, the mixing preferably comprises the steps of carrying out first mixing on the modified porous cellulose hydrogel microspheres, paclitaxel and methanol, and carrying out second mixing on the obtained mixed system and the inorganic calcium crosslinking agent to obtain a second crosslinking reaction system.
In the present invention, the first mixing is preferably performed by stirring, the first mixing is preferably performed under a dark condition, and the time for the first mixing is preferably 7.5 to 8.5 hours, and more preferably 8 to 8.5 hours. In the invention, the temperature of the second crosslinking reaction is preferably 20-30 ℃, more preferably 25-30 ℃, and the time is 40-70 min, more preferably 60-70 min.
After the second cross-linked solid is obtained, the second cross-linked solid is mixed with water, N-isopropylacrylamide, a functional monomer, paclitaxel, an initiator and an organic amine cross-linking agent to carry out a third cross-linking reaction, so as to obtain a primary paclitaxel polymer.
In the invention, the water is preferably deionized water; in the invention, the dosage ratio of the second crosslinking solid to water is preferably 500-700 mg: 15-25 mL, more preferably 550-650 mg: 15-25 mL.
In the invention, the mass ratio of the second crosslinked solid to the N-isopropylacrylamide is preferably 500-700: 80 to 100, more preferably 550 to 650: 85 to 95.
In the invention, the mass ratio of the second crosslinked solid to the paclitaxel is preferably 500-700: 40 to 50, more preferably 600 to 650: 40-45.
In the present invention, the functional monomer preferably includes 4-vinylpyridine, 2-vinylpyridine, methacrylic acid or acrylamide, and more preferably 4-vinylpyridine; in the invention, the molar ratio of the paclitaxel to the functional monomer is preferably 1: 4-10, and more preferably 1: 10.
In the present invention, the initiator preferably comprises ammonium persulfate and/or sodium persulfate; in the invention, the mass ratio of the second crosslinking solid to the initiator is preferably 500-700: 30 to 60, more preferably 550 to 650: 40-60.
The organic amine crosslinking agent preferably comprises a mixture of N, N, N ', N' -tetramethylethylenediamine and N, N-methylenebisacrylamide; the dosage ratio of N, N, N ', N' -tetramethyl ethylenediamine to N, N-methylene bisacrylamide in the organic amine crosslinking agent is preferably 100-150 mu L: 30-100 mg. In the invention, the dosage ratio of the second crosslinked solid to the N, N, N ', N' -tetramethylethylenediamine and the N, N-methylenebisacrylamide is preferably 500-700 mg: 100-150 μ L: 30-100 mg.
In the invention, the temperature of the third crosslinking reaction is preferably 20-30 ℃, more preferably 25-30 ℃, and the time is 20-25 h, more preferably 24-25 h.
After the primary paclitaxel polymer is obtained, the invention removes the paclitaxel in the primary paclitaxel polymer to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere.
In the present invention, said removing paclitaxel from said primary paclitaxel polymer preferably comprises mixing the primary paclitaxel polymer with a solution of acetic acid and methanol, and ultrasonically eluting.
In the present invention, the mass concentration of acetic acid in the acetic acid methanol solution is 8 to 15 wt%, and more preferably 10 to 15 wt%. The operation and parameters of the ultrasonic elution are not particularly limited, and the paclitaxel can be completely removed.
After the ultrasonic elution, the invention preferably utilizes a high performance liquid chromatography to detect the content of the paclitaxel, and if the paclitaxel is not completely removed, the operation of removing the paclitaxel is repeated.
The invention also provides the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere prepared by the preparation method of the technical scheme.
In the invention, the preferable pore size of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere is 6-8 nm.
The invention also provides application of the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the technical scheme as a solid-phase extraction small column filler.
The pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted polymer microspheres and the preparation method thereof provided by the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1:
4g of microcrystalline cellulose and a sodium hydroxide/urea aqueous solution (the mass ratio of sodium hydroxide to urea to water is 12:7:81) are uniformly mixed, and the mixture is subjected to centrifugal deaeration to obtain a cellulose solution.
4g of sodium alginate and a sodium hydroxide/urea aqueous solution (the mass ratio of sodium hydroxide to urea to water is 12:7:81) are uniformly mixed, and the sodium alginate solution is obtained after centrifugal deaeration.
Stirring 125mL of liquid paraffin and 2.5g of Tween 80 at the rotation speed of 300rpm and the temperature of 60 ℃ for 30min, adding a cellulose solution, a sodium alginate solution and methyl propenyl cage type polysilsesquioxane (the mass ratio is 84 g: 15 g: 1g), continuously stirring for 3h, adding hydrochloric acid to adjust the pH value to be neutral, then adding 10mL of glutaraldehyde, and carrying out a first crosslinking reaction (the crosslinking reaction temperature is 25 ℃ and the crosslinking reaction time is 30 min).
And (3) sieving the solid obtained by the crosslinking reaction with a 60-mesh sieve, and soaking in hot water for 5 hours to obtain the modified porous cellulose hydrogel microspheres (the pore diameter of the porous cellulose hydrogel microspheres is 6-8 nm).
Mixing 400mg of modified porous cellulose hydrogel microspheres, 42.4mg of paclitaxel and 20mL of methanol, stirring for 8h in a dark place, adding 50mL of calcium sulfate saturated aqueous solution, and carrying out a second crosslinking reaction (the temperature of the crosslinking reaction is 25 ℃ and the time is 1h) to obtain a second crosslinked solid.
The resultant second crosslinked solid (600 mg) was mixed with 20mL of water, 90mg of N-isopropylacrylamide, 52.9. mu.L of 4-vinylpyridine, 42.4mg of paclitaxel, 50mg of ammonium persulfate, 100. mu. L N, N, N ', N' -tetramethylethylenediamine, and 30mg of N, N-methylenebisacrylamide, and subjected to a third crosslinking reaction (under nitrogen, at a temperature of 25 ℃ C., for 24 hours) to obtain a primary paclitaxel polymer.
Mixing the obtained primary paclitaxel polymer and acetic acid methanol solution (the mass concentration of acetic acid is 10 wt%), ultrasonically eluting, detecting by high performance liquid chromatography, and completely removing paclitaxel to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere.
Comparative example 1
Comparative example 1 differed from example 1 only in that the sodium alginate solution was replaced with a chitosan solution, and as a result: the porous cellulose-based paclitaxel molecularly imprinted polymer microspheres cannot be obtained.
Comparative example 2
Comparative example 2 differs from example 1 only in that the sodium alginate solution was replaced with gelatin, and as a result: the porous cellulose-based paclitaxel molecularly imprinted polymer microspheres cannot be obtained.
Comparative example 3
Comparative example 3 differs from example 1 only in that N-isopropylacrylamide was replaced with polylactic acid, and as a result: the porous cellulose-based paclitaxel molecularly imprinted polymer microspheres cannot be obtained.
The invention also carries out infrared test on the modified porous cellulose hydrogel microspheres prepared in the example 1, as shown in figure 1, and as can be seen from figure 1: 1735cm in infrared spectrum -1 The stretching vibration peak of the Si-O bond proves the success of grafting the cellulose and the methyl propenyl cage polysilsesquioxane; 2922cm -1 Carbon-carbon double bond stretching vibration ofThe peaks demonstrate the success of the grafting of microcrystalline cellulose and sodium alginate.
FIG. 2 is a scanning electron microscope image of a pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere; as can be seen from fig. 2: the prepared porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres with double responses to pH and temperature have rich imprinted pores and can have a good adsorption effect on paclitaxel.
FIG. 3 is a physical diagram of a pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere molecularly imprinted material prepared in example 1; as can be seen from fig. 3: the prepared porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere particles with double responses of pH and temperature are uniform.
The invention also performs performance test on the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the embodiment, and the test method comprises the following steps:
(1) targeted enrichment of paclitaxel in Taxus media
Pulverizing Taxus x media into powder 2g with grinder, extracting with 20mL 80% methanol under microwave assistance for 30min for 3 times, and mixing the supernatants to obtain crude extract.
Removing methanol from the crude extract by rotary evaporation, mixing the obtained solid with 30mL of a mixed solution of ethyl acetate and water (wherein the volume ratio of ethyl acetate to water is 1:1), extracting, and taking an ethyl acetate phase; and then ethyl acetate is removed by phase rotary evaporation, and the obtained solid is subjected to constant volume to 10mL by using methanol to obtain a crude extract solution.
(2) Assembling a solid phase extraction column: 100mg of the product prepared in example 1 was loaded into a 1mL syringe type device made of polyethylene, each having a sieve plate made of polypropylene on the top and bottom, and a quark valve for controlling the flow rate was attached to the bottom of the syringe.
(3) Enrichment of
Activating a solid phase extraction column: the column was pre-washed with 30mL of methanol at a flow rate of 1 drop/sec, and the sample was applied when the liquid phase was 2mm from the upper sieve plate.
Loading: controlling the temperature of the crude extract solution to 40 ℃, adjusting the pH of the crude extract solution to 4.5 by using a PBS buffer solution, enabling all the crude extract solution to pass through a column, controlling the flow rate to be 0.5mL/min, and collecting an effluent liquid to be detected.
Leaching: the elution was carried out with 30mL of methanol at 40 ℃ and the eluate was discarded.
And (3) elution: eluting with 10mL of methanol at 30 deg.C and pH of 7.5 at a flow rate of 1mL/min, draining, and collecting all eluates. The concentration of paclitaxel in the solution of each step was determined by high performance liquid chromatography.
TABLE 1 concentration of paclitaxel in different solutions
Figure BDA0003344491960000111
The invention also tests the pH response and temperature response conditions of the obtained pH/temperature dual-response porous cellulose-based paclitaxel molecule, and the test method is to use different temperatures and pH values to probe the adsorption and desorption functions of the material, thereby obtaining the influence of the pH value and the temperature on the adsorption amount and the desorption rate.
The pH response test method is to select a pH value within the range of 1.5-10.5 and test the adsorption and desorption performance of the material, and the test result is shown in figure 4.
The temperature response test method is to select the temperature within the range of 20-55 ℃ to test the adsorption and desorption performance of the material. The test results are shown in FIGS. 5 to 6.
As can be seen from FIGS. 4 to 6, the adsorption amount of paclitaxel was the highest at 40 ℃ and pH 4.5, and the desorption efficiency was the highest at 30 ℃ and pH 7.5.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A preparation method of a pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted polymer microsphere is characterized by comprising the following steps:
mixing an oily medium and an emulsifier to obtain an emulsion;
mixing the emulsion with a cellulose solution, a sodium alginate solution and cage type polysilsesquioxane, and carrying out reversed phase suspension polymerization to obtain primary modified cellulose;
adjusting the pH value of the primary modified cellulose to be neutral, carrying out a first crosslinking reaction on an obtained product and an aldehyde crosslinking agent, and soaking the obtained first crosslinked solid in hot water to obtain modified porous cellulose hydrogel microspheres;
mixing the modified porous cellulose hydrogel microspheres, paclitaxel and an inorganic calcium crosslinking agent, and carrying out a second crosslinking reaction to obtain a second crosslinked solid;
mixing the second crosslinking solid with water, N-isopropylacrylamide, a functional monomer, paclitaxel, an initiator and an organic amine crosslinking agent, and carrying out a third crosslinking reaction to obtain a primary paclitaxel polymer;
and removing the paclitaxel in the primary paclitaxel polymer to obtain the pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres.
2. The preparation method according to claim 1, wherein the concentration of the cellulose solution is 3 to 5 wt%; the concentration of the sodium alginate solution is 2-4 wt%.
3. The preparation method of claim 1, wherein the mass ratio of the cellulose solution to the sodium alginate solution to the cage-type polysilsesquioxane is 79-84: 10-15: 1 to 2.
4. The method according to claim 1, wherein the first crosslinking reaction is carried out at a temperature of 20 to 30 ℃ for 30 to 60 minutes.
5. The preparation method according to claim 1, wherein in the raw material of the second crosslinking reaction, the mass ratio of the modified porous cellulose hydrogel microspheres to paclitaxel is 6-10: 1.
6. the method of claim 1, wherein the functional monomer comprises 4-vinylpyridine, 2-vinylpyridine, methacrylic acid, or acrylamide.
7. The method of claim 1, wherein the organic amine-based crosslinking agent comprises a mixture of N, N, N ', N' -tetramethylethylenediamine and N, N-methylenebisacrylamide.
8. The pH/temperature dual-response porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres prepared by the preparation method according to any one of claims 1 to 7.
9. The pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microsphere according to claim 8, wherein the pore size is 6-8 nm.
10. The use of the pH/temperature dual-responsive porous cellulose-based paclitaxel molecularly imprinted hydrogel microspheres of claim 8 or 9 as a filler of a solid phase extraction column for adsorbing paclitaxel.
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