CN115192542B

CN115192542B - Oral nanometer drug delivery system mediated by small molecule nutrient substances

Info

Publication number: CN115192542B
Application number: CN202210651830.5A
Authority: CN
Inventors: 黄园; 吴蕊男; 吴蕾; 周锐; 白瑜利
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2023-09-08
Anticipated expiration: 2039-11-04
Also published as: CN115040495A; CN115192542A; CN115040495B; CN112755005B; CN112755005A

Abstract

The application discloses an oral nano-carrier based on micromolecular nutrient substance mediation, actively targeting intestinal epithelial cells and a preparation method thereof. The application improves the drug delivery efficiency of the oral nano-carrier and has good research, development and application prospects in the field of medicine.

Description

Oral nanometer drug delivery system mediated by small molecule nutrient substances

The application relates to a Chinese patent with the application date of 2019, 11 and 4 entitled "oral nano drug delivery system mediated by small molecule nutrient substances", which is a divisional application of application number 201911068292.1.

Technical Field

The application relates to a strategy for modifying small molecular nutrients on the surface of nanoparticles and actively targeting intestinal epithelial cell surface receptors to overcome intestinal epithelial cell barriers and improve oral absorption of the nanoparticles, belonging to the field of pharmaceutical preparations.

Background

The nanometer drug delivery system can effectively improve the solubility of insoluble drugs, improve the gastrointestinal stability of macromolecular protein polypeptide drugs and effectively control the release of the drugs, so that the nanometer drug delivery system is widely used for the research of oral drug delivery (ACS Nano 2015,9,2345-2356).

To effectively deliver drugs into the blood circulation, nanocarriers need to overcome the intestinal epithelial cell absorption barrier (Drug Discov Today 2016,21,1155-1161). In recent years, a great deal of research has been devoted to modifying ligands on the nanoparticle surface, and by the interaction of the ligands with receptors on the surface of intestinal epithelial cells, increasing the affinity of the nanoparticles to the cell membrane and thus improving cellular uptake (Biomaterials 2018,180,78-90). Among these, many ligands have been studied as targeting polypeptides or proteins, such as RGD, transmembrane peptides, transferrin, and the like (Adv Drug Deliv Rev 2013,65,822-832).

However, notably, the complex environment and specific physiological structure of the gastrointestinal tract limit the targeting efficiency of protein polypeptide ligands (Adv Drug Deliv Rev 2013,65,833-844). On the one hand, the complex and variable pH and enzyme environment of the gastrointestinal tract may damage the structure of the protein polypeptide (Adv Drug Deliv Rev 2016,101,75-88); on the other hand, the hydrophobic and positively charged regions of the protein polypeptide ligand are susceptible to interaction with mucin in mucus, which reduces the mucus penetrability of the nanocarrier, thereby limiting the interaction of the ligand with cell membrane surface receptors (Eur J Pharm Biopharm 2014,88,518-528).

The intestinal tract is the most important absorption organ of human body, and many small-molecule nutrients such as saccharides, amino acids and the like can be rapidly absorbed by intestinal epithelial cells in large quantity, so that the energy supply of the human body is ensured (Science 2017,357,1299-1303).

Disclosure of Invention

In order to solve the problems of the active targeting oral nano-carrier in the prior study, the inventor uses small molecular nutrient substances as ligands to modify the surfaces of the nano-particles wrapped by the hydrophilic shell through creative study. On one hand, the chemical stability of the small molecular nutrient substances is higher, and the structure stability can be kept in the gastrointestinal tract environment; on the other hand, the nutrient substances with smaller molecular weight can be modified, the hydrophilic and negative electric characteristics of the surfaces of the nano particles can be maintained, the mucous penetrability of the nano particles is not influenced, and the interaction between the nano particles and epithelial cell surface receptors is improved.

The application aims at providing a method for improving oral absorption of nanoparticles, which is to modify small molecule nutrient substances on the surfaces of the nanoparticles and actively target intestinal epithelial cell surface receptors.

It is an object of the present application to provide a use of small molecule nutrients as ligands for the preparation of a pharmaceutical composition/formulation overcoming the intestinal absorption barrier.

It is an object of the present application to provide a nanoparticle that overcomes the intestinal absorption barrier, the nanoparticle having a hydrophilic shell and a hydrophobic core, wherein the hydrophilic shell is composed of a hydrophilic polymer with ends covalently linked or physically adsorbed small molecule nutrients; the hydrophobic core portion is composed of the active ingredient and a biocompatible carrier material.

As one specific embodiment, the nanoparticles have an average particle size in the range of about 10 to 1000nm; the nanoparticle with the core-shell structure is prepared from a hydrophobic core and a hydrophilic polymer shell according to the weight ratio of 1:99-95:5 (w/w); the content of the active ingredient accounts for 0.1% -90% (w/w) of the total weight of the nano-particles.

In the hydrophobic core portion, biocompatible carrier materials include, but are not limited to: a combination of one or more of lactic acid and glycolic acid, polystyrene, polysebacic acid, polyethylenimine, a mono or copolymer of lactide and glycolide, an inorganic silicon material, an inorganic carbon material, a polyalkylcyanoacrylate, a polyamino acid, cholesterol, a fatty acid, a phospholipid, a sphingolipid, a wax, and a fatty acid glyceride.

It is further preferred in the examples of the present application that the lactic acid-glycolic acid copolymer, the phospholipids and the fatty acid glycerides are used as biocompatible carrier materials with the active ingredient to prepare the nucleation core.

As one of the preferred embodiments, the active ingredient comprises at least one of protein polypeptide drugs, nucleic acid drugs and chemical drugs, and the content of the active ingredient is 0.1% -90% (w/w), preferably 1% -80% (w/w) of the total weight of the nanoparticle;

the protein polypeptide drugs include, but are not limited to: insulin, octreotide leuprorelin acetate, calcitonin, thymopentin, luteinizing hormone releasing hormone, tikeke peptide acetate, buserelin, exenatide, glucagon-like peptide-1, triptorelin acetate, leukocyte growth factor, erythrocyte growth factor, macrophage growth factor, tumor necrosis factor, epidermal growth factor, interleukins, angiostatin, bovine serum albumin, ovalbumin, parathyroid hormone, growth hormone, somatostatin, interferons, monoclonal antibodies and vaccines;

such nucleic acid agents include, but are not limited to, small interfering ribonucleic acids and plasmid DNA;

such chemical classes include, but are not limited to: antipyretic analgesics such as aspirin, acetaminophen, benorilate, ibuprofen, naproxen, diclofenac sodium, indomethacin, and the like, and non-steroidal anti-inflammatory drugs; or antibiotics such as benzoicillin sodium, tetracycline, amoxicillin, ampicillin, metronidazole, tinidazole, levofloxacin, gatifloxacin, furazolidone, gentamicin, rifamycin, erythromycin, roxithromycin, clarithromycin, azithromycin and the like, and other antibacterial agents; or antitumor agents such as doxorubicin, paclitaxel, cisplatin, 5-fluorouracil, hydroxycamptothecin, chang Chunjian, gemcitabine, and vinblastine sulfate; or misoprostol, estradiol, diethylstilbestrol, tamoxifen, levonorgestrel, norethindrone, mifepristone, hydrocortisone, dexamethasone, etc.; or central nervous system medicines such as diazepam, isopentobutyralte, phenytoin sodium, carbamazepine, sodium valproate, chlorpromazine, haloperidol, meperidine hydrochloride, levodopa and the like; or peripheral nervous system drugs such as clobetasol, bromnew stigmine, atropine sulfate, bromethazine, epinephrine, ephedrine hydrochloride, procaine, and lidocaine; or drugs of circulatory systems such as propranolol, nifedipine, captopril, losartan, digoxin, lovastatin, gemfibrozil and the like; or hypoglycemic agents and diuretics such as tolbutamide, metformin, nateglinide, hydrochlorothiazide, spironolactone, furosemide, and edemic acid.

As one of the embodiments of the present application, insulin is preferable as an active ingredient.

The nanoparticle shell consists of hydrophilic ends of amphiphilic block polymers, wherein the hydrophilic ends are subjected to covalent connection to modify small molecule nutrients (ligands) so that the ligands are exposed on the surface of the nanoparticle. Among them, hydrophilic ends include, but are not limited to, polyethylene glycol, polyamino acids, N- (2-hydroxypropyl) methacrylamide (HPMA) polymers, zwitterionic polymers, hyaluronic acid.

The small molecule nutrients of the present application include, but are not limited to: glucose, fructose, galactose, mannose, amino acids, cholesterol, sitosterol. The amino acids include, but are not limited to, glutamic acid, cysteine, histidine, lysine, threonine, leucine, isoleucine, valine, methionine, tryptophan, phenylalanine.

As one of the preferred embodiments of the present application, polyethylene glycol and polyamino acid are preferred as hydrophilic shells for the nanoparticles.

It is an object of the present application to provide a method for preparing an oral nano-drug delivery system based on small molecule nutrient-mediated enhancement of intestinal epithelial cell absorption barrier comprising the steps of:

(1) Covalently linking a small molecule nutrient or derivative thereof to the hydrophilic end of the amphiphilic polymer;

(2) Mixing the modified amphiphilic polymer, biocompatible carrier material (nanoparticle core) and active ingredient, and preparing nanoparticles by conventional methods in the pharmaceutical field such as microfluidic technology, nano precipitation method, high-pressure homogenization method, emulsion solvent volatilization method, film method, supercritical extraction method, etc.

As one of specific embodiments, the method comprises the following steps:

(1) And (3) alkynylating the D-fructose, and connecting the D-fructose with distearoyl phosphatidylethanolamine (DSPE) -polyethylene glycol (PEG) -azido through click chemistry to obtain the fructose modified DSPE-PEG.

(2) Mixing the fructose-modified DSPE-PEG with the nano particle core material, dissolving in dimethyl sulfoxide (DMSO), and slowly dripping into the stirred water phase.

It is an object of the present application to provide a carrier material modified to produce small molecule nutrients, wherein small molecule nutrients and derivatives thereof can be linked to the carrier material via amide bonds, ester bonds, etc.

As one of the specific embodiments of the application, a proper amount of 25-hydroxycholesterol (25 HC) is weighed, dissolved in dichloromethane, added with succinic anhydride, reacted for 24 hours, and extracted to obtain the succinylated 25HC; it is combined with DSPE-PEG-NH ₂ And (3) reacting for 24 hours, removing unreacted 25HC by using a dialysis method, and freeze-drying to obtain 25HC modified DSPE-PEG (DSPE-PEG-25 HC).

As one of specific embodiments of the application, the carrier material modified by the small molecular nutrient substances can be prepared into nanoparticles by adopting a conventional method in the pharmaceutical field, such as a nano precipitation method, an ion crosslinking method, a high-pressure emulsion homogenization method, an emulsion solvent volatilization method, a film method and the like.

It is an object of the present application to provide a nanoparticle for overcoming the intestinal epithelial cell absorption barrier, which can be used for oral administration, and a method for preparing the same.

Specifically, the application provides a nanoparticle preparation capable of overcoming gastrointestinal tract absorption barriers, which is prepared into oral administration preparations such as solution type liquid preparations, high polymer solutions, emulsions, suspension agents, syrups, drops, powder, granules, tablets, capsules and the like mainly from the nanoparticle and pharmaceutically acceptable auxiliary materials.

Advantageous effects

1. The active ingredients in the application are mainly positioned in the nanoparticle core, and the surface is the shell formed by the hydrophilic polymer, which is beneficial to improving the colloid stability of the nanoparticle, and can effectively reduce the leakage of the drug in the preparation and storage processes, so as to improve the stability of the drug and keep the activity of the drug.

2. The carrier material of the nanoparticle core can have various properties and can be crosslinked with the drug through covalent connection or physical adsorption, so that the drug with different properties can be encapsulated, including water-soluble drugs and fat-soluble drugs.

3. The nutrient substances have specific transport bodies in intestinal tracts and can be rapidly absorbed. The nano-carrier with the surface modified with the small molecular nutrient substances can actively target intestinal epithelial cell surface receptors by utilizing the characteristics, and effectively improve the cell uptake and transmembrane transport efficiency.

4. In the application, the small molecular nutrient substances are used as ligands, and compared with the existing protein polypeptide ligands, the ligand has higher chemical stability and is more beneficial to the function in complex gastrointestinal tract environments.

5. The application uses small molecule nutrient substances, can avoid introducing hydrophobic and positive regions of protein polypeptide ligands, and can not increase the particle size of nanoparticles, thereby effectively avoiding hydrophobic and electrostatic interactions with mucin, not affecting the mucous penetrability of nano-carriers, being beneficial to the nanoparticles reaching the surface of intestinal epithelial cells and further promoting the cell uptake.

6. The small molecule nutrient ligand has good biocompatibility, and can improve the safety of the oral nano-carrier to a certain extent compared with the existing synthesized polypeptide ligand.

Drawings

Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the synthetic pathway of fructose-modified DSPE-PEG.

FIG. 2 shows the synthesis route of glutamic acid modified amphiphilic branched polylysine.

FIG. 3 shows the particle diameter and potential change pattern of nanoparticles before and after fructose modification.

FIG. 4 shows a graph of nanoparticle mucus-penetrating rate before and after fructose modification.

FIG. 5 shows a graph of cellular uptake effects of nanoparticles before and after amino acid modification.

FIG. 6 shows a graph of active targeting ability of fructose-modified nanoparticles.

FIG. 7 shows graphs of the membrane-spanning efficiency studies of fructose-modified nanoparticles.

Fig. 8 shows a pharmacodynamic study of oral proinsulin and fructose-modified nanoparticles in rats.

Detailed Description

The following examples are further illustrative of the application but are in no way limiting of its scope. The application is further illustrated in detail below with reference to examples, but it will be understood by those skilled in the art that the application is not limited to these examples and the preparation methods used. Moreover, the present application may be equivalently replaced, combined, improved, or modified by those skilled in the art in light of the description of the present application, but are included in the scope of the present application.

EXAMPLE 1 Synthesis of fructose-modified DSPE-PEG

The synthetic route of fructose-modified DSPE-PEG is shown in figure 1. First, D-fructose (2 g, 0.0111 mol) was dissolved in acetone (40 ml), stirred at room temperature, and concentrated sulfuric acid (4 ml) was slowly added dropwise until fructose was completely dissolved. The reaction solution was added dropwise at 0℃to an aqueous sodium hydroxide (NaOH) solution (25 ml,4.5 mol/L). The reaction solution was concentrated in vacuo, extracted three times with dichloromethane, the oil phases combined and washed three times with saturated brine. Anhydrous sodium sulfate was added, dried for 30min, filtered, recrystallized from a mixed solvent of n-hexane and diethyl ether (1:1, v/v), dried and weighed to give compound 1 in 86.5% yield.

NaOH (0.87 g,0.0218 mol) was slowly added to DMF and compound 1 (2 g,0.008 mol) was added to the mixed solution with stirring at 0deg.C, and stirring was carried out for 15min, followed by dropwise addition of bromopropyne (1 ml,0.012 mol). To the reaction solution was added 75ml of water at room temperature, extracted three times with diethyl ether, and the organic phases were combined and washed successively with 10% hydrochloric acid solution and saturated NaCl. Dried over anhydrous sodium sulfate, and the reaction mixture was concentrated in vacuo to give compound 2 (2.0 g, 87.3%).

Compound 2 (0.5 g,0.0017 mol) was dissolved in trifluoroacetic acid-water (9:1), stirred at room temperature for 2h, filtered and the reaction concentrated. The crude product was washed with toluene and purified using a silica gel column (dichloromethane: methanol=15:1, v/v as mobile phase). A pale yellow oil (Compound 3) was obtained in 67.6% yield.

Compound 3 (109 mg,0.5 mmol) and DSPE-PEG-N ₃ (PEG molecular weight 5 kDa) (125 mg,0.025 mmol) was dissolved in DMF and copper sulfate solution (8 mg/ml) was added with stirring at 60 ℃. Sodium ascorbate solution (5 ml,40 mg/ml) was added dropwise at 0min, 30min, 60 min. After 2h, the reaction solution was transferred to a dialysis bag, dialyzed against disodium edetate for one day, dialyzed against ultrapure water for two days, and the dialysate was lyophilized to give fructose-modified DSPE-PEG (compound 4) in 91.2% yield. Confirmation of structure by Fourier transform infrared spectrometer, compound 4 at 2101.14cm ^-1 The characteristic peak of the azido group at 3445.65cm disappeared ^-1 A characteristic fructose peak appears.

EXAMPLE 2 Synthesis of 25-hydroxycholesterol (25 HC) modified DSPE-PEG

First, 25 hydroxycholesterol (25 HC) is succinylated to yield succinic acid-derived 25 hydroxycholesterol. The reaction was dissolved in methylene chloride (25 HC: succinic anhydride: DMAP=25:50:25 mol%) and the final 25HC concentration was 80mg/ml. The reaction was carried out at a constant temperature of 30℃and 500rpm for 24 hours. Spin-drying the product, adding pure water, and hydrolyzing at 40deg.C for 2 hr to hydrolyze unreacted succinic anhydride into succinic acid. The succinic acid 25HC is extracted by using methylene dichloride, the structure is confirmed by using nuclear magnetic resonance hydrogen spectrum, the hydrogen peak of 5.3ppm is changed into 4.6ppm (C3 position), and meanwhile, the 4 hydrogen peak of typical succinic acid appears in 2.5ppm, so that the successful connection of the succinic acid can be confirmed.

DSPE-PEG-NH by amide reaction ₂ (PEG molecular weight of 2 kDa) and 25 HC-COOH. Firstly 25HC-COOH, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) and N-hydroxysuccinimide (NHS) are activated for 0.5h at 25 ℃, and then DSPE-PEG-NH is added ₂ A small amount of triethylamine was added dropwise to adjust pH. Wherein the reactants are dissolved in DMSO (DSPE-PEG-NH) ₂ :25HC-COOH:EDCI:NHS＝16.7:33.3:25:25mol％)，DSPE-PEG-NH ₂ The final concentration was 20mg/ml. Then, the reaction was carried out at 25℃and 500rpm for 24 hours. The product was rapidly dialyzed against DMSO for 12h (25 ℃ C.), dialyzed against pure water for 24h, and lyophilized to obtain the product (DSPE-PEG-25 HC). Structural confirmation was performed using nuclear magnetic resonance hydrogen spectroscopy, and cholesterol peaks were observed at low chemical shifts (0.5-1.5 ppm), demonstrating successful 25HC ligation.

EXAMPLE 3 Synthesis of amino acid modified amphiphilic branched polylysine

The synthesis route of the gamma-glutamic acid modified amphiphilic branched polylysine is shown in figure 2, and is mainly divided into glutamic acid dodecanol ester E (C) ₁₂ ) (Compound A), amphiphilic branched polylysine (Compound B) and glutamic acid modified amphiphilic branched polylysine (Compound D).

(1) Synthesis of dodecanol ester of glutamic acid Fmoc-glutamic acid (3.69 g,10 mmol) and dodecanol (3.72 g,20 mmol) were dissolved in methylene chloride, EDCI (5.73 g,30 mmol) and 4-Dimethylaminopyridine (DMAP) (0.61 g,5 mmol) were added in sequence and left to stir at room temperature for 4 hours. The reaction solution was washed with saturated aqueous ammonium chloride and saturated aqueous sodium chloride in this order, and several layers were dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column chromatography to give the product compound A in 74% yield.

(2) Synthesis of amphiphilic branched polylysine Compound A (706 mg,1 mmol) was dissolved in dichloromethane, two drops of DBU were added dropwise, and Fmoc removal was detected by Thin Layer Chromatography (TLC). The reaction solution was washed with saturated ammonium chloride, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The resulting residue was dissolved in dichloromethane, PK-dendron (882 mg,1.1mmol; synthesized by the method of reference adv. Funct. Mater.2015,25, 5250-5260), 1-Hydroxybenzotriazole (HOBT) (135 mg,1 mmol), EDCI (287 mg,1.5 mmol), N, N-Diisopropylethylamine (DIEA) (330. Mu.L, 2 mmol) was added, and the reaction was stirred for 24h at room temperature under argon. The reaction solution was washed with saturated ammonium chloride and saturated sodium chloride solution in this order, and dried over anhydrous sodium sulfate. Recrystallisation from diethyl ether and acetonitrile gives the product compound B in 62% yield. Compound B has C12 as the hydrophobic end and polylysine as the hydrophilic end.

(3) The synthesis of glutamic acid modified amphiphilic branched polylysine comprises dissolving compound B (317 mg,0.25 mmol) in 3mL of dichloromethane, stirring at room temperature, dropwise adding 3mL of trifluoroacetic acid, and stirring for 4h. The solvent and TFA were distilled off under reduced pressure, diethyl ether was added, and a solid was precipitated at a low temperature of-10 ℃. The supernatant was removed by centrifugation, the solid was dissolved in dichloromethane, boc-Glu-OtBu (463 mg,1.2 mmol), HOBT (135 mg,1 mmol), EDCI (287 mg,1.5 mmol), DIEA (330. Mu.L, 2 mmol) was added and the reaction was stirred for 24h at room temperature under argon. The reaction solution was then washed with saturated ammonium chloride and saturated aqueous sodium chloride solution in this order, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Acetonitrile was added to precipitate out a precipitate, which was filtered and washed with acetonitrile to give compound C in 64% yield.

The resulting compound C was dissolved in 4mL of methylene chloride, stirred at room temperature, and TFA 4mL was added dropwise, followed by stirring for 6 hours. The solvent and TFA were distilled off under reduced pressure, acetonitrile was added to precipitate out, the supernatant was centrifuged, and the solid was washed with acetonitrile and diethyl ether to give the final product compound D in 87% yield. The synthesis reaction of the compound D is analyzed by utilizing nuclear magnetic resonance hydrogen spectrum, and the successful synthesis of the product is confirmed. The spectrum analysis is as follows: ¹ H NMR(400MHz,DMSO-d ₆ )δ9.71(m,4H),8.06(m,11H),4.30–3.95(m,12H),3.01(m,8H),2.38–2.05(m,16H),1.93(m,10H),1.53(m,10H),1.24(m,44H),0.85(t,J＝6.7Hz,6H)。

for the synthesis of cysteine and alpha-glutamic acid modified amphiphilic branched polylysine, compound B is respectively and covalently connected with cysteine and alpha-glutamic acid according to a similar method to obtain Cys-B and alpha-Glu-B. Analysis by nuclear magnetic resonance hydrogen spectroscopy confirmed successful synthesis of the product. Cys-B spectra were resolved as follows: ¹ H NMR(400MHz,DMSO-d ₆ ) Delta 8.28-7.74 (m, 11H), 4.46-3.87 (m, 12H), 3.13-2.89 (m, 6H), 2.87-2.54 (m, 8H), 2.46-1.91 (m, 8H), 1.87 (s, 12H), 1.73-1.44 (m, 10H), 1.42-1.07 (m, 48H), 0.85 (t, j=6.4 hz, 6H). The α -Glu-B spectrum is resolved as follows: ¹ H NMR(400MHz,DMSO-d ₆ )δ9.86–9.51(m,4H),8.64–7.70(m,11H),δ4.36–4.14(m,4H),4.06–3.92(m,4H),3.83–3.66(m,4H),3.27–2.76(m,8H),2.44–2.11(m,10H),2.05–1.75(m,10H),1.72–1.39(m,16H),1.31–1.03(m,44H),0.85(t,J＝6.4Hz,6H)。

EXAMPLE 4 preparation of fructose-modified polylactic-co-glycolic acid (PLGA) nanoparticles

A suitable amount of PLGA (50/50, viscosity: 0.26-0.54 dL/g) was weighed and dissolved in DMSO at a concentration of 40mg/ml, and fructose-modified DSPE-PEG and unmodified DSPE-PEG of example 1 were dissolved in DMSO, respectively, at a concentration of 20mg/ml. Insulin (INS) and Phospholipid (PC) are dissolved in DMSO and methanol respectively at the concentration of 12mg/ml and 96mg/ml respectively, and the mixture is uniformly mixed according to the volume ratio of 1:1, and magnetically stirred (500 rpm) at room temperature for 1h to prepare a phospholipid complex (IPC). Nanoparticles with different fructose modification degrees are prepared by adjusting the mass fractions of the two DSPE-PEG. The specific method comprises mixing the above materials at the ratio of Table 1, slowly dripping into 2ml of water phase (700 rpm), stirring for 5min, removing DMSO by ultrafiltration, adding ultrapure water, and dispersing again.

Table 1: ratio of materials of fructose modified nanoparticle

EXAMPLE 5 preparation of 25HC modified PLGA nanoparticles and cellular uptake

PLGA, DSPE-PEG-25HC and phospholipid were dissolved in DMSO to prepare stock solution (16 mg/ml), and fluorochrome coumarin 6 was dissolved in DMSO at a concentration of 2mg/ml. PLGA, DSPE-PEG-25HC, phospholipid: coumarin 6=4:3:1:0.05, and the stock solution was mixed well as the organic phase. The organic phase and 10-15 times of deionized water are uniformly mixed through a microfluidic device, the flow rate of the organic phase is 160 mu l/min, the flow rate of the aqueous phase is 1ml/min, and the 25HC modified PLGA nanoparticles are prepared. And then removing DMSO by adopting an ultrafiltration method to obtain the 25HC modified PLGA nanoparticles.

After digestion of Caco-2 cells, 1X 10 cells per well ⁴ Cell density was seeded in 96-well plates, after 4 days of cell growth differentiation, the medium was removed and the cells were rinsed with fresh PBS. The above nanoparticles were incubated with cells for 3h, the nanoparticles were removed, the cells were rinsed three times with fresh PBS, cells and nanoparticles were destroyed by adding 0.1ml DMSO per well, and the DiI fluorescence value was determined with an enzyme-labeled instrument. The cell number per well was corrected by the resazurin method to obtain the relative cell uptake. We found that the uptake of 25HC modified PLGA nanoparticles into cells is 3.45 times that of unmodified nanoparticles, indicating that the use of 25HC as ligand can effectively increase the cell membrane affinity of the nanoparticles.

EXAMPLE 6 investigation of the ability of nanoparticles to penetrate mucus before and after fructose modification

Dye DiI-loaded fructose nanoparticles with different degrees of fructose modification were prepared as in example 4. IPC was replaced with DiI, which was dissolved in DMSO at a stock concentration of 1mg/ml and the final nanoparticle dispersion at a DiI concentration of 5. Mu.g/ml. The particle size and zeta potential are used for characterizing each nanoparticle, and the result is shown in figure 3 of the specification.

As can be seen from fig. 3, after fructose modification, the particle size of the nanoparticle is not changed significantly, and as the modification degree of fructose increases, the negative charge on the surface of the nanoparticle increases, and the surface fructose is modified on the surface of the nanoparticle successfully.

Fresh pig intestinal mucus was spread in a Transwell chamber (100 μl of mucus per well) with a membrane area of 0.33cm ² The pore size of the polycarbonate semipermeable membrane was 3. Mu.m. 200 μl of nanoparticle dispersion was carefully added dropwise over the mucus, 800 μl of blank buffer was added to the receiving chamber, 50 μl of the blank buffer was sampled from the receiving chamber for fluorescence analysis at 15, 30, 60 and 120min, respectively, and an equal volume of blank buffer was immediately added to the receiving chamber. The apparent permeability coefficient (Papp) value of the nanoparticle is calculated as follows: papp= (dQ/dt) x [ 1/(A×C) ₀ )](dQ/dt represents the diffusion rate of nanoparticles, A is the membrane area, C ₀ Initial concentration of drug), the results are shown in figure 4 of the specification.

As can be seen from the figure 4, after fructose modification, the Papp value of the mucus penetrating PEG nanoparticle has no obvious change, which indicates that the fructose with negative electricity of small molecules can not increase the electrostatic and hydrophobic acting force of the nanoparticle and mucin, thereby ensuring the mucus penetrating rate.

EXAMPLE 7 examination of cellular uptake effects of nanoparticles before and after amino acid modification

PLGA, DSPE-PEG (PEG molecular weight of 2 kDa), the three amino acid modified compounds B of example 3 (alpha-Glu-B, gamma-Glu-B and Cys-B) and hydrophobic dye DiI were dissolved in DMSO at concentrations of 40mg/mL, 20mg/mL and 1mg/mL, respectively; the phospholipids were dissolved in methanol at a concentration of 20mg/mL. According to the method of example 4, a series of nanoparticles with different amino acid modification ratios were prepared by substituting compound B modified with DSPE-PEG in a certain ratio. For 100% amino acid modified nanoparticles, according to the volume ratio of PLGA to amino acid modified compound B to phospholipid to DiI=5:2:2:2, uniformly mixing the materials, slowly dripping an organic phase into deionized water (organic phase: water phase=1:20, v/v) which is rapidly stirred (900 rpm) at room temperature, removing DMSO by an ultrafiltration method, and adding Phosphate Buffer Solution (PBS) (pH=7.4) for re-dispersing to obtain nanoparticle dispersion. The cell uptake experiments were performed according to the method of example 5, we preferred 100% α -Glu nanoparticles, 100% γ -Glu nanoparticles and 50% Cys nanoparticles, compared to the cell uptake of unmodified PEG nanoparticles, as shown in FIG. 5 of the specification.

Figure 5 shows that the alpha-glutamic acid, gamma-glutamic acid and cysteine are modified on the surface of the nanoparticle, so that the affinity between the nanoparticle and a cell membrane can be increased, and the cellular uptake of the nanoparticle can be remarkably improved.

Example 8 active targeting capability investigation of fructose modified nanoparticles

DiI-loaded PEG nanoparticles and 100% fructose nanoparticles were prepared as in example 6. The nanoparticles were incubated with antibodies to glucose transporter 2 (GLUT 2) and glucose transporter 5 (GLUT 5), respectively, and after 3 hours cellular uptake of each group of nanoparticles was determined as described in example 5, and the results are shown in fig. 6 of the specification.

Fig. 6 shows that specific blocking of glucose transporter with GLUT2 and GLUT5 antibodies can significantly inhibit the uptake of fructose nanoparticles without affecting the uptake of PEG nanoparticles. GLUT2 and GLUT5 mediate the uptake of free fructose, and therefore, fructose modification enables active targeting of nanoparticles to glucose transporters to increase nanoparticle affinity to cell membranes.

EXAMPLE 9 examination of transmembrane transport efficiency of nanoparticles before and after fructose modification

After digestion of Caco-2 cells, the cells were digested at 3X 10 per well ⁴ Is inoculated into a Transwell chamber (upper chamber), and 0.6mL of complete medium is added to a receiving chamber (lower chamber). The membrane area of the cell was 0.33cm ² The pore size of the polycarbonate semipermeable membrane was 3. Mu.m. The medium was changed every other day for the first 12 days, and every other day thereafter. Meanwhile, from day 8, the transmembrane resistance (TEER) of the cell monolayer was measured every two days with a resistance meter, and the growth of the cells and the integrity of the cell monolayer were examined.

The TEER value is more than 500 Ω cm ² Is used for determining the transmembrane transport of the nanoparticle. The medium in the upper and lower chambers was removed prior to the experiment, equilibrated for 30min with an equal volume of pre-warmed blank medium, and then the blank medium was removed. 200. Mu.l of fluorescent-labeled nanoparticles dispersed in a blank medium was added to the upper chamber, and 800. Mu.l of blank medium was added to the lower chamber. 50 μl was sampled from the receiving chamber for fluorescence analysis at 0, 15, 30, 60, 90, 150, 240 and 360min, respectively, and the receiving chamber was immediately replenished with an equal volume of blank medium. Papp values were calculated as described in example 6, and the results are shown in FIG. 7. From fig. 7, the Papp value of 100% fructose nanoparticle is 5.2 times of that of PEG nanoparticle, which indicates that fructose modification can significantly improve the transmembrane transport efficiency of nanoparticle through active targeting effect, and helps to overcome intestinal epithelial cell absorption barrier.

EXAMPLE 10 in vivo pharmacodynamics investigation of fructose-modified PLGA nanoparticles

SD rats (180-220 g) fasted for 12 hours were randomly picked up and randomly divided into 3 groups of 5 each of the free INS solution group, the INS-entrapped PEG nanoparticle and the fructose nanoparticle group (nanoparticles prepared in example 4). 2.0ml (containing 50IU/kg of insulin) of INS crude drug and each group of nanoparticles were respectively administered by stomach infusion, and the blood glucose level of rats was measured at predetermined time points (0, 1, 2, 4, 6, 8 and 10 hours) and the percent of blood glucose reduction at each time point was calculated by the following formula, taking the blood glucose level of the rats before administration as 100%: percent change in blood glucose =gt/g0×100 (Gt and G0 represent the blood glucose level of the rat at time t and the blood glucose level of the rat before administration, respectively), and the percent change in blood glucose is plotted against time t to obtain a percent change in blood glucose versus time curve, and the results are shown in fig. 8 of the specification.

As can be seen from fig. 8, the hypoglycemic effect of the fructose nanoparticle group in 1 to 6 hours is obviously better than that of the original medicine group, and meanwhile, the blood sugar value in 2 hours is obviously lower than that of the PEG nanoparticle group, which indicates that the fructose modification can obviously improve the oral absorption of the nanoparticle.

Claims

1. An oral administration preparation for overcoming intestinal absorption barrier is characterized by being prepared from nanoparticles and pharmaceutically acceptable auxiliary materials, wherein the nanoparticles are prepared by taking small molecular nutrient substances as ligands, and modifying the small molecular nutrient substances on the surfaces of the nanoparticles to actively target intestinal epithelial cell surface receptors; the small molecule nutrient is 25 hydroxyl cholesterol; the nanoparticle has a hydrophilic shell and a hydrophobic core structure, wherein the hydrophilic shell is formed by a hydrophilic end of an amphiphilic polymer and a small molecular nutrient substance which is covalently connected with the hydrophilic end, the core is formed by a hydrophobic end of the amphiphilic polymer, an active ingredient and a biocompatible carrier material, and the hydrophilic end of the amphiphilic polymer is covalently connected with the small molecular nutrient substance to form 25 hydroxyl cholesterol modified DSPE-PEG.

2. The formulation of claim 1, wherein the 25-hydroxycholesterol-modified DSPE-PEG is prepared by: weighing a proper amount of 25-hydroxy cholesterol, dissolving in dichloromethane, adding succinic anhydride, reacting for 24 hours, and extracting to obtain succinylated 25-hydroxy cholesterol; it is combined with DSPE-PEG-NH ₂ And (3) reacting for 24 hours, removing unreacted 25-hydroxy cholesterol by using a dialysis method, and freeze-drying to obtain the 25-hydroxy cholesterol modified DSPE-PEG.

3. The formulation of claim 2, wherein the nanoparticle is made from a hydrophobic core to a hydrophilic shell in a mass ratio of 1:99-95:5; the mass of the active ingredient accounts for 0.1-90% of the total weight of the nano-particles.

4. The formulation of claim 2, wherein the biocompatible carrier material is selected from at least one of a mono-or co-polymer of lactic acid and glycolic acid, polystyrene, polysebacic acid, polyethylenimine, inorganic silicon material, inorganic carbon material, polyalkylcyanoacrylate, polyamino acid, cholesterol, fatty acid, phospholipid, sphingolipid, waxy and glyceride of fatty acid.

5. The formulation of claim 2, wherein the active ingredient is selected from at least one of a protein polypeptide drug, a nucleic acid drug, and a small molecule drug.

6. The formulation of claim 5, wherein the active ingredient is selected from the group consisting of:

(1) The protein polypeptide medicine is at least one selected from insulin, octreotide, leuprorelin acetate, calcitonin, thyme pentapeptide, luteinizing hormone releasing hormone, tecatide acetate, buserelin, exenatide, glucagon-like peptide-1, triptorelin acetate, leukocyte growth factor, erythrocyte growth factor, macrophage growth factor, tumor necrosis factor, epidermal growth factor, interleukin, angiostatin, bovine serum albumin, ovalbumin, parathyroid hormone, growth hormone, somatostatin, interferon and monoclonal antibody;

(2) The nucleic acid medicine is selected from at least one of small molecule interfering ribonucleic acid and plasmid DNA;

(3) The small molecule medicine is selected from antipyretic analgesic, non-steroidal anti-inflammatory, antibacterial, antitumor, hormone, central nervous system, peripheral nervous system, circulatory system, blood sugar lowering and diuretic.

7. The formulation of claim 5, wherein the active ingredient is selected from one or more of the following: vaccines, somatostatin acetate, aspirin, acetaminophen, benorilate, ibuprofen, naproxen, diclofenac sodium, indomethacin, benzocilin sodium, tetracycline, amoxicillin, ampicillin, metronidazole, tinidazole, levofloxacin, gatifloxacin, furazolidone, gentamicin, rifamycin, erythromycin, roxithromycin, clarithromycin, azithromycin, doxorubicin, taxol, cisplatin, 5-fluorouracil, hydroxycamptothecin, chang Chunjian, gemcitabine, vinblastine sulfate, misoprostol, estradiol, diethylstilbestrol, tamoxifen, levonorgestrel, norethindrone, mifepristone, hydrocortisone, dexamethasone, diazepam, isopentobutyralte, phenytoin sodium, carbamazepine, sodium valproate, chlorpromazine, haloperidol, meperidine hydrochloride, levodopa, clobetaline, bromhexine, atropine sulfate, bromethaline, epinephrine, ephedrine hydrochloride, procaine, lidocaine, propranolol, nifedipine, captopril, losartan, digoxin, lovastatin, gemfibrozil, tolbutamide, metformin, nateglinide, hydrochlorothiazide, spironolactone, furosemide, and edemic acid.

8. A method of preparing a formulation according to any one of claims 1 to 7, comprising the steps of:

(1) Covalently connecting a small molecule nutrient ligand with the hydrophilic end of the amphiphilic polymer;

(2) The polymer and the hydrophobic core material are dissolved in an organic solvent together to be used as an organic phase;

(3) Dropping the organic phase into the stirred water phase, and preparing nanoparticles by a nano precipitation method.

9. Use of a formulation according to any one of claims 1 to 7 for the preparation of a formulation for overcoming the intestinal absorption barrier.