CN112755005B

CN112755005B - Oral nano drug delivery system mediated by small molecular nutrient substances

Info

Publication number: CN112755005B
Application number: CN201911068292.1A
Authority: CN
Inventors: 黄园; 吴蕾; 周锐; 白瑜利; 吴蕊男
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2022-05-13
Anticipated expiration: 2039-11-04
Also published as: CN115192542B; CN115040495A; CN115192542A; CN112755005A; CN115040495B

Abstract

An oral nano-drug delivery system mediated by small molecule nutrients. The invention discloses an oral nano-carrier based on micromolecular nutrient substance mediation and actively targeting intestinal epithelial cells and a preparation method thereof. The invention 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 nano drug delivery system mediated by small molecular nutrient substances

Technical Field

The invention relates to a strategy for modifying micromolecular nutrient substances on the surface of nanoparticles to actively target intestinal epithelial cell surface receptors so as to overcome intestinal epithelial cell barriers and improve oral absorption of the nanoparticles, belonging to the field of pharmaceutical preparations.

Background

The Nano 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 the Nano drug delivery system is widely used for the research of oral drug delivery (ACS Nano 2015,9, 2345-.

In order 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 is devoted to modify a ligand on the surface of a nanoparticle, and the affinity of the nanoparticle and a cell membrane is increased by the interaction of the ligand and a receptor on the surface of an intestinal epithelial cell, so that the cell uptake is improved (Biomaterials 2018,180, 78-90). Among the ligands that have been studied more are targeting polypeptides or proteins, such as RGD, transmembrane peptides, transferrin, etc. (Adv Drug Deliv Rev 2013,65, 822-832).

However, it is noteworthy that the complex environment and the 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 enzymatic environment of the gastrointestinal tract may destroy the structure of protein polypeptides (Adv Drug Deliv Rev 2016,101, 75-88); on the other hand, the hydrophobic and positive regions in the protein polypeptide ligand are easy to interact with mucin in mucus, so that the mucus penetrability of the nanocarrier is reduced, and the interaction between the ligand and the cell membrane surface receptor is limited (Eur J Pharm Biopharm 2014,88, 518-.

The intestinal tract is the most important absorption organ of human body, and many small molecular nutrients, such as saccharides, amino acids, etc., can be rapidly absorbed by intestinal epithelial cells in large quantities, thereby ensuring the energy supply of human body (Science 2017,357, 1299-.

Disclosure of Invention

In order to solve the problems of the active targeting oral nano-carrier in the existing research, the inventor takes a small molecular nutrient substance as a ligand through creative research to modify the surface of the nano-particle coated by a hydrophilic shell. On one hand, the chemical stability of the micromolecule nutrient substance is higher, and the structure can be kept stable in the gastrointestinal tract environment; on the other hand, the modified nutrient substances with smaller molecular weight can keep the characteristics of hydrophilicity and negative electricity on the surface of the nanoparticle, do not influence the mucus penetrability of the nanoparticle, and improve the interaction between the nanoparticle and the epithelial cell surface receptor.

One of the purposes of the invention is to provide a method for improving the oral absorption of nanoparticles, which is to modify small molecular nutrients on the surface of the nanoparticles to actively target intestinal epithelial cell surface receptors.

One of the objects of the present invention is to provide the use of small molecule nutrients as ligands for the preparation of pharmaceutical compositions/preparations for overcoming intestinal absorption barriers.

One of the objectives of the present invention is to provide a nanoparticle for overcoming intestinal absorption barrier, which has a hydrophilic shell and a hydrophobic core, wherein the hydrophilic shell is composed of a hydrophilic polymer with terminals covalently linked or physically adsorbing small molecular nutrients; the hydrophobic core portion is composed of an active ingredient and a biocompatible carrier material.

In one specific embodiment, the average particle size of the nanoparticles is in the range of about 10-1000 nm; the nanoparticle with the core-shell structure is prepared from a hydrophobic core and a shell of a hydrophilic polymer according to a 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 nanoparticles.

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

In the embodiment of the invention, lactic acid-glycolic acid copolymer, phospholipid and fatty glyceride are further preferably used as biocompatible carrier materials and active ingredients to prepare the core.

As one of the preferred embodiments, the active ingredient comprises at least one of protein polypeptide drug, nucleic acid drug and chemical drug, and the content thereof 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 acetate leuprolide, calcitonin, thymopentin, luteinizing hormone releasing hormone, tecekide 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, monoclonal antibodies, and vaccines;

the nucleic acid drugs include but are not limited to small interfering ribonucleic acid and plasmid DNA;

such chemical classes include, but are not limited to: antipyretic analgesic and nonsteroidal anti-inflammatory drugs such as aspirin, acetaminophen, benorilate, ibuprofen, naproxen, diclofenac sodium, indomethacin, etc.; or antibiotic such as oxacillin sodium, tetracycline, amoxicillin, ampicillin, metronidazole, tinidazole, levofloxacin, gatifloxacin, furazolidone, gentamicin, rifamycin, erythromycin, roxithromycin, clarithromycin, azithromycin and other antibacterial drugs; or antineoplastic agents such as adriamycin, paclitaxel, cisplatin, 5-fluorouracil, hydroxycamptothecin, hederin, gemcitabine, vinblastine sulfate, etc.; or hormone drugs such as misoprostol, estradiol, diethylstilbestrol, tamoxifen, levonorgestrel, norethindrone, mifepristone, hydrocortisone, dexamethasone, etc.; or central nervous system drugs such as diazepam, amobarbital, phenytoin sodium, carbamazepine, sodium valproate, chlorpromazine, haloperidol, pethidine hydrochloride, levodopa, etc.; or peripheral nervous system drugs such as clobecholine, neostigmine bromide, atropine sulfate, propantheline bromide, epinephrine, ephedrine hydrochloride, procaine, and lidocaine; or circulatory system medicaments such as propranolol, nifedipine, captopril, losartan, digoxin, lovastatin, gemfibrozil and the like; or hypoglycemic and diuretic agents such as tolbutamide, metformin, nateglinide, hydrochlorothiazide, spironolactone, furosemide, and edetic acid.

As one of the embodiments of the present invention, insulin is preferred as an active ingredient.

The nanoparticle shell consists of hydrophilic ends of amphiphilic block polymers, wherein the hydrophilic ends are covalently connected and modified with micromolecular nutrients (ligands), so that the ligands are exposed on the surface of the nanoparticles. Wherein, the hydrophilic end includes but is not limited to polyethylene glycol, polyamino acid, N- (2-hydroxypropyl) methacrylamide (HPMA) polymer, zwitterion polymer and hyaluronic acid.

The small molecule nutrient substances of the invention include but are not limited to: one or more of glucose, fructose, galactose, mannose, amino acids, cholesterol, and 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 invention, polyethylene glycol and polyamino acids are preferred as the hydrophilic shell of the nanoparticle in the present invention.

One of the objects of the present invention is to provide a method for preparing a small molecule nutrient-mediated oral nano drug delivery system based on overcoming intestinal epithelial cell absorption barrier, comprising the following steps:

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

(2) the modified amphiphilic polymer, the biocompatible carrier material (nanoparticle core) and the active ingredient are mixed, and the nanoparticles are prepared 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 and the like.

As one specific embodiment, the method comprises the following steps:

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

(2) Mixing the prescription amount of fructose modified DSPE-PEG with the nanoparticle core material, dissolving in dimethyl sulfoxide (DMSO), and slowly dripping into a stirred water phase to obtain the final product.

One of the purposes of the invention is to provide a carrier material modified by preparing small molecule nutrient substances, wherein the small molecule nutrient substances and derivatives thereof can be connected with the carrier material through amido bonds, ester bonds and the like.

As one specific embodiment of the invention, a proper amount of 25-hydroxycholesterol (25HC) is weighed, dissolved in dichloromethane, added with succinic anhydride, reacted for 24 hours, and extracted to obtain succinated 25 HC; mixing it with DSPE-PEG-NH₂Reacting for 24h, removing unreacted 25HC by dialysis, and freeze drying to obtain 25HC modified DSPE-PEG (DSPE-PEG-25HC)。

As one specific embodiment of the invention, the carrier material modified by the small molecule nutrient substances can be used for preparing the nano particles by adopting a conventional method in the field of pharmacy, 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.

The invention aims to provide a nanoparticle for overcoming intestinal epithelial cell absorption barriers and a preparation method thereof, wherein the nanoparticle can be used for oral administration.

The invention provides a nanoparticle preparation capable of overcoming gastrointestinal absorption barriers, which is mainly prepared into oral administration preparations such as solution type liquid preparations, high molecular solution, emulsions, suspensions, syrups, drops, powders, granules, tablets, capsules and the like by using the nanoparticles and pharmaceutically acceptable auxiliary materials.

Has the advantages that:

1. the active ingredients in the invention are mainly positioned in the core of the nanoparticle, and the surface of the active ingredients is a shell formed by hydrophilic polymers, thus being beneficial to improving the colloidal stability of the nanoparticle, and effectively reducing the leakage of the medicament in the preparation and storage processes so as to improve the stability of the medicament and keep the activity of the medicament.

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

3. The nutrient substance has specific transporter in intestinal tract and can be rapidly absorbed. The nano-carrier of the surface modified micromolecular nutrient substance can actively target the intestinal epithelial cell surface receptor by utilizing the characteristics, and effectively improve the cell uptake and transmembrane transport efficiency.

4. The invention takes the micromolecule nutrient substance as the ligand, compared with the existing protein polypeptide ligand, the chemical stability is higher, and the invention is more beneficial to the function of the small molecule nutrient substance in the complex gastrointestinal tract environment.

5. According to the invention, the micromolecular nutrient substances are used, so that hydrophobic and positive charge regions of protein polypeptide ligands can be prevented from being introduced, and the particle size of the nanoparticles can not be increased, therefore, hydrophobic and electrostatic interaction with mucin can be effectively avoided, mucus penetrability of a nano carrier is not influenced, the nanoparticles can reach the surface of intestinal epithelial cells, and further, cell uptake is promoted.

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

Description of the drawings:

embodiments of the invention 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 synthetic pathway of glutamic acid-modified amphiphilic branched polylysine.

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

FIG. 4 shows a graph of nanoparticle transmucosal rates before and after fructose modification.

FIG. 5 is a graph showing the effect of cellular uptake of nanoparticles before and after amino acid modification.

Fig. 6 shows a graph of active targeting ability study of fructose modified nanoparticles.

FIG. 7 shows the transmembrane efficiency of nanoparticles after fructose modification.

FIG. 8 shows the pharmacodynamic study of nanoparticles before and after oral administration of proinsulin drug and fructose modification in rats.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are in no way intended to limit the scope of the invention. The present invention is further illustrated in detail below with reference to examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Also, equivalent alterations, combinations, improvements or modifications of the present invention will occur to those skilled in the art based on the description of the invention, but are intended to be within the scope of the invention.

Example 1 Synthesis of fructose-modified DSPE-PEG

The synthetic route of the fructose modified DSPE-PEG is shown in figure 1. First, D-fructose (2g, 0.011mol) was dissolved in acetone (40ml), stirred at room temperature, and concentrated sulfuric acid (4ml) was slowly dropped until fructose was completely dissolved. The reaction solution was added dropwise to an aqueous solution of sodium hydroxide (NaOH) (25ml, 4.5mol/L) at 0 ℃. The reaction was concentrated in vacuo, extracted three times with dichloromethane, the oil phases combined and washed three times with saturated brine. Adding anhydrous sodium sulfate, drying for 30min, filtering, recrystallizing with mixed solvent of n-hexane and diethyl ether (1:1, v/v), drying, and weighing to obtain compound 1 with yield of 86.5%.

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

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

Compound 3(109mg,0.5mmol) and DSPE-PEG-N₃(PEG molecular weight 5kDa) (125mg,0.025mmol) in DMF and copper sulphate solution (8mg/ml) was added with stirring at 60 ℃. Sodium ascorbate solution (5ml, 40mg/ml) was added dropwise at 0min, 30min, 60 min. After 2h, the reaction solution was transferred to a dialysis bag, dialyzed against disodium ethylenediaminetetraacetate for one day, dialyzed against ultrapure water for two days, and the dialyzate was lyophilized to obtain fructose-modified DSPE-PEG (compound 4) with a yield of 91.2%. The structure was confirmed by Fourier transform infrared spectroscopy, and Compound 4 was 2101.14cm^-1The characteristic peak of azide group disappears at 3445.65cm^-1A characteristic fructose peak appeared.

EXAMPLE 225 Synthesis of HydroxyCholesterol (25HC) modified DSPE-PEG

First, 25 hydroxycholesterol (25HC) is succinated to provide succinic acid-derived 25 hydroxycholesterol. In a specific scheme, the reaction mixture was dissolved in dichloromethane (25HC: succinic anhydride: DMAP 25:50:25 mol%) to a final concentration of 25HC of 80 mg/ml. The reaction is carried out for 24h at the constant temperature of 30 ℃ and the speed of 500 rpm. Spin-drying the product, adding pure water, and hydrolyzing at 40 deg.C for 2h to hydrolyze unreacted succinic anhydride to succinic acid. The successful connection of succinic acid can be confirmed by extracting succinated 25HC with dichloromethane and performing structure confirmation by NMR, and finding that 5.3ppm of hydrogen peak is changed to 4.6ppm (C3 site), and 2.5ppm of 4 hydrogen peak of typical succinic acid appears.

By amide reaction of DSPE-PEG-NH₂(PEG molecular weight 2kDa) and 25HC-COOH were covalently linked. 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 added₂And dropwise adding a small amount of triethylamine to adjust the pH value. Wherein the reaction mixture is dissolved in DMSO (DSPE-PEG-NH)₂:25HC-COOH:EDCI:NHS＝16.7:33.3:25:25mol％)，DSPE-PEG-NH₂The final concentration was 20 mg/ml. Then reacted at 25 ℃ for 24h at 500 rpm. The product is dialyzed rapidly with DMSO for 12h (25 deg.C), dialyzed with pure water for 24h, and lyophilized to obtain product (DSPE-PEG-25 HC). The successful connection of 25HC was confirmed by the structural confirmation using NMR spectroscopy, where a cholesterol peak was observed at low chemical shifts (0.5-1.5 ppm).

Example 3 Synthesis of amino acid-modified amphiphilic branched polylysine

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

(1) Synthesis of Decaglucol glutamate Fmoc-glutamic acid (3.69g,10mmol) and dodecanol (3.72g,20mmol) were dissolved in dichloromethane, EDCI (5.73g,30mmol) and 4-Dimethylaminopyridine (DMAP) (0.61g,5mmol) were added in this order, and the mixture was stirred at room temperature for 4 hours. Washing the reaction solution with saturated ammonium chloride aqueous solution and saturated sodium chloride aqueous solution in sequence, drying several layers with anhydrous sodium sulfate, concentrating, and separating by silica gel column chromatography to obtain the product compound A with the yield of 74%.

(2) Synthesis of amphiphilic branched polylysine Compound A (706mg,1mmol) 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 and PK-dendron (882mg,1.1 mmol; synthesized by the method of reference adv. Funct. Mater.2015,25, 5250-5260), 1-Hydroxybenzotriazole (HOBT) (135mg,1mmol), EDCI (287mg,1.5mmol), N, N-Diisopropylethylamine (DIEA) (330. mu.L, 2mmol) were added and the reaction was stirred at room temperature for 24h 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. Recrystallizing with ether and acetonitrile to obtain the product compound B with the yield of 62 percent. Compound B has C12 as the hydrophobic end and polylysine as the hydrophilic end.

(3) Synthesis of glutamic acid-modified amphiphilic branched polylysine Compound B (317mg,0.25mmol) was dissolved in 3mL of dichloromethane, stirred at room temperature, 3mL of trifluoroacetic acid was added dropwise, and stirring was continued for 4 h. The solvent and TFA were distilled off under reduced pressure, diethyl ether was added and the solid was precipitated at-10 ℃ C. The supernatant was centrifuged off, the solid was dissolved in dichloromethane, Boc-Glu-OtBu (463mg,1.2mmol), HOBT (135mg,1mmol), EDCI (287mg,1.5mmol), DIEA (330. mu.L, 2mmol) were added, and the reaction was stirred at room temperature for 24h under argon. The reaction mixture was washed successively with saturated ammonium chloride and saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. Adding acetonitrile to separate out a precipitate, filtering, and washing with acetonitrile to obtain a compound C with the yield of 64%.

The resulting compound C was dissolved in 4mL of dichloromethane, stirred at room temperature, TFA4mL was added dropwise, and stirring was continued for 6 h. Vacuum distilling to remove solvent and TFA, adding acetonitrile to precipitate, centrifuging to separate supernatant, and washing solid with acetonitrile and diethyl ether to obtain final product compound D with yield of 87%. The successful synthesis of the product is confirmed by analyzing the synthesis reaction of the compound D by utilizing nuclear magnetic resonance hydrogen spectrum. The spectrum was resolved as follows:¹H NMR(400MHz,DMSO-d₆)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, the compound B is covalently linked with cysteine and alpha-glutamic acid respectively according to a similar method to obtain Cys-B and alpha-Glu-B. The successful synthesis of the product was confirmed by analysis using hydrogen nuclear magnetic resonance spectroscopy. Cys-B spectrum was resolved as follows:¹H NMR(400MHz,DMSO-d₆) δ 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 ═ 6.4Hz, 6H). The spectrum of alpha-Glu-B was analyzed as follows:¹H NMR(400MHz,DMSO-d₆)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

An appropriate amount of PLGA (50/50, viscosity: 0.26-0.54dL/g) was weighed out and dissolved in DMSO at a concentration of 40mg/ml, and the fructose-modified DSPE-PEG and the unmodified DSPE-PEG in example 1 were dissolved in DMSO at concentrations of 20mg/ml, respectively. Dissolving Insulin (INS) and Phospholipid (PC) in DMSO and methanol respectively at concentrations of 12mg/ml and 96mg/ml, mixing at a volume ratio of 1:1, and magnetically stirring at room temperature (500rpm) for 1h to prepare phospholipid complex (IPC). By adjusting the mass fractions of the two DSPE-PEG, nanoparticles with different fructose modification degrees are prepared. The specific method comprises the steps of uniformly mixing the materials according to the proportion shown in the table 1, slowly dripping 2ml of water phase (700rpm), continuously stirring for 5min, removing DMSO by an ultrafiltration method, and adding ultrapure water for re-dispersing to obtain the product.

Table 1: proportion of each material of fructose modified nano-particle

Example 525 preparation and cellular uptake of HC-modified PLGA nanoparticles

PLGA, DSPE-PEG-25HC and phospholipid were dissolved in DMSO to prepare stock solutions (16mg/ml), and coumarin 6, a fluorescent dye, was dissolved in DMSO at a concentration of 2 mg/ml. PLGA, DSPE-PEG-25HC, phospholipid according to the volume ratio: and (3) uniformly mixing the stock solution to obtain an organic phase, wherein the ratio of coumarin 6 to coumarin 6 is 4:3:1: 0.05. And uniformly mixing the organic phase and 10-15 times of deionized water by volume through a microfluidic device, wherein the flow rate of the organic phase is 160 mu l/min, and the flow rate of the water phase is 1ml/min, and preparing the 25HC modified PLGA nano-particles. And then removing DMSO by adopting an ultrafiltration method to obtain the 25HC modified PLGA nano-particle.

After digestion of Caco-2 cells, the cells were placed at 1X 10 per well⁴The density of cells was plated in 96-well plates, and after the cells had grown for 4 days to differentiate, the medium was removed and the cells were rinsed with fresh PBS. Incubating the nanoparticles and cells for 3h, removing the nanoparticles, rinsing the cells with fresh PBS for three times, adding 0.1ml of DMSO into each hole to destroy the cells and the nanoparticles, and measuring the DiI fluorescence value by using a microplate reader. The number of cells per well was corrected by resazurin to obtain the relative amount of cellular uptake. We find that the cellular uptake of 25HC modified PLGA nanoparticles is 3.45 times that of unmodified nanoparticles, which indicates that the 25HC serving as a ligand can effectively improve the cell membrane affinity of the nanoparticles.

Example 6 examination of nanoparticle transmucosal Capacity before and after fructose modification

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

As shown in the attached figure 3, after fructose modification, the particle size of the nanoparticles is not obviously changed, and with the increase of the fructose modification degree, the negative charge on the surfaces of the nanoparticles is increased, and the surface fructose is successfully modified on the surfaces of the nanoparticles.

Fresh porcine intestinal mucus was collected and spread in a Transwell chamber (100. mu.l mucus per well) with a membrane area of 0.33cm²The pore size of the polycarbonate semipermeable membrane was 3 μm. 200 μ l of nanoparticle dispersion was carefully added dropwise to the mucus top, and 800 μ l of blank buffer was added to the receiving chamber at 15, 30, 6, respectivelyAt 0 and 120min, 50. mu.l of sample was taken from the receiving chamber for fluorescence analysis and the receiving chamber was immediately replenished with an equal volume of blank buffer. The apparent permeability coefficient (Papp) values of the nanoparticles were calculated according to the following formula: papp ═ dQ/dt × 1/(A × C)₀)](dQ/dt represents the diffusion rate of nanoparticles, A is the membrane area, C₀The initial concentration of the drug) and the results are shown in figure 4 of the specification.

As can be seen from the attached figure 4, after fructose modification, the Papp value of the mucus penetrating type PEG nanoparticle has no obvious change, which indicates that the micromolecule negatively charged fructose can not increase the electrostatic and hydrophobic acting force between the nanoparticle and mucin, thereby ensuring the mucus penetrating rate.

Example 7 examination of cellular uptake Effect of nanoparticles before and after amino acid modification

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

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

Example 8 active targeting ability study of fructose modified nanoparticles

DiI-loaded PEG nanoparticles and 100% fructose nanoparticles were prepared as in example 6. The nanoparticles were incubated with the antibodies of glucose transporter 2(GLUT2) and glucose transporter 5(GLUT5), respectively, and after 3h, the cellular uptake of each group of nanoparticles was determined as described in example 5, with the results shown in FIG. 6.

Fig. 6 shows that the specific blocking of glucose transporters by GLUT2 and GLUT5 antibodies can significantly inhibit the uptake of fructose nanoparticles without affecting the uptake of PEG nanoparticles. GLUT2 and GLUT5 mediate the absorption of free fructose, therefore, fructose modification can enable nanoparticles to actively target glucose transporters to increase the affinity of the nanoparticles with cell membranes.

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

After digestion of Caco-2 cells, 3X 10 cells per well were used⁴Was inoculated into a Transwell chamber (upper chamber) and 0.6mL of complete medium was added to the receiving chamber (lower chamber). The membrane area of the cell was 0.33cm²The pore size of the polycarbonate semipermeable membrane was 3 μm. The culture medium was changed every other day for the first 12 days, and every other day thereafter. Meanwhile, from day 8 onwards, the transmembrane resistance value (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.

Taking the TEER value of more than 500 omega cm²Transwell cell of (2) to determine the transmembrane transport of nanoparticles. The medium in the upper and lower chambers was removed before the experiment, an equal volume of pre-warmed blank medium was added for equilibration for 30min, 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. At 0, 15, 30, 60, 90, 150, 240 and 360min, 50 μ l of sample from the receiving chamber was taken for fluorescence analysis and the receiving chamber was immediately supplemented with an equal volume of blank medium. The Papp values were calculated according to the method of example 6, and the results are shown in FIG. 7. As can be seen from the attached figure 7, the Papp value of 100% fructose nanoparticles is 5.2 times that of PEG nanoparticles, which shows that fructose modification can significantly improve transmembrane transport efficiency of nanoparticles through active targeting effect, and is helpful for promoting transmembrane transport efficiency of nanoparticlesOvercoming the intestinal epithelial cell absorption barrier.

Example 10 in vivo pharmacodynamic study of fructose-modified PLGA nanoparticles

15 SD rats (180-220g) fasted for 12 hours were randomly selected and divided into 3 groups, 5 each of the free INS solution group, INS-loaded PEG nanoparticles and fructose nanoparticles group (nanoparticles prepared in example 4). 2.0ml (containing 50IU/kg of insulin) of INS technical product and each group of nano-particles are respectively administered by intragastric administration, the blood sugar value of a rat is measured according to preset time points (0, 1, 2, 4, 6, 8 and 10h), the blood sugar value of the rat before administration is taken as 100%, and the percentage of blood sugar reduction at each time point is calculated according to the following formula: percent change in blood glucose (% Gt/G0 × 100(Gt and G0 represent blood glucose levels in rats at time t and in rats before administration, respectively), and a plot of percent change in blood glucose versus time t was made to obtain a percent blood glucose-time curve, the results of which are shown in fig. 8 of the specification.

As can be seen from the figure 8, the fructose nanoparticle group has a blood glucose reduction effect obviously superior to that of the original medicine group within 1 to 6 hours, and the blood glucose value within 2 hours is obviously lower than that of the PEG nanoparticle group, so that the fructose modification can obviously improve the oral absorption of the nanoparticles.

Claims

1. An oral administration preparation for overcoming intestinal absorption barriers is characterized by being prepared from nanoparticles and pharmaceutically acceptable auxiliary materials, wherein the nanoparticles are modified on the surfaces of the nanoparticles by taking micromolecular nutrient substances as ligands, so that the nanoparticles actively target intestinal epithelial cell surface receptors; the micromolecule nutrient substance is selected from fructose, the nanoparticle is provided with a hydrophilic shell and a hydrophobic core, wherein the hydrophilic shell is composed of a hydrophilic end of an amphiphilic polymer and a micromolecule nutrient substance of which the hydrophilic end is covalently connected; the hydrophobic core part consists of hydrophobic ends of amphiphilic polymer, active components and biocompatible carrier material.

2. The preparation according to claim 1, wherein the nanoparticle is prepared from a hydrophobic core and a hydrophilic shell according to a mass ratio of 1: 99-95: 5; the active ingredient accounts for 0.1-90% of the total weight of the nanoparticles.

3. The formulation of claim 1, wherein the biocompatible carrier material is selected from at least one of a mono-or copolymer of lactic acid and glycolic acid, polystyrene, polysebacic acid, polyethyleneimine, a mono-or copolymer of lactide and glycolide, an inorganic silicon material, an inorganic carbon material, an alkyl polycyanoacrylate, a polyamino acid, cholesterol, a fatty acid, a phospholipid, a sphingolipid, a wax and a fatty acid glyceride.

4. The formulation of claim 1, wherein the active ingredient is selected from at least one of a protein polypeptide drug, a nucleic acid drug, and a small molecule drug.

5. The formulation according to claim 4, characterized in that the active ingredient is selected from:

(1) the protein polypeptide drug is at least one of insulin, octreotide, leuprorelin acetate, calcitonin, thymopentin, luteinizing hormone releasing hormone, techocin 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 drug is at least one selected from small molecule interfering ribonucleic acid and plasmid DNA;

(3) the small molecule drug is selected from antipyretic analgesics, non-steroidal anti-inflammatory drugs, antibacterial drugs, antitumor drugs, hormone drugs, central nervous system drugs, peripheral nervous system drugs, circulatory system drugs, hypoglycemic drugs and diuretic drugs.

6. The formulation according to claim 4, wherein the active ingredient is selected from one or more of the following drugs: vaccines, somatostatin acetate, aspirin, acetaminophen, benorilate, ibuprofen, naproxen, diclofenac sodium, indomethacin, oxacillin sodium, tetracycline, amoxicillin, ampicillin, metronidazole, tinidazole, levofloxacin, gatifloxacin, furazolidone, gentamycin, rifamycin, erythromycin, roxithromycin, clarithromycin, azithromycin, doxorubicin, taxol, cisplatin, 5-fluorouracil, hydroxycamptothecin, hederin, gemcitabine, vinblastine sulfate, misoprostol, estradiol, diethylstilbestrol, tamoxifen, levonorgestrel, norethindrone, mifepristone, hydrocortisone, dexamethasone, diazepam, amobarbital, phenytoin sodium, carbamazepine, sodium valproate, chlorpromazine, haloperidol, pethidine hydrochloride, levodopa, clobecholine, neostigmine, bromoneostigmine, doxycycline, and doxycycline, Atropine sulfate, bromalantopalin, epinephrine, ephedrine hydrochloride, procaine, lidocaine, propranolol, nifedipine, captopril, losartan, digoxin, lovastatin, gemfibrozil, tolbutamide, metformin, nateglinide, hydrochlorothiazide, spironolactone, furosemide and edenic acid.

7. A method of preparing a formulation according to any one of claims 1 to 6, comprising the steps of:

(1) covalently linking a small molecule nutrient ligand to the hydrophilic end of an amphiphilic polymer;

(2) dissolving the polymer and the hydrophobic core material in an organic solvent together to obtain an organic phase;

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

8. Use of a formulation according to any one of claims 1 to 6 in the manufacture of a formulation for overcoming intestinal absorption barriers.