CN112191232A

CN112191232A - Cellulose microsphere adsorbent, preparation method thereof and blood perfusion apparatus

Info

Publication number: CN112191232A
Application number: CN202010972006.0A
Authority: CN
Inventors: 董凡; 蔡礼桃; 毕大武
Original assignee: Zhuhai Jianke Medical Materials Co ltd
Current assignee: Jafron Biomedical Co Ltd
Priority date: 2020-09-16
Filing date: 2020-09-16
Publication date: 2021-01-08
Anticipated expiration: 2040-09-16
Also published as: CN112191232B

Abstract

The invention relates to a cellulose microsphere adsorbent, a preparation method thereof and a hemoperfusion device. The preparation method comprises the steps of primary pore forming, secondary pore forming and the like. The cellulose microsphere adsorbent can adsorb beta 2-microglobulin, can reduce the adsorption of beneficial components in blood such as albumin and globulin with large molecular weight, and improves the treatment safety.

Description

Cellulose microsphere adsorbent, preparation method thereof and blood perfusion apparatus

Technical Field

The invention relates to the technical field of blood purification, in particular to a cellulose microsphere adsorbent, a preparation method thereof and a blood perfusion device.

Background

Uremia is a series of complex clinical complex diseases generated by irreversible decline of renal function gradually caused by various renal diseases in the body and final loss of renal function, incapability of timely discharging and massive aggregation of in vivo metabolic wastes such as beta 2-microglobulin (beta 2-MG) and the like, wherein beta 2-MG accumulation causes amyloidosis and carpal tunnel syndrome, PTH accumulation causes renal bone diseases and ectopic calcification, IL-6 accumulation increases and causes systemic chronic inflammatory reactions, and more researches show that the inflammatory reactions are related to arteriosclerosis and malnutrition.

Blood purification is still the main current clinical treatment method for uremia, including Hemodialysis (HD), Hemoperfusion (HP), and hemodialysis combined hemoperfusion (HD + HP). Compared with hemodialysis for removing solute mainly through a diffusion mode, hemoperfusion not only has higher removing effect on poison and fat-soluble poison with high protein binding rate, but also has certain effect of removing free poison in blood, can effectively remove middle and large molecular toxins in the body of a uremia patient, such as beta 2-MG, PTH, AGEs, Hcy, IL-6 and the like, has better application value, and is worthy of clinical popularization and application.

The blood perfusion requires good blood compatibility, strong specificity, large adsorption capacity and the like of the adsorbent, wherein the key points are the selection of a carrier, the control of a pore structure, the selection of a ligand and the grafting modification. The adsorbent carriers mainly used for removing beta 2-MG by blood perfusion at the present stage comprise active carbon, polystyrene-divinylbenzene resin, chitosan, cellulose and the like. The activated carbon adsorbent has poor selectivity, poor mechanical strength and easy breakage, and particles fall off during use to cause safety risk; the most common is polystyrene-divinylbenzene resin, such as that related to chinese patent applications CN108371945A, CN105504131A, CN104941607A, and CN104174386A, but polystyrene-divinylbenzene resin uses toxic styrene and divinylbenzene as monomers in the synthesis process, commonly uses reagents such as toluene, liquid paraffin, and solvent oil as pore-forming agents, and uses highly toxic chloromethyl ether as chloromethylation reagent, and the organic reagent residue will also cause safety risk, and meanwhile, a large amount of waste water and waste gas are generated in the adsorbent production process, which causes serious damage to ecology and environment, and the production personnel also have the problem of safety accident caused by contacting and inhaling toxic steam. Cellulose is the most abundant and reproducible natural high molecular compound in nature, has the advantages of low price, degradability, strong environmental friendliness, good biocompatibility, blood compatibility and the like, and the spherical adsorbent prepared by using the cellulose and the derivatives thereof can solve the problems.

In the aspect of the pore structure of the adsorbent, solid pore-foaming agent or liquid pore-foaming agent is adopted to perform pore-foaming in one step in the prior art, and the pore structures of the outer layer and the inner layer of the adsorbent at different positions have no obvious difference. If the pore size is large, macromolecular substances in blood such as albumin, globulin and even blood cells can enter the pore channel in the using process, so that the normal blood composition is influenced, and a large safety risk exists. If the pore diameter is smaller, the pore space is limited after the substances to be adsorbed enter the pore channel through the action of the molecular sieve, and the adsorption capacity is influenced.

Disclosure of Invention

In view of the defects of the prior art, the first object of the present invention is to provide a cellulose microsphere adsorbent, which can adsorb beta 2-microglobulin, and at the same time, can reduce the adsorption of beneficial components in blood, such as albumin and globulin with large molecular weight, and improve the treatment safety; the second object of the present invention is to provide a method for preparing the above cellulose microsphere adsorbent; the third purpose of the invention is to provide a hemoperfusion apparatus containing the cellulose microsphere adsorbent.

In order to achieve the first object of the invention, the invention provides a cellulose microsphere adsorbent, which is of a core-shell structure and comprises an inner core and an outer shell layer wrapping the inner core, wherein the outer shell layer and the inner core have a continuous through hole structure, and the aperture of the through hole of the outer shell layer is smaller than that of the through hole of the inner core.

Therefore, the invention provides the cellulose microsphere adsorbent with the core-shell structure, the aperture of the through hole of the outer shell layer is smaller than that of the through hole of the inner core, and the beneficial protein and blood cells with larger molecular weight can be blocked by the molecular sieving function of the pore canal of the shell layer, so that the adsorption caused by entering the hole is reduced; the pore size of the inner core is large, which is beneficial to the diffusion and adsorption of beta 2-microglobulin in the pore canal, and the adsorption capacity is higher. Through the screening effect of different pore diameters of the outer shell and the inner core, the cellulose microsphere adsorbent can selectively adsorb beta 2-microglobulin in blood, reduces beneficial ingredients such as albumin and globulin with large molecular weight in the blood, and has high treatment safety.

The further technical proposal is that the aperture of the through hole of the inner core is 50nm to 3000nm, preferably 200nm to 1500nm, and more preferably 200nm to 600 nm.

From the above, it can be seen that the adsorption performance of the cellulose microsphere adsorbent of the present invention on β 2-MG can be further improved within the above-mentioned range of the size of the through-core pores. When the core through-hole size is less than the above range value, the adsorption amount decreases. When the pore diameter of the core through-hole exceeds the above range, the specific surface area of the adsorbent decreases, and the adsorption sites decrease.

Further, the pore diameter of the through-hole of the outer shell layer is 1nm to 500nm, preferably 10nm to 100nm, and more preferably 10nm to 50 nm.

From the above, when the size of the through hole of the shell layer is within the range, the adsorption performance of the cellulose microsphere adsorbent of the invention on beta 2-MG can be further improved, and the adsorption on beneficial macromolecular proteins in blood plasma, such as albumin and globulin, can be reduced. When the shell layer through-hole is less than the above range, the entry of β 2-microglobulin into the pore channel is blocked, and the adsorption rate of β 2-MG decreases. When the through hole of the shell layer exceeds the range, macromolecular protein is easier to enter the pore channel to be adsorbed, so that beta 2-microglobulin is prevented from entering the pore channel, and the normal blood composition is influenced.

The further technical proposal is that the thickness of the outer shell layer is 0.2 to 50 percent of the diameter of the inner core, and preferably 1 to 10 percent.

From the above, within the range of the ratio of the thickness of the outer shell layer to the diameter of the inner core, the adsorption performance of the cellulose microsphere adsorbent of the invention on beta 2-microglobulin can be further improved, and the adsorption on beneficial macromolecular proteins in blood plasma, such as albumin and globulin, can be reduced. When the ratio of the thickness of the outer shell layer to the diameter of the inner core is lower than the range, the outer shell layer has poor screening effect on albumin and globulin in blood plasma, and albumin, globulin and the like easily enter the pore channel. When the ratio of the thickness of the shell layer to the diameter of the core exceeds the range, the thickness of the shell layer is increased, the diameter of the core is reduced, the macroporous adsorption capacity is reduced, the difficulty of beta 2-microglobulin entering the core is increased, and the adsorption rate of the adsorbent to the beta 2-microglobulin is reduced.

The further technical proposal is that the inner core and the outer shell are both cellulose or cellulose derivatives; the inner surfaces of the inner core through hole and the outer shell through hole are grafted with hydrophobic ligands, and the outer surface of the outer shell is not grafted with hydrophobic ligands; the outer surface of the outer shell layer is connected with hydrophilic groups.

Therefore, the invention further grafts hydrophobic aglucon in the pore canal, and the hydrophobic aglucon can adsorb beta 2-microglobulin through hydrophobic effect. The adsorption of beta 2-microglobulin can be further improved by the screening function of the small pore canal of the shell layer of the microsphere adsorbent and the grafting of the ligand on the inner surface of the pore canal. Hydrophobic ligand is not grafted on the surface of the microsphere shell layer, so that the adsorption of the surface on beneficial proteins in blood is avoided, and the safety is improved. Preferably, the outer surface of the shell layer is connected with hydrophilic groups, so that the blood compatibility of the microsphere is improved, and the adsorption of albumin and globulin is reduced.

The further technical proposal is that the outer surface of the outer shell layer is also coated with a cellulose or cellulose derivative semipermeable membrane, and the cellulose derivative is selected from at least one of cellulose acetate and nitrocellulose.

As can be seen from the above, the outer surface of the adsorbent of the present invention may be coated with a cellulose or cellulose derivative semipermeable membrane, the semipermeable membrane is introduced between the activation step and the ligand grafting step by means of coating, and the cellulose or cellulose derivative semipermeable membrane may be eluted and removed or not, preferably, eluted and removed by a solvent capable of dissolving the membrane layer. When the cellulose derivative semipermeable membrane is not removed, it is preferable that the hydrophobic groups on the cellulose derivative, e.g., cellulose ester semipermeable membrane, are removed with water to become hydrophilic groups, which improves the blood compatibility of the microspheres and reduces adsorption of beneficial proteins such as albumin, globulin, etc.

In order to achieve the second object of the present invention, the present invention provides a preparation method of a cellulose microsphere adsorbent, comprising the following steps:

primary pore forming: preparing a cellulose or cellulose derivative solution, adding a first pore-forming agent, dispersing, heating and curing to form balls, and cleaning to obtain primary pore-forming cellulose microspheres; when the cellulose derivative is cellulose ester, a saponification step is also included after heating and curing to form balls and before cleaning;

secondary pore forming: preparing a cellulose or cellulose derivative solution, adding a second pore-foaming agent, adding primary pore-foaming cellulose microspheres, dispersing, heating and curing to form balls, cleaning, and screening to obtain secondary pore-foaming cellulose microspheres; when the cellulose derivative is cellulose ester, the hydrolysis is carried out after the cellulose derivative is heated and cured into spheres; the secondary pore-forming cellulose microsphere comprises a core with a through hole formed in the primary pore-forming step and an outer shell layer with a through hole formed in the secondary pore-forming step and covering the core, wherein the core through hole is communicated with the outer shell layer through hole; the first porogen has a molecular weight not less than the second porogen.

From the above, the cellulose microsphere adsorbent with core-shell structure with different pore diameters is prepared by two pore-forming steps. Among these, the cellulose derivative may be a cellulose ester, and the cellulose ester may be, for example, cellulose acetate or other cellulose esterification reaction product. When the cellulose ester is adopted, the outer layer of the microsphere is saponified after primary pore forming, and secondary pore forming is carried out, so that the phenomenon that the pores deform and collapse to influence the adsorption performance due to swelling and dissolution of the microsphere when the microsphere is cleaned by using an organic solvent and when the organic solvent is used for secondary pore forming is avoided. The larger the molecular weight of the porogen, the larger the pore diameter of the resulting through-hole, and by selecting the first porogen molecular weight and the second porogen molecular weight, the desired core through-hole diameter and shell layer through-hole diameter can be obtained.

The further technical scheme is that the method also comprises the following steps after the secondary pore-forming step:

and (3) activation: connecting the secondary pore-forming cellulose microspheres with reactive groups;

grafting ligand: grafting a hydrophobic ligand onto the reactive group;

end capping: an end-capping reagent is used to react with the remaining reactive groups.

Therefore, the hydrophobic ligand is further grafted on the secondary pore-forming cellulose microspheres, particularly in the through holes, and can adsorb beta 2-microglobulin through hydrophobic effect.

The further technical proposal is that an enveloping step is also included between the step of activating and the step of grafting the ligand: and forming a cellulose or cellulose derivative semipermeable membrane on the surface of the activated secondary pore-forming cellulose microsphere by using a cellulose or cellulose derivative solution.

Therefore, the invention also comprises an enveloping step, which avoids the hydrophobic ligand from being connected to the surface of the shell layer, avoids the adsorption of the microsphere surface to the blood beneficial protein and improves the treatment safety.

The further technical proposal is that a step of eluting a membrane layer is also included between the step of grafting the ligand and the step of blocking, and the step of eluting the membrane layer comprises eluting a cellulose or cellulose derivative semipermeable membrane; or, when the cellulose derivative in the coating step is cellulose ester, a secondary hydrolysis step is also included between the grafting ligand step and the end capping step, and the secondary hydrolysis step comprises hydrolyzing the ester group of the esterified cellulose; or between the grafting ligand step and the capping step, without an elution membrane layer step or a re-hydrolysis step.

Therefore, after coating and grafting the ligand, the cellulose or cellulose derivative semipermeable membrane is preferably eluted and then blocked, so that the influence of the coating layer on the adsorption performance is avoided; the cellulose or cellulose derivative semipermeable membrane may be left, and when the cellulose derivative semipermeable membrane is a cellulose ester semipermeable membrane, it is preferable to hydrolyze the ester group of the cellulose ester to increase the hydrophilicity of the semipermeable membrane.

The further technical proposal is that the first pore former is selected from one or more of ethyl acetate, butyl acetate, ethylene glycol diacetate, n-hexanol and n-octanol; the second pore-foaming agent is selected from one or more of ethyl acetate, butyl acetate, ethylene glycol diacetate, n-hexanol and n-octanol.

From the above, the first pore-forming agent and the second pore-forming agent of the present invention are selected from common liquid esters and higher alcohols, and have low toxicity.

The further technical scheme is that the activation step comprises the step of reacting the secondary pore-forming cellulose microspheres with an etherification reagent, wherein one end of the etherification reagent and the secondary pore-forming cellulose microspheres form an ether bond, and the other end of the etherification reagent is a reactive group. Therefore, after the microsphere is activated, the reactive group is introduced. Preferably, the ether linkage is formed by etherification reaction and is chemically stable.

The hydrophobic ligand is formed by the reaction of aliphatic long-chain alkylamine and reactive groups, and the carbon number of the aliphatic long-chain alkyl group is more than 8.

When the carbon number of the aliphatic long-chain alkyl group in the hydrophobic ligand is less than 8, for example, when the heptamine (fatty chain contains 7 carbon atoms) is used for grafting, the hydrophobicity of the adsorbent is not obvious, and the adsorption performance is poor; the number of carbon atoms of the aliphatic long-chain alkyl group in the hydrophobic ligand is not less than 8, for example. After n-nonanamine (fatty chain containing 9 carbon atoms) is used for grafting, the adsorption performance is obviously improved.

Therefore, the hydrophobic ligand can be aliphatic long-chain alkyl, can provide hydrophobic performance, and has relatively easily obtained raw materials. The hydrophobic ligand is preferably aliphatic long-chain alkylamine, and amino has high reactivity and can react with reactive groups such as epoxy groups.

The further technical solution is that the capping reagent has a first group capable of reacting with the reactive group and a second group having hydrophilicity.

Therefore, the end capping reagent of the present invention preferably has a hydrophilic group, and after the end capping reagent reacts with the residual reactive group, the hydrophilic group can be introduced to improve the hydrophilicity of the microsphere, especially the surface of the microsphere.

The further technical scheme is that the preparation method comprises the following steps:

primary pore forming: preparing a cellulose diacetate solution with the mass fraction of 0.06g/mL to 0.20g/mL, adding a first pore-forming agent, uniformly mixing, adding the solution into an aqueous solution containing a dispersing agent, stirring, dispersing, heating, solidifying into balls, adding alkali liquor, saponifying, cleaning and drying to obtain primary pore-forming cellulose microspheres;

secondary pore forming: preparing 0.06g/mL to 0.20g/mL cellulose diacetate solution, adding a second pore-forming agent, adding primary pore-forming cellulose microspheres, stirring, dispersing, heating, curing to form balls, adding alkali liquor in any order to hydrolyze the cellulose diacetate microspheres, cleaning, and screening by using a screen to obtain secondary pore-forming cellulose microspheres with required particle size;

and (3) activation: activating and etherifying the secondary pore-forming cellulose microspheres by using sodium hydroxide and epoxy chloropropane;

coating a film: adding the dried activated microspheres into a cellulose diacetate solution, continuously stirring and coating under a sealed condition, then pumping the solution, drying the microspheres by hot air vibration, and volatilizing a solvent to form a cellulose acetate semipermeable membrane on the surfaces of the microspheres; the solid content of the cellulose diacetate solution is 0.5-2.5%, the dosage of the cellulose diacetate solution is 1-4 times of the volume of the microsphere, and the stirring and coating time is 10-60 minutes;

grafting ligand: adding a hexadecylamine solution into the coated microspheres, and heating and grafting under an alkaline condition; the dosage of the hexadecylamine is 1 to 10 percent of the dry weight of the microsphere, the pH value of a reaction system is controlled to be in a range of 9 to 12 by adding alkaline substances under the alkaline condition, the reaction temperature is 35 to 65 ℃, and the reaction time is 4 to 24 hours;

and (3) eluting a membrane layer: eluting the cellulose acetate semipermeable membrane with an elution solvent capable of dissolving cellulose diacetate;

end capping: and adding an end-capping reagent with amino and hydroxyl into the microspheres after the membrane layer is eluted, and reacting with the residual epoxy groups to obtain the cellulose adsorbent.

From the above, the present invention further provides a detailed preparation method of the adsorbent. Wherein, cellulose diacetate is adopted to prepare the microspheres, and the cellulose diacetate has good dissolution and forming properties. After the secondary pore-forming and heating solidification to form the sphere, the steps of adding alkali liquor to hydrolyze the cellulose diacetate microsphere, cleaning and screening by using a screen can be carried out in any order, and preferably, the alkali liquor is added firstly to hydrolyze the cellulose diacetate microsphere. The elution solvent capable of dissolving cellulose diacetate may be, for example, methylene chloride, acetone or the like. The compound having an amino group and a hydroxyl group may be, for example, ethanolamine.

In order to achieve the third object of the present invention, the present invention provides a hemoperfusion apparatus, which contains the cellulose microsphere adsorbent described in any one of the above aspects or the cellulose microsphere adsorbent prepared by the preparation method described in any one of the above aspects.

Therefore, the invention also provides the blood perfusion device, the perfusion device can comprise a shell, and the adsorbent can be filled in the shell to realize the blood perfusion function.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1

The preparation method of the cellulose microsphere adsorbent comprises the steps of primary pore forming, secondary pore forming, purification and screening, hydrolysis, activation, coating, ligand grafting, elution of a membrane layer and end capping, and specifically comprises the following steps:

primary pore forming: weighing 10g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 50mL of dichloromethane and 12.5mL of absolute ethanol to prepare a cellulose diacetate solution with the mass fraction of 0.16g/mL, adding 20mL of ethylene glycol diacetate and 20mL of n-octanol, and uniformly stirring and mixing. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier with deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral with a large amount of deionized water, screening with a screen to obtain the microspheres with the diameter of 0.50mm to 0.71mm, replacing water with absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: weighing 2g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 10mL of dichloromethane and 2.5mL of absolute ethyl alcohol to prepare a cellulose diacetate solution with the mass fraction of 0.16g/mL, adding 4mL of ethyl acetate and 3mL of n-octanol, and uniformly stirring and mixing. Then, 20mL of the dried microspheres obtained in the primary pore-forming step are weighed and added into the solution, and the solution is sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

Purifying and screening: purifying the microspheres by using 60% of methanol by mass, removing the pore-forming agent, and then screening by using a screen to obtain the microspheres with the diameter of 0.60mm to 0.85 mm.

Hydrolysis: to the microspheres was added 2mol/L sodium hydroxide solution twice the volume of the microspheres, stirred at room temperature for 4 hours, and then washed with a large amount of deionized water to neutrality.

And (3) activation: weighing 20mL of microspheres, adding 10mL of DMSO, 20mL of epichlorohydrin and 10mL of 3mol/L sodium hydroxide solution, reacting at constant temperature of 40 ℃ for 3 hours, and purifying the microspheres by using deionized water and ethanol.

Coating a film: and (3) drying the activated microspheres in vacuum at room temperature, adding a cellulose diacetate solution with the solid content of 1.0 percent and the volume of 4 times of the microspheres, sealing and stirring for 30 minutes to carry out coating, then draining the solution, and putting the coated microspheres into a vibration dryer for drying.

Grafting ligand: 40mL of 5% hexadecylamine-ethanol solution (the amount of hexadecylamine is 1.6% of the dry weight of the microsphere) and 4mL of 0.01mol/L sodium hydroxide solution (the pH value is 12) are added into the coated microsphere, and the mixture is reacted for 10 hours at the constant temperature of 45 ℃. The unreacted hexadecylamine was then purged with ethanol.

And (3) eluting a membrane layer: and washing the grafted microspheres for several times by using acetone to remove the cellulose diacetate on the surface.

End capping: 40mL of 0.5mol/L ethanolamine solution is added into the microspheres after the membrane layer is eluted, the reaction is carried out for 12 hours at room temperature, and then the microspheres are washed to be neutral by deionized water.

The cellulose microsphere adsorbent prepared in this example has a continuous through-hole structure, and is a core-shell structure. The diameter of the through hole of the adsorbent shell layer is between 20nm and 50nm, the diameter of the through hole of the inner core is between 200nm and 600nm, the diameter of the through hole of the adsorbent shell layer is smaller than that of the through hole of the inner core, and the thickness of the adsorbent shell layer is 6% of the diameter of the inner core.

Example 2

The preparation method of the cellulose microsphere adsorbent comprises the steps of primary pore-forming, purification and screening, hydrolysis, activation, ligand grafting and end capping, and specifically comprises the following steps:

primary pore forming: 10g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol, 20mL of ethylene glycol diacetate and 20mL of n-octanol were added thereto, and the mixture was stirred and mixed uniformly. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. And (4) washing the carrier by using deionized water to remove the PVA on the surface.

Purifying and screening: purifying the microspheres by using methanol or ethanol with the mass fraction of 60-80%, removing a pore-forming agent, and then screening by using a screen to obtain the microspheres with the diameter of 0.4-0.8 mm.

Grafting ligand: and adding 40mL of 5% hexadecylamine-ethanol solution and 4mL of 0.01mol/L sodium hydroxide solution into the activated microspheres, and reacting at constant temperature of 45 ℃ for 10 hours. The unreacted hexadecylamine was then purged with ethanol.

End capping: 40mL of 0.5mol/L ethanolamine solution is added into the microspheres after the ligand grafting, the reaction is carried out for 12 hours at room temperature, and then the microspheres are washed to be neutral by deionized water.

The cellulose microsphere adsorbent prepared in the embodiment has a continuous through hole structure, and the aperture of the through hole of the adsorbent is between 200nm and 600 nm.

Example 3

The preparation method of the cellulose microsphere adsorbent comprises the steps of primary pore forming, secondary pore forming, purification and screening, hydrolysis, activation, ligand grafting and end capping, and specifically comprises the following steps:

primary pore forming: 10g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol, 20mL of ethylene glycol diacetate and 20mL of n-octanol were added thereto, and the mixture was stirred and mixed uniformly. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier by deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral by a large amount of deionized water, screening by a screen to obtain the microspheres with the diameter of 0.3mm to 0.7mm, replacing water by absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: 2g of cellulose diacetate was dissolved in a mixed solution of 10mL of methylene chloride and 2.5mL of absolute ethanol, and 4mL of ethyl acetate and 3mL of n-octanol were added thereto and mixed well with stirring. Then, 20mL of the dried microspheres obtained in the primary pore-forming step are weighed and added into the solution, and the solution is sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

Purifying and screening: purifying the microspheres by using methanol or ethanol with the mass fraction of 60-80%, removing a pore-forming agent, and then screening by using a screen to obtain the microspheres with the diameter of 0.60-0.85 mm.

The cellulose microsphere adsorbent prepared in the embodiment has a continuous through hole structure and a core-shell structure; the aperture of the through hole of the adsorbent shell layer is between 20nm and 50nm, the aperture of the through hole of the inner core is between 200nm and 600nm, and the aperture of the through hole of the adsorbent shell layer is smaller than that of the through hole of the inner core; the thickness of the adsorbent shell layer is 6% of the diameter of the inner core.

Example 4

The preparation method of the cellulose microsphere adsorbent of this embodiment includes steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating, ligand grafting, secondary hydrolysis and end capping, where operations of the steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating and ligand grafting are the same as those in embodiment 1, the embodiment does not include the step of eluting the membrane layer in embodiment 1, and the steps of secondary hydrolysis and end capping of this embodiment are as follows:

and (3) secondary hydrolysis: to the microspheres was added 2mol/L sodium hydroxide solution twice the volume of the microspheres, stirred at room temperature for 4 hours, and then washed with a large amount of deionized water to neutrality.

End capping: 40mL of 0.5mol/L ethanolamine solution was added to the microspheres after the re-hydrolysis, reacted at room temperature for 12 hours, and then washed with deionized water to neutrality.

The cellulose microsphere adsorbent prepared in the embodiment has a continuous through hole structure and a core-shell structure; the pore diameter of the through hole of the adsorbent outer shell is between 5nm and 50nm, the pore diameter of the through hole of the inner core is between 200nm and 600nm, and the pore diameter of the through hole of the adsorbent outer shell is smaller than that of the through hole of the inner core; the thickness of the adsorbent shell layer is 6% of the diameter of the inner core.

Example 5

The preparation method of the cellulose microsphere adsorbent of this embodiment includes steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating, grafting ligand and end capping, where operations of the steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating and grafting ligand are the same as those in embodiment 1, the embodiment does not include the step of eluting a membrane layer in embodiment 1, and does not include the step of hydrolyzing again in embodiment 4, and the end capping step of this embodiment is as follows:

The cellulose microsphere adsorbent prepared in the embodiment has a continuous through hole structure and a core-shell structure; the aperture of the through hole of the adsorbent outer shell is between 10nm and 50nm, the aperture of the through hole of the inner core is between 200nm and 600nm, and the aperture of the through hole of the adsorbent outer shell is smaller than that of the through hole of the inner core; the thickness of the adsorbent shell layer is 6% of the diameter of the inner core.

Example 6

The adsorption rates of the cellulose microsphere adsorbents prepared in examples 1 to 5 were examined.

The method for measuring the adsorption rate comprises the following steps: 1mL of the capped adsorbent prepared in examples 1 to 5 was accurately weighed into a conical flask, and the water was removed by a syringe, and two samples of each example were taken in parallel. Respectively and accurately measuring 10mL of blood plasma with beta 2-microglobulin concentration of about 50 mug/L and adding the blood plasma into corresponding conical flasks containing 1mL of adsorbent, and respectively measuring 10mL of the blood plasma and adding the blood plasma into two clean conical flasks to serve as blank controls. After adsorbing at a constant temperature of 37 ℃ for 2 hours at 140rpm, plasma was taken, and the concentrations of β 2-MG, total protein and albumin were measured to calculate the adsorption rate. The results are shown in table 1 below:

TABLE 1 adsorption rates of examples 1 to 5 adsorbents for beta 2-MG, total protein, albumin

As can be seen from table 1: (1) in the aspect of the screening effect of the pore structure, compared with the examples 2 and 3, the cellulose microsphere adsorbent prepared in the example 3 after secondary pore forming has a core-shell structure, and the pore diameter of the outer layer is smaller than that of the inner core; the cellulose microsphere adsorbent prepared in example 2 is of a monolayer pore structure. From the adsorption rate results of the cellulose microsphere adsorbents prepared in the two examples, it can be seen that the adsorption rate of the cellulose microsphere adsorbent prepared in example 3 to total protein and albumin is lower than that of the cellulose microsphere adsorbent prepared in example 2, which may be caused by the fact that the cellulose microsphere adsorbent prepared in example 3 has the outer shell layer through hole with small pore size to prevent albumin and globulin with large molecular weight from entering the inside of the adsorbent, so that the adsorption of albumin and globulin with large molecular weight is reduced, the influence on beneficial components in blood during blood purification treatment is reduced, and the treatment safety is higher.

(2) In terms of adsorbent grafting, comparing examples 1 and 3, and example 3 has no coating step, and the outer layer surface of the prepared cellulose microsphere adsorbent is grafted with hydrophobic groups; in the step of coating and eluting the film layer of the cellulose microsphere adsorbent prepared in the embodiment 1, no hydrophobic group is grafted on the surface of the outer shell layer, the epoxy group generated on the outer shell layer in the activation step of the cellulose microsphere in the embodiment 1 is protected by the coating, and after the coating is eluted, the epoxy group remained on the surface of the outer shell layer reacts with ethanolamine in the blocking step to generate a hydrophilic group. Compared with the cellulose microsphere adsorbent prepared in example 3, the cellulose microsphere adsorbent prepared in example 1 has no grafted hydrophobic groups on the surface and generates part of hydrophilic groups, and the cellulose microsphere adsorbent shell prepared in example 1 has better hydrophilicity than that of the cellulose microsphere adsorbent shell prepared in example 3, so that the adsorption rate of the cellulose microsphere adsorbent shell prepared in example 1 on total protein and albumin is lower than that of the cellulose microsphere adsorbent shell prepared in example 3, and the coating step is favorable for reducing the adsorption of the adsorbent on total protein and albumin in blood in the blood purification treatment process, and the treatment safety is higher.

(3) In the aspect of adsorption rate on pathogenic substance beta 2-microglobulin, comparing examples 1, 4 and 5, in example 1, in the step of eluting a membrane layer, acetone is used for cleaning and removing a surface cellulose diacetate coating layer, and the prepared cellulose microsphere adsorbent is of a core-shell two-layer structure; example 4 does not include the step of eluting the membrane layer, but hydrolyzes the cellulose diacetate of the membrane layer into cellulose by using sodium hydroxide solution in the step of secondary hydrolysis, and the coating layer with the component of cellulose is reserved, and the prepared cellulose microsphere adsorbent sequentially comprises a cellulose core, a cellulose shell and a cellulose coating layer from inside to outside; example 5 does not include the step of eluting the membrane layer and the step of hydrolyzing again, the coating layer with the component of the cellulose diacetate is reserved, and the prepared cellulose microsphere adsorbent is sequentially cellulose core-cellulose shell-cellulose diacetate coating layer from inside to outside. The cellulose microsphere adsorbent prepared in example 1 is of a core-shell two-layer structure without a film layer, and compared with examples 4 and 5, the beta 2 microglobulin is easier to enter the inner core, and the adsorption rate is higher. Comparing examples 4 and 5, the cellulose microsphere adsorbent prepared in example 4 has a cellulose semipermeable membrane on the surface, the cellulose microsphere adsorbent prepared in example 5 has a cellulose diacetate semipermeable membrane on the surface, and the hydrophobicity of the cellulose diacetate semipermeable membrane is stronger than that of the cellulose semipermeable membrane, so that the adsorption rates of the cellulose microsphere adsorbent prepared in example 5 on beta 2 microglobulin, albumin and total protein are all higher than those of example 4.

(4) The adsorbent of the embodiment 1 has a two-layer pore structure, the pore diameter of the outer shell layer is small, and albumin, globulin and blood cells with large molecular weight are prevented from entering a pore channel; the pore size of the inner core is large, which is beneficial to the diffusion and adsorption of beta 2-microglobulin in the pore canal, and the adsorption quantity is improved. In the embodiment 1, the coating step is adopted to avoid grafting hydrophobic groups on the surfaces of the microspheres, so that the microspheres have low adsorption rate on albumin and globulin, and the safety is ensured; a large number of hydrophobic groups are grafted in the adsorbent, and beta 2-microglobulin is adsorbed by hydrophobic effect. The beta 2-microglobulin in blood can be better and selectively adsorbed and removed by combining the screening effect of the small pore channel of the shell layer of the microsphere adsorbent and the hydrophobic effect of the grafting ligand on the inner surface of the pore channel.

Example 7

The preparation method of the cellulose microsphere adsorbent of the embodiment includes steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping, wherein the steps of secondary pore-forming, purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping are the same as those of embodiment 1, compared with embodiment 1, the step of primary pore-forming of the embodiment does not use sodium hydroxide for saponification, and specifically the step of primary pore-forming is as follows:

primary pore forming: 10g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol, 20mL of ethylene glycol diacetate and 20mL of n-octanol were added thereto, and the mixture was stirred and mixed uniformly. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. And (3) washing the carrier by using deionized water, removing PVA on the surface, replacing water by using ethanol, and airing at room temperature.

Although the cellulose microsphere prepared in the embodiment is of a core-shell structure, a compact area appears between the inner core and the outer shell, and the pore shape is irregular, because the surface of the microsphere swells and the pores collapse in the process of replacing water by ethanol after primary pore forming, and the microsphere further swells and dissolves under the action of dichloromethane and absolute ethanol in the secondary pore forming process, so that the pore structure of the outer layer of the inner core cannot be maintained, the shape is also changed, the surface smoothness is reduced, and the outer layer of the inner core is not spherical any more. The appearance of the compact layer influences the diffusion of target toxin to internal pore channels in the adsorption process, so that the adsorption performance of the adsorbent is reduced, and after the roughness of the surface of the adsorbent is increased, the coagulation risk is increased when the adsorbent is used for whole blood perfusion, and the treatment safety is influenced.

Example 8

The preparation method of the cellulose microsphere adsorbent comprises the steps of primary pore forming, secondary pore forming, purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping, wherein the steps of purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping are the same as those in embodiment 1, the steps of primary pore forming and secondary pore forming are different from those in embodiment 1, and specifically, the steps of primary pore forming and secondary pore forming are as follows:

primary pore forming: 10g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol, and 20mL of ethyl acetate and 15mL of n-octanol were added thereto and mixed well with stirring. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier by deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral by a large amount of deionized water, screening by a screen to obtain the microspheres with the diameter of 0.3mm to 0.7mm, replacing water by absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: 2g of cellulose diacetate was dissolved in a mixed solution of 10mL of methylene chloride and 2.5mL of absolute ethanol, 5mL of ethylene glycol diacetate and 5mL of n-octanol were added thereto, and the mixture was stirred and mixed uniformly. Then, 20mL of the dried microspheres prepared in the primary pore-forming step were weighed into the solution, and sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

The cellulose microsphere adsorbent prepared in the embodiment has a continuous through hole structure and is of a core-shell structure; the aperture of the through hole of the adsorbent outer shell is 500nm to 1500nm, the aperture of the through hole of the inner core is 30nm to 100nm, and the aperture of the through hole of the adsorbent outer shell is larger than that of the through hole of the inner core; the thickness of the adsorbent shell layer is 4% of the diameter of the inner core.

The adsorption rate of the cellulose microsphere adsorbent prepared in example 8 was measured, as shown in table 2 below:

TABLE 2 adsorption rates of the adsorbent of example 8 for beta 2-MG, total protein, albumin

Substance(s)	β2－MG	Total protein	Albumin
				Adsorption rate	33.25％	4.36％	5.23％

Comparing the cellulose microsphere adsorbents prepared in example 8 and example 1, the cellulose microsphere adsorbent prepared in example 1 has better performance of adsorbing and removing beta 2-MG and lower adsorption rate to albumin and globulin, because the albumin and the globulin are blocked by sieving through outer layer pores to enter into pore channels, and hydrophobic groups in the pore channels are mainly combined with the entering beta 2-MG. In example 8, the outer layer has a large pore size, and albumin and globulin enter the outer layer pores and occupy ligand binding sites, thereby increasing the albumin and globulin adsorption rate, preventing beta 2-microglobulin from diffusing into the inner layer pore channels, and reducing the beta 2 microglobulin adsorption rate.

Example 9

The preparation method of the cellulose microsphere adsorbent comprises the steps of primary pore-forming, secondary pore-forming, purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping, wherein the steps of secondary pore-forming, purification screening, hydrolysis, activation, coating, grafting ligand, elution membrane layer and end capping are the same as those in embodiment 1, the step of primary pore-forming is different from that in embodiment 1, and specifically, the step of primary pore-forming is as follows:

primary pore forming: 10g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol, 20mL of tributyrin and 20mL of n-octanol were added thereto, and the mixture was stirred and mixed uniformly. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier by deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral by a large amount of deionized water, screening by a screen to obtain the microspheres with the diameter of 0.3mm to 0.7mm, replacing water by absolute ethyl alcohol, and airing at room temperature.

Compared with the cellulose microsphere adsorbent prepared in example 1, the cellulose microsphere adsorbent prepared in this example has a shell layer pore structure similar to that of example 1, but the pore diameter of the inner core is larger and mainly distributed in the range of 3 μm to 10 μm, which shows that the molecular weight of the mixed pore-forming agent affects the pore diameter of the adsorbent, and the larger the molecular weight, the larger the pore diameter, the smaller the molecular weight and the smaller the pore diameter.

Example 10

The operation is carried out according to the steps of the embodiment 1, the concentration, the temperature, the type of the pore-forming agent, the dosage of the pore-forming agent and the like of the cellulose diacetate solution in the secondary pore-forming process are adjusted to obtain the cellulose microsphere adsorbent with different shell layer through hole diameters, the adsorption rate is detected, and the experimental results are shown in the following table 3:

table 3 example 10 pore size and adsorption rate of adsorbents from experimental groups 1 to 6

As can be seen from table 3: the cellulose microsphere adsorbents of experimental groups 1 to 6 have similar pore sizes of the through holes of the inner core and different through holes of the outer shell. In terms of adsorption rate, when the pore size of the through hole of the inner core is similar, the pore size of the through hole of the outer shell layer of the cellulose microsphere adsorbent affects the adsorption rate of the adsorbent. Specifically, in the aspect of adsorption of beneficial macromolecular proteins such as albumin and globulin in blood plasma, the larger the external pore diameter is, the easier the macromolecular proteins enter a pore channel to be adsorbed, and the higher the adsorption rate is; in the aspect of adsorbing the target toxin beta 2-microglobulin, when the pore diameter of the outer layer is not more than 50nm, the larger the pore diameter is, the more the toxin can enter the pore channel, and the higher the adsorption rate is. When the pore diameter of the outer layer exceeds 100nm, the larger the pore diameter is, the lower the adsorption rate of the beta 2-microglobulin is, because macromolecular proteins with higher concentration in blood plasma, such as albumin and globulin, enter the pore channel and are adsorbed by hydrophobic effect to occupy adsorption sites. Thus, the adsorbent shell layer preferably has a pore size of between 10nm and 100nm, and an optimal pore size of between 10nm and 50 nm.

Example 11

The operation is performed according to the steps of example 1, the concentration, the temperature, the type of the pore-forming agent, the amount of the pore-forming agent and the like of the cellulose diacetate solution in the primary pore-forming process are adjusted to obtain cellulose microsphere adsorbents with different core through-hole diameters, the adsorption rate is detected, and the experimental results are shown in the following table 4:

table 4 example 11 pore size and adsorption rate of adsorbents from experimental groups 1 to 6

As can be seen from table 4: the pore sizes of the through holes of the outer shell layer of the cellulose microsphere adsorbent in the experimental groups 1 to 6 are similar and are all between 10nm and 100nm, and the through holes of the inner core have differences. From the adsorption rate, when the pore size of the through hole of the outer shell layer is similar, the pore size of the through hole of the inner core of the cellulose microsphere adsorbent also influences the adsorption rate of the adsorbent, and particularly, the difference in the adsorption performance of substances with the molecular size smaller than that of the outer shell layer, such as beta 2-MG, is obvious, and the difference in the adsorption performance of substances with the molecular size larger than that of the outer shell layer, such as total protein and albumin, is not obvious. Specifically, when the through pore diameter of the inner core is not more than 600nm, the diffusion of beta 2-MG in the pore channel is facilitated along with the increase of the pore diameter, the utilization rate of hexadecyl binding sites in the pore channel is improved, and the adsorption rate is improved. When the pore diameter of the inner core exceeds 1500nm, the specific surface area of the adsorbent is reduced, adsorption sites are reduced, and the adsorption rate to beta 2-MG is reduced. Thus, the preferred pore size of the adsorbent core is between 200nm and 1500nm, and the preferred pore size is between 200nm and 600 nm.

Example 12

The operation is performed according to the steps of example 1, the stirring rotation speed in the primary pore-forming process, the screening of the target particle size after balling, the dosage ratio of the core microsphere to the solution in the secondary pore-forming process, the stirring rotation speed and the screening of the target particle size after balling are adjusted to obtain the cellulose microsphere adsorbent with the ratio of the shell layer thickness to the core diameter being changed, the adsorption rate is detected, and the experimental results are shown in the following table 5:

table 5 example 12 pore size and adsorption rate of adsorbents from experimental groups 1 to 6

As can be seen from Table 5: when the ratio of the thickness of the outer layer to the diameter of the inner core is not more than 10%, the thickness of the outer layer is increased along with the increase of the ratio, the screening effect of the outer layer pores on beneficial macromolecular proteins such as albumin and globulin in blood plasma is better, the beneficial macromolecular proteins can be prevented from entering the pore channels to be adsorbed, and the adsorption rate is reduced. When the ratio of the thickness of the shell layer to the diameter of the inner core exceeds 10 percent, the thickness of the outer surface layer is increased along with the increase of the ratio, the diameter of the inner core is reduced, the adsorption capacity of the macropores is reduced, the difficulty of the beta 2-microglobulin entering the inner core is increased, and the adsorption rate of the adsorbent to the beta 2-microglobulin is reduced. Therefore, the ratio of the thickness of the outer shell layer to the diameter of the inner core is controlled to be optimal between 1% and 10%, the adsorption rate of the beta 2-microglobulin adsorbent for the target toxin is highest, and the adsorption rate of beneficial macromolecular proteins such as albumin and globulin in blood plasma is at a lower level.

Example 13

Primary pore forming: weighing 3.7g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 50mL of dichloromethane and 12.5mL of absolute ethanol to prepare a cellulose diacetate solution with the mass fraction of 0.06g/mL, adding 20mL of ethylene glycol diacetate and 20mL of n-octanol, and uniformly stirring and mixing. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier with deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral with a large amount of deionized water, screening with a screen to obtain the microspheres with the diameter of 0.50mm to 0.71mm, replacing water with absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: 0.75g of cellulose diacetate was weighed and dissolved in a mixed solution of 10mL of methylene chloride and 2.5mL of absolute ethanol to prepare a cellulose diacetate solution with a mass fraction of 0.06g/mL, 4mL of ethyl acetate and 3mL of n-octanol were added, and the mixture was stirred and mixed uniformly. Then, 20mL of the dried microspheres obtained in the primary pore-forming step are weighed and added into the solution, and the solution is sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

Purifying and screening: purifying the microspheres by using 80% of ethanol by mass fraction, removing a pore-forming agent, and then screening by using a screen to obtain the microspheres with the diameter of 0.60mm to 0.85 mm.

Coating a film: and (3) drying the activated microspheres in vacuum at room temperature, adding a cellulose diacetate solution with the solid content of 0.5 percent and the volume of 4 times of the microspheres, sealing and stirring for 40 minutes to carry out coating, then draining the solution, and putting the coated microspheres into a vibration dryer for drying.

Grafting ligand: 40mL of 5% hexadecylamine-ethanol solution (the amount of hexadecylamine is 1.6% of the dry weight of the microsphere) and 4mL of 0.01mol/L sodium hydroxide solution (the pH value is 12) are added into the coated microsphere, and the mixture is reacted for 24 hours at the constant temperature of 35 ℃. The unreacted hexadecylamine was then purged with ethanol.

The cellulose microsphere adsorbent prepared in this example has a continuous through-hole structure, and is a core-shell structure. The diameter of the through hole of the adsorbent shell layer is between 10nm and 100nm, the diameter of the through hole of the inner core is between 200nm and 1500nm, the diameter of the through hole of the adsorbent shell layer is smaller than that of the through hole of the inner core, and the thickness of the adsorbent shell layer is 3% of the diameter of the inner core.

Example 14

Primary pore forming: weighing 12.5g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 50mL of dichloromethane and 12.5mL of absolute ethanol to prepare a cellulose diacetate solution with the mass fraction of 0.20g/mL, adding 20mL of ethylene glycol diacetate and 20mL of n-octanol, and uniformly stirring and mixing. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier with deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral with a large amount of deionized water, screening with a screen to obtain the microspheres with the diameter of 0.50mm to 0.71mm, replacing water with absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: weighing 2.5g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 10mL of dichloromethane and 2.5mL of absolute ethyl alcohol to prepare a cellulose diacetate solution with the mass fraction of 0.20g/mL, adding 4mL of ethyl acetate and 3mL of n-octanol, and uniformly stirring and mixing. Then, 20mL of the dried microspheres obtained in the primary pore-forming step are weighed and added into the solution, and the solution is sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

Coating a film: and (3) drying the activated microspheres in vacuum at room temperature, adding a cellulose diacetate solution with the solid content of 2.5 percent and the volume of 1 time of the microspheres, sealing and stirring for 60 minutes to carry out coating, then draining the solution, and putting the coated microspheres into a vibration dryer for drying.

Grafting ligand: 40mL of 5% hexadecylamine-ethanol solution (the amount of hexadecylamine is 1.6% of the dry weight of the microsphere) and 4mL of 0.01mol/L sodium hydroxide solution (the pH value is 12) are added into the coated microsphere, and the mixture is reacted for 4 hours at a constant temperature of 65 ℃. The unreacted hexadecylamine was then purged with ethanol.

The cellulose microsphere adsorbent prepared in this example has a continuous through-hole structure, and is a core-shell structure. The diameter of the through hole of the adsorbent shell layer is between 10nm and 100nm, the diameter of the through hole of the inner core is between 200nm and 1500nm, the diameter of the through hole of the adsorbent shell layer is smaller than that of the through hole of the inner core, and the thickness of the adsorbent shell layer is 10% of the diameter of the inner core.

Example 15

Primary pore forming: 6.25g of cellulose diacetate was weighed and dissolved in a mixed solution of 50mL of methylene chloride and 12.5mL of absolute ethanol to prepare a cellulose diacetate solution with a mass fraction of 0.10g/mL, 20mL of ethylene glycol diacetate and 20mL of n-octanol were added, and the mixture was stirred and mixed uniformly. And (2) dropwise adding the solution into 400mL of PVA solution with the mass fraction of 2% at a constant speed at room temperature, continuously stirring at the rotating speed of 140 rpm-160 rpm for 2 hours at room temperature, and heating to 35 ℃ and stirring for 6 hours to obtain the cellulose acetate microspheres. Washing a carrier with deionized water, removing PVA on the surface, adding 2mol/L sodium hydroxide solution with the volume being twice that of the microspheres into the microspheres, stirring for 4 hours at room temperature, washing to be neutral with a large amount of deionized water, screening with a screen to obtain the microspheres with the diameter of 0.50mm to 0.71mm, replacing water with absolute ethyl alcohol, and airing at room temperature.

Secondary pore forming: weighing 1.25g of cellulose diacetate, dissolving the cellulose diacetate in a mixed solution of 10mL of dichloromethane and 2.5mL of absolute ethyl alcohol to prepare a cellulose diacetate solution with the mass fraction of 0.10g/mL, adding 4mL of ethyl acetate and 3mL of n-octanol, and uniformly stirring and mixing. Then, 20mL of the dried microspheres obtained in the primary pore-forming step are weighed and added into the solution, and the solution is sealed and stirred for 30 minutes. And slowly pouring the mixture into 400mL of PVA solution with the mass fraction of 2%, continuously stirring at the rotating speed of 140rpm to 160rpm, stirring for 2 hours at room temperature, heating to 35 ℃, and stirring for 6 hours to obtain the secondary pore-forming cellulose acetate microspheres.

Coating a film: and (3) drying the activated microspheres in vacuum at room temperature, adding a cellulose diacetate solution with the solid content of 2% and the volume of 2 times of the microspheres, sealing and stirring for 20 minutes to carry out coating, draining the solution, and drying the coated microspheres in a vibration dryer.

The cellulose microsphere adsorbent prepared in this example has a continuous through-hole structure, and is a core-shell structure. The diameter of the through hole of the adsorbent shell layer is between 10nm and 100nm, the diameter of the through hole of the inner core is between 200nm and 1500nm, the diameter of the through hole of the adsorbent shell layer is smaller than that of the through hole of the inner core, and the thickness of the adsorbent shell layer is 4% of the diameter of the inner core.

Finally, it should be emphasized that the above-described embodiments are merely preferred examples of the invention, which is not intended to limit the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A cellulose microsphere adsorbent characterized by:

the cellulose microsphere adsorbent is of a core-shell structure and comprises an inner core and an outer shell layer wrapping the inner core, the outer shell layer and the inner core are of a continuous through hole structure, and the aperture of the through hole of the outer shell layer is smaller than that of the through hole of the inner core.

2. The cellulose microsphere adsorbent of claim 1, wherein:

both the inner core and the outer shell are cellulose or cellulose derivatives;

hydrophobic ligands are grafted on the inner surfaces of the inner core through hole and the outer shell through hole, and the hydrophobic ligands are not grafted on the outer surface of the outer shell; the outer surface of the outer shell layer is connected with a hydrophilic group.

3. A cellulose microsphere adsorbent according to claim 1 or 2, characterized in that:

the aperture of the inner core through hole is 50nm to 3000nm, preferably 200nm to 1500nm, and more preferably 200nm to 600 nm;

the pore diameter of the through-hole of the outer shell layer is 1nm to 500nm, preferably 10nm to 100nm, and more preferably 10nm to 50 nm.

4. A cellulose microsphere adsorbent according to claim 1 or 2, characterized in that:

the shell layer thickness is 0.2% to 50%, preferably 1% to 10%, of the core diameter.

5. A preparation method of a cellulose microsphere adsorbent is characterized by comprising the following steps:

secondary pore forming: preparing a cellulose or cellulose derivative solution, adding a second pore-forming agent, adding the primary pore-forming cellulose microspheres, dispersing, heating and curing to form spheres, cleaning, and screening to obtain secondary pore-forming cellulose microspheres; when the cellulose derivative is cellulose ester, the hydrolysis step is further included after heating and curing to form balls; the secondary pore-forming cellulose microsphere is provided with an inner core with through holes formed in the primary pore-forming step and an outer shell layer with through holes formed in the secondary pore-forming step and coating the inner core, and the inner core through holes are communicated with the outer shell layer through holes; the first porogen has a molecular weight not less than the second porogen.

6. The preparation method according to claim 5, characterized in that the method further comprises the following steps after the secondary pore-forming step:

grafting ligand: grafting a hydrophobic ligand onto the reactive group;

7. The method of claim 6, wherein:

and an enveloping step is further included between the activating step and the grafting ligand step: forming a cellulose or cellulose derivative semipermeable membrane on the surface of the secondary pore-forming cellulose microsphere by using a cellulose or cellulose derivative solution;

between the grafting ligand step and the capping step, further comprising an elution membrane layer step comprising eluting the cellulose or cellulose derivative semipermeable membrane; or, when the cellulose derivative in the coating step is a cellulose ester, a secondary hydrolysis step is further included between the grafting ligand step and the end capping step, and the secondary hydrolysis step includes hydrolyzing an ester group of the cellulose ester; or the capping step may be performed directly after the grafting ligand step.

8. The production method according to any one of claims 5 to 7, characterized in that:

the first pore-forming agent is selected from one or more of ethyl acetate, butyl acetate, ethylene glycol diacetate, n-hexanol and n-octanol; the second pore-foaming agent is selected from one or more of ethyl acetate, butyl acetate, ethylene glycol diacetate, n-hexanol and n-octanol.

9. The method of claim 6, wherein: the activating step comprises the step of reacting the secondary pore-forming cellulose microspheres with an etherification reagent, wherein one end of the etherification reagent and the secondary pore-forming cellulose microspheres form an ether bond, and the other end of the etherification reagent is a reactive group.

10. Blood perfusion ware, its characterized in that:

the hemoperfusion apparatus contains a cellulose microsphere adsorbent according to any one of claims 1 to 4 or a cellulose microsphere adsorbent prepared by the preparation method according to any one of claims 5 to 9.