CN112569366B - Oral nano polymer targeted delivery system for encapsulating biological macromolecule medicine - Google Patents

Oral nano polymer targeted delivery system for encapsulating biological macromolecule medicine Download PDF

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CN112569366B
CN112569366B CN201910927139.3A CN201910927139A CN112569366B CN 112569366 B CN112569366 B CN 112569366B CN 201910927139 A CN201910927139 A CN 201910927139A CN 112569366 B CN112569366 B CN 112569366B
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cholic acid
acid
hydrophobic
polyethylene glycol
freeze
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CN112569366A (en
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高钟镐
韩颖
黄伟
陈丽青
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Institute of Materia Medica of CAMS
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Abstract

The invention belongs to the technical field of biological medicine, and in particular relates to a novel nano polymer targeted delivery system for oral administration, which comprises a composition for entrapping biological macromolecular drugs and a preparation method, wherein the biological macromolecular drugs are nucleic acid, peptide, vaccine, antibody or glycan. Two cholic acids with different properties are selected: the hydrophobic cholic acid and the hydrophilic cholic acid are respectively an inner target and an outer target, firstly, a cationic polymer-hydrophobic cholic acid core compound for encapsulating biological macromolecular medicaments is prepared through charge action and hydrophobic action, then the core compound is encapsulated in an anionic polymer-polyethylene glycol-hydrophilic cholic acid through film hydration action and charge action to form nanoparticles, and the nanoparticles are prepared into freeze-dried powder and filled into enteric capsules to construct a self-assembled oral nano polymer targeting delivery system. After oral administration, the carrier enters lymph and then absorbs blood, so that the bioavailability of the oral medicine can be improved.

Description

Oral nano polymer targeted delivery system for encapsulating biological macromolecule medicine
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a nano polymer targeting delivery system for oral administration, which comprises a composition for entrapping biological macromolecule medicines and a preparation method. Two cholic acids with different properties are selected: the hydrophobic cholic acid and the hydrophilic cholic acid are respectively an inner target and an outer target, firstly, a cationic polymer-hydrophobic cholic acid core compound for encapsulating biological macromolecular medicaments is prepared through charge action and hydrophobic action, then the core compound is wrapped in an anionic polymer-polyethylene glycol-hydrophilic cholic acid through film hydration action and charge action to form nanoparticles, and the nanoparticles are prepared into freeze-dried powder and filled into enteric capsules to construct a self-assembled oral nano polymer targeting delivery system. After oral administration, the cholic acid transporter enters lymph and then absorbs blood, so that the bioavailability of the oral medicine can be improved. Drugs include nucleic acids, peptides, vaccines, antibodies or glycans.
Background
Oral delivery of biomacromolecule drugs requires overcoming various physical and chemical barriers of the gastrointestinal tract, including complex pH and enzyme environments, mucous barriers and intestinal epithelial barriers, and biomacromolecule drugs often have characteristics of poor lipid solubility, large molecular weight, unstable physical and chemical properties, etc., which in turn increases the difficulty of delivery. Some chronic disease therapeutic drugs such as diabetes drugs, anticancer drugs, nerve drugs and the like need to be orally taken for a long time, and injection can bring a plurality of side effects and inconvenience. Therefore, there is an urgent need to develop an economical and practical oral pharmaceutical preparation which can effectively improve the drug effect, reduce the dosage and reduce the toxic and side effects.
Cholic acid transporter is an important absorption transporter in intestinal tract, and cholic acid secreted by liver enters lymph through a series of transporter such as sodium-dependent bile salt transporter at ileum top of distal small intestine, bile salt binding protein in cytoplasm, organic protein transporter at bottom, etc., and then is absorbed into blood and transported back to liver. Under the action of cholic acid, digested fat molecules are taken up into small intestinal cells and reassembled into triglycerides, treated via the endoplasmic reticulum and golgi apparatus, and finally homolipoproteins and cholesterol are released from small intestinal epithelial cells in the form of chylomicrons, and then transferred into the systemic circulation via the lymphatic system (i.e., chylomicron and chest catheter) and the left subclavian vein. Therefore, the medicine carrying compound with the surface modified cholic acid can promote the absorption of medicine particles into blood and improve the absorption availability in vivo under the action of cholic acid transport proteins at the tail end of the ileum.
Disclosure of Invention
The invention provides an oral drug nano polymer, which aims to improve the oral delivery capacity of biological macromolecular drugs. The method comprises the steps of selecting cholic acid with two properties of hydrophobicity and hydrophilicity, adopting hydrophilic cholic acid as a target to modify covalently-connected negative charged anionic polymer with good biocompatibility and polyethylene glycol material with excellent mucous membrane penetration effect, modifying positive charged low-toxicity cationic polymer by the hydrophobic cholic acid, coating biomacromolecule medicine with the cationic polymer-hydrophobic cholic acid to form a core compound through charge effect and hydrophobic effect, coating the core compound in the anionic polymer-polyethylene glycol-hydrophilic cholic acid through film hydration effect and charge effect to form nanoparticles, preparing lyophilized powder from the nanoparticles, and filling the lyophilized powder into enteric capsules to construct the self-assembled oral nanometer polymer targeted delivery system.
The modification of cholic acid in the polymer increases the bioavailability of the medicine, and provides possibility of oral administration for biological macromolecular medicines such as peptide, gene, oligonucleotide, vaccine and the like. The invention is characterized in that the properties of different cholic acids are skillfully utilized, and firstly, the characteristics of the biological macromolecular medicament are considered: the capacity of entrapping biological macromolecular drugs is low and easy to agglomerate only through the charge effect, and the entrapping capacity and stability can be greatly improved through the hydrophobic effect of hydrophobic cholic acid; the second hydrophobic cholic acid can play a further role in intracellular targeting in the inner layer, and the second hydrophobic cholic acid and the hydrophilic cholic acid in the outer layer together greatly improve the oral bioavailability of the biological macromolecule medicine. The self-assembled nano targeted delivery system provides an oral technology platform, and provides a technology and a method for oral administration of biological macromolecular drugs which are poor in intestinal absorption, unstable and enzymatically degraded and can not be taken orally.
In order to solve the technical problems, the invention adopts the following technical scheme.
In a first aspect of the technical scheme of the invention, the synthesis and purification of the carrier material cationic polymer-hydrophobic cholic acid and anionic polymer-polyethylene glycol-hydrophilic cholic acid are provided.
Wherein the cationic polymer is at least one selected from low molecular weight protamine, polyarginine and polylysine. The anionic polymer is a high polymer, is negatively charged under the condition of neutral pH value, and is selected from the following components: natural anionic polymers, synthetic anionic polymers or polyanionic polymers. The natural anionic polymer is polysialic acid, hyaluronic acid, alginic acid, chondroitin sulfate, dextran sulfate, heparin and alginic acid; the synthetic anionic polymer is sulfonic acid, carboxylic acid polymer, phosphate or sulfonamide; the polyanionic polymer is a copolymer of one natural or synthetic polymer, or any type of polymer copolymer of two or more. Cholic acid includes cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid or cholic acid of any type, and has structure shown in figure 1.
The method comprises the following steps:
in the first step, a cationic polymer-hydrophobic cholic acid functional carrier material is synthesized:
1) From the C end to the N end of the sequence, the steps are as follows: weighing N equivalents of resin, putting the resin into a reactor, adding dichloromethane to swell for half an hour, then pumping out the dichloromethane, adding 2N equivalents of first amino acid in the sequence, adding 2N equivalents of diisopropylethylamine, an appropriate amount of N, N-dimethylformamide, and dichloromethane (the appropriate amount is suitable for enabling the resin to fully swell), and carrying out nitrogen bubbling reaction for 60 minutes. Then adding about 5N equivalent of methanol, reacting for half an hour, pumping out the reaction solution, and cleaning with N, N-dimethylformamide and methanol;
2) The second amino acid in the sequence (also 2n equivalents), 2n equivalents of 1-hydroxy, benzo, trichloraz tetramethyl hexafluorophosphate and diisopropylethylamine were added to the reactor and the liquid was purged with nitrogen for half an hour, and the ninhydrin was detected and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of uncapping liquid to remove the 9-fluorenylmethoxycarbonyl protecting group, cleaning, and detecting ninhydrin;
3) Sequentially adding different amino acids in the sequence according to the mode of the step 2), and adding hydrophobic cholic acid at the N end of the last amino acid;
4) The resin is taken off from the reaction column after being dried by nitrogen, poured into a flask, then cutting fluid and cutting fluid (the composition is 95 percent trifluoroacetic acid, 2 percent ethanedithiol, 2 percent triisopropylsilane and 1 percent water) with the ratio of 10ml/g are added into the flask, the mixture is vibrated, and the resin is filtered;
5) Obtaining filtrate, adding a large amount of diethyl ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain a crude product of the sequence;
6) Purifying the crude product to the required purity by high performance liquid chromatography, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain the cationic polymer-hydrophobic cholic acid white powder.
Secondly, synthesizing an anionic polymer-polyethylene glycol-hydrophilic cholic acid functional carrier material:
1) Dissolving hydrophilic cholic acid in N, N-dimethylformamide, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents, and activating at room temperature;
2) Adding an equal proportion of amino polyethylene glycol with one end protected by tertiary butyl into the solution, adding a small amount of triethylamine, and continuing to react at room temperature;
3) After the reaction is finished, dialyzing, freeze-drying, and 1 H-NMR verification to obtain white polyethylene glycol-hydrophilic cholic acid powder with one end protected by tert-butyl;
4) Adding polyethylene glycol-hydrophilic cholic acid with one end protected by tertiary butyl into a dichloromethane solvent, slowly dropwise adding trifluoroacetic acid, stirring at room temperature, removing trifluoroacetic acid and dichloromethane by rotary evaporation to obtain deprotected polyethylene glycol-hydrophilic cholic acid, and dissolving in water;
5) Dissolving an anionic polymer with carboxyl into an ethanesulfonic acid buffer solution, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents, and activating at room temperature;
6) Adding deprotected polyethylene glycol-hydrophilic cholic acid into the ethanesulfonic acid buffer in the step 5), adding sodium hydroxide to adjust the pH to about 8, and continuing the reaction at room temperature;
7) After the reaction is finished, dialyzing, freeze-drying, and 1 H-NMR verification to obtain white powder of anionic polymer-polyglycol-hydrophilic cholic acid;
wherein in step 1)
Hydrophilic cholic acid mass: mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride: n-hydroxysuccinimide mass: the volume ratio of N, N-dimethylformamide is 2:2:1:1, a step of;
the preferred mass of hydrophilic cholic acid is: 50mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, 50mg of N-hydroxysuccinimide, mass: 25mg of N, N-dimethylformamide was 25ml in volume;
the preferred reaction temperatures are: 30 ℃;
preferred activation times are: 2h.
Wherein in step 2)
The molecular weight of the polyethylene glycol ranges from 2000 to 20000;
polyethylene glycol: hydrophilic cholic acid: the molar ratio of triethylamine is 1:1:400;
the preferred polyethylene glycol 2000 mass is: 215mg of hydrophilic cholic acid, the mass of which is: the dosage of triethylamine was 50 mg: 15 μl;
the preferred reaction temperatures are: 30 ℃;
the preferred reaction times are: 24h.
Wherein in step 3)
Molecular weight cut-off range for dialysis bags: 1 k-10 kDa;
preferred dialysate is: ethanol and deionized water;
preferred dialysis time: 72h.
Wherein in step 4)
Dichloromethane: the volume ratio of trifluoroacetic acid is 2:1;
the preferred stirring times are: 2h;
the preferred reaction temperatures are: 30 ℃.
Wherein in step 5)
Preferred anionic polymers: the molar ratio of polyethylene glycol to hydrophilic cholic acid is: 1:20 to 1:40;
mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride: mass of N-hydroxysuccinimide: the volume of the ethanesulfonic acid solution is: 10:1:1 to 100:1:1;
the preferred mass of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is: 100mg of N-hydroxysuccinimide, the mass of which is: the volume of the ethanesulfonic acid solution was 10 mg: 10ml;
the preferred activation time is 25min.
Wherein in step 6)
The preferred reaction time is 24 hours.
Wherein in step 7)
Molecular weight cut-off range for dialysis bags: 5 k-50 kDa;
preferred dialysate is: deionized water;
the preferred dialysis times are: 72h.
In a second aspect, the present invention provides the use of a cationic polymer-hydrophobic cholic acid and an anionic polymer-polyethylene glycol-hydrophilic cholic acid as a delivery vehicle for a biomacromolecule drug in vitro and in vivo.
The biological macromolecular medicine comprises peptide, nucleic acid, vaccine, antibody and glycan.
The method comprises the following steps:
1) In the aqueous solution, the molar ratio of the cationic polymer-hydrophobic cholic acid to the biomacromolecule drug is 1:1 to 9:1, so as to form a core complex;
2) Dissolving anionic polymer-polyethylene glycol-hydrophilic cholic acid in methanol, removing methanol by rotary evaporation to form a layer of film, dissolving the film with water, and adding the core compound in the step 1) into water to spontaneously form nanoparticles, wherein the molar ratio of the anionic polymer-polyethylene glycol-hydrophilic cholic acid to the biomacromolecule medicine is 0.1:1 to 0.9:1;
3) And (3) preparing the nano particles in the step (2) into freeze-dried powder, and filling the freeze-dried powder into an enteric capsule to obtain the self-assembled oral nano polymer targeted delivery system, wherein the freeze-dried protective agent is trehalose with the concentration of 1-4% (w/v).
Wherein in step 1)
Preferred molar ratio of cationic polymer-hydrophobic cholic acid to biomacromolecule drug is 3:1 to 5:1;
the preferred stirring speeds are: 400rpm;
the preferred reaction temperatures are: 4 ℃;
the preferred stirring times are: 2h.
Wherein in step 3)
The preferred molar ratio of anionic polymer-polyethylene glycol-hydrophilic cholic acid to biomacromolecule drug is 0.3:1 to 0.6:1;
the preferred trehalose doses are: 2% (w/v).
The beneficial technical effects are as follows:
the invention mainly provides an oral preparation for increasing the solubility of biological macromolecular drugs and improving the bioavailability, which can effectively improve the drug effect, reduce the dosage and reduce the toxic and side effects. Oral delivery of biomacromolecule drugs requires overcoming various complicated physical and chemical barriers of the gastrointestinal tract, and biomacromolecule drugs have the characteristics of poor lipid solubility, large molecular weight, unstable physical and chemical properties and the like, which in turn increases the difficulty of delivery. Some chronic disease treatment medicines need to be orally taken for a long time, and injection can bring a plurality of side effects and inconvenience.
In order to solve the defects of the products, the invention skillfully utilizes the properties of different cholic acids, and firstly considers the characteristics of biological macromolecular medicaments: the capacity of entrapping biological macromolecular drugs is low and easy to agglomerate only through the charge effect, and the entrapping capacity and stability can be greatly improved through the hydrophobic effect of hydrophobic cholic acid; the second hydrophobic cholic acid can play a further role in intracellular targeting in the inner layer, and the second hydrophobic cholic acid and the hydrophilic cholic acid in the outer layer together greatly improve the oral bioavailability of the biological macromolecule medicine. The self-assembled nano targeted delivery system provides an oral technology platform, provides a technology and a method for oral administration of biological macromolecular drugs which are poor in intestinal absorption, unstable and enzymatically degraded and cannot be taken orally, has the advantages that the in vivo bioavailability is obviously higher than that of nanoparticles without cholic acid modification, the carrier has low cytotoxicity, the in vivo and in vitro application is good, the particle size range is 100-300 nm, and the encapsulation rate is more than 70%.
Drawings
FIG. 1 Structure of several cholic acids
FIG. 2 several representative cationic polymer-hydrophobic cholic acid synthesis equations
FIG. 3 several representative anionic polymer-PEG-hydrophilic cholic acid Synthesis equations
FIG. 4. Nanoparticle preparation Process
FIG. 5 nanoparticle electron microscopy
FIG. 6 nanoparticle storage stability
FIG. 7 uptake of nanoparticles on SK-BR-3 cells with high expression of bile salt transport
FIG. 8. Transport of nanoparticles on in vitro Caco-2 model
FIG. 9 absorption of nanoparticles in the small intestine
FIG. 10 in vivo blood glucose concentration variation of exenatide drug-loaded nano-polymer
FIG. 11 in vivo pharmacokinetics of exenatide drug-loaded nano-polymer
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present invention, the present invention will be described in as much detail herein. The following examples further illustrate the invention, but do not limit it.
Example 1:synthesis of octapoly arginine-chenodeoxycholic acid
1) 275mg of the resin are weighed into a reactor, 4ml of dichloromethane are added to swell for half an hour, then the dichloromethane is pumped off, 35mg of the first arginine in the sequence is added, 26mg of diisopropylethylamine, 5ml of N, N-dimethylformamide, 5ml of dichloromethane and nitrogen are added to carry out a bubbling reaction for 60 minutes. Then adding 4ml equivalent methanol, reacting for half an hour, pumping out the reaction liquid, and cleaning with N, N-dimethylformamide and methanol;
2) The reactor was charged with 35mg of the second arginine in sequence, 76mg of 1-hydroxy, benzo, trichloraz tetramethyl hexafluorophosphate and 26mg of diisopropylethylamine, nitrogen sparged for half an hour, the liquid was washed off, ninhydrin detected, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of uncapping liquid to remove the 9-fluorenylmethoxycarbonyl protecting group, cleaning, and detecting ninhydrin;
3) Sequentially adding the rest arginine, and adding 78.5mg of chenodeoxycholic acid at the N end of the last arginine;
4) Drying the resin with nitrogen, taking off from the reaction column, pouring the resin into a flask, adding a cutting fluid and a cutting fluid (the composition is 95% trifluoroacetic acid, 2% ethanedithiol, 2% triisopropylsilane and 1% water) of the resin in a ratio of 10ml/g into the flask, vibrating, and filtering the resin;
5) Obtaining filtrate, adding a large amount of diethyl ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain a crude product of the sequence;
6) Purifying the crude product to the required purity by high performance liquid chromatography, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain the cationic polymer-hydrophobic cholic acid white powder.
The schematic diagram is shown in fig. 2-1.
Example 2:synthesis of low molecular weight protamine-deoxycholic acid
1) 275mg (0.1 mmol) of the resin are weighed into a reactor, 4ml of dichloromethane are added to swell for half an hour, then the dichloromethane is pumped off, 35mg (0.2 mmol) of the first arginine in the sequence is added, 26mg of diisopropylethylamine, 5ml of N, N-dimethylformamide, 5ml of dichloromethane, and nitrogen is bubbled for 60min. Then adding 4ml equivalent methanol, reacting for half an hour, pumping out the reaction liquid, and cleaning with N, N-dimethylformamide and methanol;
2) The reactor was charged with 0.2mmol of the second amino acid in sequence, 76mg of 1-hydroxy, benzo, trichloraz-tetramethyl hexafluorophosphate and 26mg of diisopropylethylamine, nitrogen sparged for half an hour, the liquid was washed off, ninhydrin detected, and then capped with pyridine and acetic anhydride. Finally, cleaning, adding a proper amount of uncapping liquid to remove the 9-fluorenylmethoxycarbonyl protecting group, cleaning, and detecting ninhydrin;
3) Sequentially adding the rest amino acids, and adding 78.5mg deoxycholic acid at the N end of the last valine;
4) Drying the resin with nitrogen, taking off from the reaction column, pouring the resin into a flask, adding a cutting fluid and a cutting fluid (the composition is 95% trifluoroacetic acid, 2% ethanedithiol, 2% triisopropylsilane and 1% water) of the resin in a ratio of 10ml/g into the flask, vibrating, and filtering the resin;
5) Obtaining filtrate, adding a large amount of diethyl ether into the filtrate to separate out a crude product, centrifuging, and cleaning to obtain a crude product of the sequence;
6) Purifying the crude product to the required purity by high performance liquid chromatography, concentrating the purified liquid in a freeze dryer, and freeze-drying to obtain the cationic polymer-hydrophobic cholic acid white powder.
The schematic diagram is shown in fig. 2-2.
Example 3:synthesis of polysialic acid-polyethylene glycol 2000-glycocholic acid
1) In a round bottom flask, 109mg of glycocholic acid was dissolved in 40ml of N, N-dimethylformamide, 100mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 50mg of N-hydroxysuccinimide were added as condensing agents, activated at room temperature for 2 hours,
2) 270mg of amino polyethylene glycol 2000 protected by one end of tert-butyl is added into the solution, 200 μl of triethylamine is added, the reaction is continued for 24 hours at room temperature,
3) After the reaction, dialyzing and freeze-drying.
4) Adding 12mg of polyethylene glycol 2000-glycocholic acid with one end protected by tert-butyl into 2ml of dichloromethane, slowly dropwise adding 1ml of trifluoroacetic acid, stirring for 2 hours at room temperature, removing the trifluoroacetic acid and the dichloromethane by rotary evaporation to obtain amino polyethylene glycol 2000-glycocholic acid, dissolving in water,
5) 60mg of polysialic acid is dissolved in 10ml of ethanesulfonic acid buffer, 300mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 6mg of N-hydroxysuccinimide are added as condensing agents, activated for 25min at room temperature,
6) Adding aminopolyethylene glycol 2000-glycocholic acid into ethanesulfonic acid buffer solution, adding 5N sodium hydroxide to adjust pH to about 8, continuing to react for 12h at room temperature,
7) After the reaction is finished, dialyzing, freeze-drying, and verifying by 1H-NMR to obtain white powder of the polysialic acid-polyethylene glycol 2000-glycocholic acid.
The schematic diagram is shown in fig. 3-1.
Example 4:synthesis of chondroitin sulfate-polyethylene glycol 7500-taurocholate
1) In a round bottom flask, 60mg of taurocholate was dissolved in 20ml of N, N-dimethylformamide, 50mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 25mg of N-hydroxysuccinimide were added as condensing agents, activated at room temperature for 2 hours,
2) 500mg of amino polyethylene glycol 7500 protected by one end tertiary butyl is added into the solution, 100 mu l of triethylamine is added, the reaction is continued for 24 hours at room temperature,
3) After the reaction, dialyzing and freeze-drying.
4) Adding 20mg of polyethylene glycol 7500-taurocholate with one end protected by tertiary butyl into 2ml of dichloromethane, slowly dripping 1ml of trifluoroacetic acid, stirring for 2 hours at room temperature, removing the trifluoroacetic acid and the dichloromethane by rotary evaporation to obtain amino polyethylene glycol 7500-taurocholate, adding water for dissolution,
5) 100mg of chondroitin sulfate is dissolved in 10ml of ethanesulfonic acid buffer, 600mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 12mg of N-hydroxysuccinimide are added as condensing agents, activated for 25min at room temperature,
6) Adding amino polyethylene glycol 7500-taurocholate into ethanesulfonic acid buffer solution, adding 5N sodium hydroxide to adjust pH to about 8, continuing to react for 12h at room temperature,
7) After the reaction is finished, dialyzing, freeze-drying, and verifying by 1H-NMR to obtain chondroitin sulfate-polyethylene glycol 7500-taurocholate white powder.
The schematic diagram is shown in fig. 3-2.
Example 5:preparation of exenatide drug-loaded nano polymer targeted delivery system
1) In a round bottom flask, 600 μg exenatide and 1.2mg low molecular weight protamine-deoxycholic acid are dissolved in aqueous solution and stirred at 400rpm for 2h at 4 ℃ to form a positively charged core complex;
2) Dissolving 4mg of polysialic acid-polyethylene glycol 2000-glycocholic acid in 5ml of methanol, removing an organic solvent by rotary evaporation to form a layer of film, dissolving with water, and adding the core compound into water to spontaneously form nanoparticles;
3) 2% of trehalose is used as a freeze-drying protective agent, and the nano particles are prepared into freeze-dried powder and filled into enteric capsules.
The preparation schematic diagram is shown in fig. 4.
Uptake of the nanoparticle with a test on SK-BR-3 cells with high expression of bile salt transport, quantitative determination by flow experiment, and the SK-BR-3 cells with a ratio of 3×10 5 The density of each/well was inoculated in 6-well plates, cultured for 48h, then incubated with different nanoparticles for 1.5h, centrifuged by pancreatin digestion, the supernatant was discarded, the cells were washed 3 times with PBS, and the analysis was re-suspended, demonstrating the best uptake of the cholic acid-bearing nano-polymer in vitro, and the results are shown in fig. 5.
After Cy7 labeling of the nanoparticles, mice were subjected to gastric lavage, and the absorption in the small intestine was examined by real-time imaging of the small animals, and the results demonstrate that the main absorption site of the nanoparticles with cholate is in the ileum, see FIG. 6.
In-vivo hypoglycemic effect of the nanoparticle is considered, in a db/db mouse model, the nanoparticle is divided into a subcutaneous administration group (dosage is 20 mug/kg) and a gastric administration group, the gastric administration group is an exenatide free drug group, a drug-loaded nano-polymer group and a drug-loaded cholic acid nano-polymer group (dosage is 300 mug/kg), and the result proves that the hypoglycemic effect of the drug-loaded cholic acid nano-polymer is better, as shown in figure 7. In vivo pharmacokinetic experiments are carried out on rats, and the rats are divided into subcutaneous administration groups (the dosage is 20 mug/kg), drug-loaded nano-polymer stomach-filling groups and drug-loaded cholic acid nano-polymer stomach-filling groups, and the results prove that the bioavailability of the drug-loaded cholic acid nano-polymer stomach-filling groups is 2.2 times that of the drug-loaded nano-polymer stomach-filling groups, and the results are shown in fig. 8 and table 1.
TABLE 1 in vivo pharmacokinetics of exenatide-loaded nanoparticles
Example 6:preparation of insulin drug-loaded nano polymer targeted delivery system
1) In a round bottom flask, 600 mug of insulin and 1.5mg of octapoly arginine-chenodeoxycholic acid are dissolved in water solution and stirred at 400rpm for 2 hours at 4 ℃ to form positively charged drug complex;
2) Dissolving 12mg of chondroitin sulfate-polyethylene glycol 7500-taurine in 5ml of methanol, removing an organic solvent by rotary evaporation to form a layer of film, dissolving with water, and adding the core compound into water to spontaneously form nanoparticles;
3) 2% of trehalose is used as a freeze-drying protective agent, and the nano particles are prepared into freeze-dried powder and filled into enteric capsules.
The morphology of the nanoparticles was seen under electron microscopy and was uniformly rounded, see fig. 9.
Establishing an in vitro Caco-2 cell model with a cell density of 2 multiplied by 10 5 Inoculating each cell/well into a polycarbonate membrane 12-well plate, adding 0.5ml of cell suspension into each well at the top end of the cell, adding 1.5ml of fresh culture medium into each well at the bottom end of the substrate, changing the liquid every two days for the first two weeks, changing the liquid every day, continuously culturing for 21 days, and considering that the transmembrane resistance is more than 800 ohms. Fluorescence labeling of fluorescein isothiocyanate is carried out on the nano polymer, qualitative measurement is carried out through a laser confocal microscope, nano particles with different concentrations are added for incubation and fixation, phenylindole nuclei are dyed, and the nano polymer with cholic acid is found to have the highest transfer efficiency through observation under a mirror, and the result is shown in figure 10.
Example 7:preparation of siRNA gene drug-loaded nano polymer targeted delivery system
1) 200 mug of siRNA gene and 2.5mg of low molecular weight protamine-ursodeoxycholic acid are dissolved in an aqueous solution in a round bottom flask, and stirred at 400rpm for 2 hours at 4 ℃ to form a positively charged core complex;
2) Dissolving 6mg of hyaluronic acid-polyethylene glycol 12000-taurine-cholic acid in 5ml of methanol, removing an organic solvent by rotary evaporation to form a layer of film, dissolving with water, and adding the core compound into water to spontaneously form a nano polymer;
3) The freeze-dried powder is prepared by using 4% of trehalose as a freeze-drying protective agent, and the freeze-dried powder is filled into enteric capsules.
The storage stability of the nano polymer was examined, the nano suspension was stored at 4℃for a certain period of time, and the storage stability of the nanoparticles was examined by the particle size and the degree of dispersion, and the stability was good, and the results are shown in FIG. 11.
While the present patent has been described in terms of a detailed general description and various embodiments, modifications, additions, permutations and certain sub-combinations thereof are possible. Accordingly, modifications, additions, permutations, and sub-combinations based on the teachings of the present patent are within the scope of the invention as claimed.

Claims (4)

1. A biopolymer targeted delivery system for oral administration, wherein the construction of the biopolymer targeted delivery system comprises:
the cationic polymer-hydrophobic cholic acid is used for encapsulating biological macromolecular drugs to obtain a core compound, the core compound is further coupled with the anionic polymer-polyethylene glycol-hydrophilic cholic acid to form nanoparticles, and the nanoparticles are prepared into freeze-dried powder and filled into enteric capsules;
wherein the cationic polymer is selected from at least one of low molecular weight protamine, polyarginine and polylysine;
the biological macromolecular drug is selected from siRNA, miRNA, peptide, vaccine, antibody or glycan;
the anionic polymer is selected from polysialic acid, hyaluronic acid, alginic acid, chondroitin sulfate, dextran sulfate, heparin or alginic acid;
the hydrophobic cholic acid is at least one selected from cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid and lithocholic acid;
the hydrophilic cholic acid is selected from at least one of glycocholic acid and taurocholic acid;
the molecular weight of the polyethylene glycol ranges from 2000 to 20000.
2. The oral biopolymer targeted delivery system of claim 1, wherein,
the peptide is selected from insulin, glucagon-like peptide-1 analogue exenatide, glucagon-like peptide-1 analogue cable Ma Lutai, glucagon-like peptide-1 analogue liraglutide, calcitonin, parathyroid hormone, octreotide, leuprorelin, human growth hormone, urokinase, lysozyme, ovalbumin, bovine serum albumin, superoxide dismutase or anti-ovulation peptide.
3. A method of preparing an oral biopolymer targeted delivery system according to any of claims 1-2, characterized in that,
1) Firstly preparing a core compound of a cationic polymer-hydrophobic cholic acid which is used for encapsulating a biomacromolecule drug through charge action and hydrophobic action,
2) Then the core compound is wrapped in anionic polymer-polyethylene glycol-hydrophilic cholic acid by the hydration of the film and the charge action to form nano particles,
3) And (3) preparing the nano particles into freeze-dried powder and filling the freeze-dried powder into enteric capsules.
4. A method according to claim 3, characterized in that the method comprises the steps of:
(1) Synthesis of cationic Polymer-hydrophobic cholic acid functional Carrier Material: the ratio of the cationic polymer to the hydrophobic cholic acid is 1:1, a solid-phase polypeptide synthesis method is adopted, the synthesis sequence is from the C end to the N end of the sequence, 1-hydroxybenzotrichloraz tetramethyl hexafluorophosphate and diisopropylethylamine are used as coupling agents of amino acids, different amino acids in the sequence are sequentially added, after the cationic polymer is synthesized, the hydrophobic cholic acid is coupled to the N end, the crude product is purified to more than 90% by using a high performance liquid chromatography method, and the purified liquid is put into a freeze dryer for concentration and freeze drying to white powder;
(2) Synthesis of anionic Polymer-polyethylene glycol-hydrophilic cholic acid functional Carrier Material: firstly, adopting 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide as condensing agents for amidation reaction to synthesize polyethylene glycol-hydrophilic cholic acid material with one end protected by tertiary butyl, dialyzing, purifying and freeze-drying to white powder; secondly, deprotecting polyethylene glycol-hydrophilic cholic acid material with one end protected by tertiary butyl, connecting the polyethylene glycol-hydrophilic cholic acid material to an anionic polymer through amidation reaction, and freeze-drying the polyethylene glycol-hydrophilic cholic acid material into white powder;
(3) Forming a core compound by the action of the synthesized cationic polymer-hydrophobic cholic acid material and the biomacromolecule drug through charge action and hydrophobic action, wherein the molar ratio of the cationic polymer-hydrophobic cholic acid to the biomacromolecule drug is 1:1 to 9:1;
(4) Coating the core complex in the step (3) in anionic polymer-polyethylene glycol-hydrophilic cholic acid through film hydration and charge to form nanoparticles, wherein the molar ratio of the anionic polymer-polyethylene glycol-hydrophilic cholic acid to the biological macromolecular medicament is 0.1:1 to 0.9:1;
(5) Preparing the nano particles in the step (4) into freeze-dried powder, wherein the freeze-drying protective agent is trehalose with the concentration of 1-4% (w/v), and filling the freeze-dried powder into enteric capsules.
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