CN106581645B

CN106581645B - Vitamin B loaded with medicine12Derivative self-assembled nano-particles, preparation method and application

Info

Publication number: CN106581645B
Application number: CN201610992427.3A
Authority: CN
Inventors: 李弘剑; 冉艳红; 刘天祥; 陈美云; 李万维; 杨晓苹
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2016-11-11
Filing date: 2016-11-11
Publication date: 2020-11-13
Anticipated expiration: 2036-11-11
Also published as: CN106581645A

Abstract

The invention discloses a vitamin B loaded with medicine₁₂Derivative self-assembly nanometer particles, a preparation method and application. The invention respectively dissolves the drug and the phenylalanine dipeptide in hexafluoroisopropanol to obtain a solution A and a solution B; mixing vitamin B as shown in formula I₁₂Dissolving the derivative in hexafluoroisopropanol to obtain a solution C; uniformly mixing an organic solvent or a solution thereof with the solution A to obtain a solution D; uniformly mixing the solution B and the solution C, adding the mixture into the solution D, and uniformly mixing to obtain a solution E; mixing the solution E with water, adding PEG200, and mixing to obtain solution F; freeze drying, adding water, and resuspending to obtain vitamin B loaded with medicine₁₂The derivatives self-assemble nanoparticles. The nanometer particle contains vitamin B₁₂Therefore, a plurality of drug molecules can pass through intestinal mucosa to enter portal circulation in an active transportation mode, and the treatment effect of the administration dosage is ensured.

Description

Vitamin B loaded with medicine12Derivative self-assembled nano-particles, preparation method and application

Technical Field

The invention belongs to the field of nano-drug carriers, and particularly relates to drug-loaded vitamin B₁₂Derivative self-assembly nanometer particles, a preparation method and application.

Background

Polypeptide protein biological drugs are more and more shared in the drug market at present, and common drugs comprise monoclonal antibodies for treating cancers and autoimmune diseases, hepatitis A and B resistant vaccines, diabetes therapeutic agents such as insulin and incretin, human growth hormone for treating hormone deficiency and the like. However, the wider application of such drugs is limited by their own properties, such as: low stability, high molecular weight and hydrophilicity, resulting in difficulty in crossing biological membranes resulting in low bioavailability, etc. Polypeptide and protein drugs are currently administered primarily by drug injection. However, this administration mode has low patient compliance due to pain, and the therapeutic effect of some drugs is affected by the difference between the injection administration and the physiological utilization route. The needle-free administration route, including various non-invasive administration modes such as oral administration, nasal administration, pulmonary administration and the like, is receiving wide attention.

Of all administration, oral administration is the most highly compliant, convenient and safe for the patient. Oral administration of polypeptide protein drugs is mainly limited by two aspects: firstly, the protease and peptidase in intestines and stomach are easy to degrade the medicine; secondly, the drugs are generally of relatively large molecular weight and have no specific absorption pathway on small intestinal epithelial cells. In view of the above limitations, methods such as prescription composition, encapsulation technology, macromolecule compounding and chemical modification are available at the present stage to improve oral absorption of polypeptide protein drugs. These methods all improve the bioavailability of oral protein drugs, but each has drawbacks. The fatty acid microemulsion in the formula can change the integrity of the gastrointestinal mucosal surface, the polysaccharide structure in the encapsulation technology needs to be improved in absorption efficiency, and the macromolecular compound and chemical modification can change the molecular structure form of the polypeptide protein medicine.

Vitamin B₁₂There is a specific absorption pathway in the intestinal wall, which cannot be absorbed by the intestine and stomach by simple diffusion due to its relatively large molecular weight (1355 Da). Vitamin B in food₁₂Is released by digestion with gastric juice and forms a complex with pepsin, an endogenous factor (IF), vitamin B₁₂the-IF complex has specific receptor proteins on small intestinal epithelial cells, and thus permeates the intestinal tract by means of receptor endocytosis. After absorption by small intestinal epithelial cells, IF reacts with vitamin B₁₂Separating and separating vitamin B₁₂Released into the portal circulation. Vitamin B has been reported₁₂Can directly guide protein or polypeptide to pass through intestinal wall, and improve oral absorption rate of medicine. However, this way of chemically modifying polypeptide protein drugs is also deficient: firstly, the polypeptide protein medicine is chemically modified and vitamin B₁₂Linked, which can affect drug activity; second, vitamin B in intestinal membrane₁₂The receptor of the-IF complex is limited and the receptor binds vitamin B₁₂Vitamin B in receptor plasma₁₂Regulation of concentration, thus, vitamin B₁₂The dosage of the drug carrying protein or polypeptide often does not meet the treatment requirement.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a vitamin B loaded with a medicament₁₂A method for preparing derivative self-assembly nanometer particles.

Another object of the present invention is to provide a drug-loaded vitamin B obtained by the above-mentioned preparation method₁₂The derivatives self-assemble nanoparticles.

It is still another object of the present invention to provide the drug-loaded vitamin B₁₂The application of the derivative self-assembly nanometer particles.

The purpose of the invention is realized by the following technical scheme: vitamin B loaded with medicine₁₂The preparation method of the derivative self-assembly nano particles comprises the following steps:

(A) dissolving a drug in hexafluoroisopropanol to obtain a solution A; dissolving phenylalanine dipeptide in hexafluoroisopropanol to obtain solution B; vitamin B which can be self-assembled into nano particles and is shown as formula I₁₂Dissolving the derivative in hexafluoroisopropanol to obtain a solution C;

(B) uniformly mixing an organic solvent X or an organic solvent X solution with the solution A to obtain a solution D; the organic solvent X is ethanol, acetone or ethyl acetate;

(C) uniformly mixing the solution B and the solution C, adding the mixture into the solution D, and uniformly mixing to obtain a solution E;

(D) mixing solution E with water, and mixingAdding PEG200, and mixing to obtain solution F; freeze drying, adding water for re-dissolving and re-suspending to obtain vitamin B loaded with medicine₁₂Derivative self-assembled nanoparticles;

the concentration of the drug in the solution A is preferably 20 mg/ml.

The concentration of the phenylalanine dipeptide in the solution B is preferably 100 mg/ml.

The solution C can be self-contained with vitamin B which forms nano particles₁₂The concentration of the derivative is preferably 1 mg/ml.

In the solution D, the concentration of the drug is preferably 4mg/ml, and the concentration of the organic solvent X is preferably 40 percent by volume.

The concentration of the organic solvent X solution is preferably 50% by volume.

In the solution E, the concentration of the medicament is preferably 3.3mg/ml, the concentration of the organic solvent X is preferably 33 percent by volume, the concentration of the phenylalanine dipeptide is preferably 13.3mg/ml, and the vitamin B which can be automatically filled into nano-particles₁₂The concentration of the derivative is preferably 0.03 mg/ml.

In the solution F, the concentration of the medicine is preferably 0.1mg/ml, the concentration of the organic solvent X is preferably 10 percent by volume, the concentration of the phenylalanine dipeptide is preferably 4mg/ml, and the vitamin B which can be automatically filled into nano particles₁₂The concentration of the derivative is preferably 0.01mg/ml, and the final concentration of PEG200 is preferably 1% by volume.

The medicament is preferably a polypeptide medicament and/or a protein medicament; more preferred are glucagon-like peptides and derivatives thereof, such as 6-KTP.

The vitamin B capable of being self-assembled to form nano particles₁₂The derivative is prepared by the following steps:

(1) preparation of amide intermediate:

firstly, coupling agent of phosphate onium salt, DMF (dimethyl formamide), DIEA (dimethyl amine-dimethyl ether)Isopropyl ethylamine) and Boc-phenylalanine dipeptide, and carrying out ultrasonic water bath reaction to obtain amide intermediate product X₁；

② mixing DMF, carbodiimide coupling agent, HOAT (1-hydroxy-7-azobenzotriazole) and Boc-phenylalanine dipeptide, and making them undergo the process of ultrasonic water-bath reaction to obtain amide intermediate product X₂；

(2) Vitamin B₁₂-preparation of OP:

amide intermediate product X obtained in the previous step (1)₁Or the amide intermediate product X obtained in the step (1)₂Adding vitamin B₁₂-carbonyl-putrescine (vitamin B)₁₂-OD), ultrasonic water bath reaction, adding ethyl acetate to stop reaction, centrifuging, removing supernatant, collecting precipitate, and drying to obtain vitamin B₁₂-OP (vitamin B)₁₂-carbonyl-putrescine-phenylalanine dipeptide-Boc);

wherein, DMF is solvent, and the raw materials participating in the reaction are proportioned according to the following molar ratio:

by amide intermediates X₁Obtaining vitamin B₁₂Vitamin B in the case of carbonyl-putrescine₁₂-carbonyl-putrescine: coupling agent of phosphate salt: boc-phenylalanine dipeptide: DIEA 1: 1-4: 0.75-3: 0.5 to 2;

by amide intermediates X₂Obtaining vitamin B₁₂Carbonyl-putrescine, carbodiimide-based coupling agents: HOAT: boc-phenylalanine dipeptide: vitamin B₁₂-carbonyl-putrescine ═ 0.75 to 3: 0.75-3: 0.75-3: 1.

The raw materials participating in the reaction are more preferably prepared according to the following molar ratio:

vitamin B₁₂-carbonyl-putrescine: coupling agent of phosphate salt: boc-phenylalanine dipeptide: DIEA 17:34:25: 34;

carbodiimide coupling agent: HOAT: boc-phenylalanine dipeptide: vitamin B₁₂-carbonyl-putrescine ═ 25:25:25: 17.

The phosphate coupling agent in the step (1) is preferably HATU (2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) or HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate).

The carbodiimide coupling agent in the step (1) is preferably DIC (N, N-diisopropylcarbodiimide).

The conditions of the ultrasonic water bath reaction I in the first step and the second step are preferably as follows: carrying out ultrasonic reaction at 30 ℃ and 70w for 5 min.

The DMF in the step (1) is used as a solvent (reaction medium) and the dosage of the DMF is enough to dissolve all reaction raw materials.

The conditions of the ultrasonic water bath reaction II in the step (2) are preferably as follows: carrying out ultrasonic reaction at 30 ℃ and 70w for 2 h.

The dosage of the ethyl acetate in the step (2) is that the volume ratio of the ethyl acetate to DMF is 2-8: 1, preferably, the volume ratio of ethyl acetate to DMF is 2.5-4: 1, calculating the mixture ratio.

The conditions for the centrifugation in step (2) are preferably: centrifuging at 8000r/min for 5 min.

The drying in step (2) is preferably vacuum drying or freeze drying.

The vitamin B capable of being self-assembled to form nano particles₁₂The preparation method of the derivative also comprises the step of mixing the product (vitamin B) obtained in the step (2)₁₂OP) desalting via a C18 reverse phase column.

And the C18 reverse phase column is desalted and eluted by acetonitrile solution.

The desalting is preferably carried out by:

(I) mixing vitamin B₁₂-OP is dissolved in aqueous acetonitrile containing TFA (trifluoroacetic acid) to obtain vitamin B₁₂-an OP solution;

(II) gradient elution with C18 reverse phase column: the reverse phase column was equilibrated with 20% (v/v) acetonitrile in water containing 0.1% (v/v) TFA, then desalted with 20% (v/v) acetonitrile (without TFA) and the vitamin B was eluted with 50% (v/v) acetonitrile (without TFA)₁₂-OP, collecting the red eluted product under this gradient;

(III) concentrating, freezing and drying the red elution product obtained in the step (ii) to obtain desalted vitamin B₁₂-OP。

Vitamin B as described in step (I)₁₂The concentration of the-OP solution is preferably 2 mg/ml.

The equilibration time in step (II) is preferably 12 min.

The flow rate for desalting described in step (II) is preferably 10 ml/min.

The time for desalting in step (II) is preferably 12 min.

Elution of vitamin B as described in step (II)₁₂The degree of-OP was such that it eluted to a red-free solution.

The concentration in step (III) is preferably carried out by vacuum rotary evaporation at 45 ℃.

The drying in step (III) is preferably performed in a freeze dryer.

The drying time in step (III) is preferably 48 h.

The desalting further comprises regenerating the reverse phase column with an 80% (v/v) acetonitrile solution containing 0.1% (v/v) TFA after the elution is completed.

The vitamin B capable of being self-assembled to form nano particles₁₂The preparation method of the derivative further comprises desalting the product (desalted vitamin B)₁₂-OP) was further purified by semi-preparative high performance liquid chromatography.

The purification is preferably achieved by:

(i) desalting vitamin B₁₂Dissolving OP in 20% (v/v) acetonitrile to obtain desalted vitamin B₁₂-an OP solution;

(ii) gradient eluting with semi-preparative column of C18 reverse phase column, collecting eluate, freezing, and drying to obtain purified vitamin B₁₂-OP。

(ii) the desalted vitamin B as described in step (i)₁₂The concentration of the-OP solution is preferably 10 mg/ml.

The rate of elution described in step (ii) is preferably 3 ml/min.

The drying time in step (ii) is preferably 48 h.

Vitamin B as described in step (2)₁₂-carbonyl-putrescine (vitamin B)₁₂-OD) is preferably prepared by:

(a) mixing vitamin B₁₂Dissolving in DMSO (dimethyl sulfoxide), adding CDT with 8 times of molar weight, performing ultrasonic water bath reaction, adding ethyl acetate to stop reaction, centrifuging, and removing supernatant to obtain an ester imidazole intermediate product;

(b) dissolving the intermediate product of the esterimidazole obtained in the step (1) in DMSO, adding DIEA (N, N-diisopropylethylamine) and putrescine, carrying out ultrasonic water bath reaction, adding ethyl acetate to stop the reaction, centrifuging, removing supernatant, taking precipitate, and drying to obtain vitamin B₁₂-OD。

The amount of ethyl acetate used in step (a) is preferably an amount corresponding to 4 times the volume of DMSO used in this step.

The ultrasonic water bath reaction conditions in the step (a) are as follows: carrying out ultrasonic reaction at 30-33 ℃ and 70w for 30 min.

The conditions for the centrifugation in step (a) and step (b) are: centrifuging at 8000r/min for 5 min.

The amount of ethyl acetate used in step (b) is preferably an amount corresponding to 4 times the volume of DMSO used in this step.

The ultrasonic water bath reaction conditions in the step (b) are as follows: carrying out ultrasonic reaction at 30-33 ℃ for 2h at 70 w.

The drying in the step (b) is drying by using a nitrogen blower.

Vitamin B as described in step (B)₁₂-OD may also be further purified by silica gel column chromatography.

The purification step is preferably: mixing vitamin B₁₂-OD is dissolved in a preformed eluent to obtain vitamin B₁₂Loading the OD solution on a silica gel column, and eluting to obtain purified vitamin B₁₂-OD。

The particle size of the silica gel is 5-10 μm.

The prefabricated eluent is obtained by mixing n-butanol, isopropanol and deionized water according to the volume ratio of 45:30: 25.

The vitamin B₁₂The concentration of the OD solution was 15 mg/ml.

The eluted eluent is obtained by mixing ammonia water and a prefabricated eluent according to the volume ratio of 2: 98.

Vitamin B loaded with medicine₁₂The derivative self-assembled nano particles are obtained by the preparation method.

The vitamin B loaded with the medicine₁₂The diameter of the derivative self-assembled nanoparticles is preferably 100 to 400 nm.

The vitamin B loaded with the medicine₁₂The shape of the derivative self-assembled nanoparticles is preferably spherical.

The vitamin B loaded with the medicine₁₂The application of the derivative self-assembly nano particles in preparing intestinal absorption medicines.

Compared with the prior art, the invention has the following advantages and effects:

1. the biological medicine can utilize vitamin B without any modification₁₂The absorption route of (2) enters into the portal circulation to keep the vitality of the medicine.

2. Vitamin B per molecule₁₂The derivative can carry a plurality of drug molecules to pass through intestinal mucosa to enter portal circulation in an active transportation mode, and the treatment effect of the dosage of the drug is ensured.

3. Vitamin B₁₂The derivative combined with IF protects the nanoparticles from degradation and together with the nanoparticles resists degradation by gastrointestinal proteases of the biopharmaceutical.

4. The phenylalanine dipeptide nanometer particle entering into portal circulation can be slowly degraded by blood protease to release medicine.

5. Vitamin B₁₂The phenylalanine dipeptide is a biomolecule, the side effect of the drug carrier is very low, and the phenylalanine dipeptide is a friendly carrier of the drug.

6. Active molecules, such as incretins for the treatment of diabetes, are absorbed through the intestines and stomach and exert the drug effect in a physiological manner.

Drawings

FIG. 1 is vitamin B₁₂-carbonyl-putrescine-phenylalanine dipeptide-Boc (VB)₁₂-OP) synthetic route scheme.

FIG. 2 is vitamin B₁₂-mass spectrum of carbonyl-putrescine.

FIG. 3 is a synthesis of VB using HBTU as coupling agent₁₂-HPLC analytical profile of OP.

FIG. 4 is the synthesis of VB using HATU as coupling agent₁₂-HPLC analytical profile of OP.

FIG. 5 Synthesis of VB Using DIC as coupling agent₁₂-HPLC analytical profile of OP.

FIG. 6 is vitamin B₁₂-absorption spectrum at 361nm before and after desalting of OP synthesis product, wherein: a is an absorption chart spectrogram before desalination; b is an absorption chart after desalination.

FIG. 7 shows the HPLC purification of vitamin B₁₂-mass spectrometric detection of OP profile.

FIG. 8 shows the purified vitamin B₁₂-spectrum of HPLC purity analysis of OP.

FIG. 9 is vitamin B₁₂The particle size distribution map of the mediated nano-particle after drug loading.

FIG. 10 shows vitamin B₁₂Field emission electron microscopy images of mediated nanoparticle drug loading.

FIG. 11 shows vitamin B₁₂HPLC analysis chart of the mediated ultrafiltration filtrate after nanoparticle loading.

FIG. 12 is a graph showing the results of apparent permeability coefficients (Papp) of the drug on Caco-2 cell monolayer membranes at various time points.

FIG. 13 is a graph of the concentration of drug in the blood of mice at various time points.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 vitamin B₁₂Synthesis and purification preparation of-OP (scheme shown in FIG. 1)

(1) Vitamin B₁₂-carbonyl-putrescine (vitamin B)₁₂-OD) Synthesis

Collecting 0.2mg vitamin B₁₂Dissolved in 5ml of DMSO (dimethyl sulfoxide),adding vitamin B ₁₂8 times molar amount of CDT. CDT and vitamin B₁₂The reaction is carried out in an ultrasonic water bath with the ultrasonic power of 70w and the temperature of the water bath kept between 30 ℃ and 33 ℃ for half an hour to obtain an ester imidazole intermediate product, and then 4 times of volume of ethyl acetate is added to stop the reaction. Transferring the reaction solution into a centrifuge tube, centrifuging for 5min at 8000r/min, removing supernatant, adding 5ml DMSO to dissolve the centrifugal precipitate, and rapidly adding 300. mu.l DIEA (N, N-diisopropylethylamine) and 600. mu.l putrescine. Ultrasonic water bath assisted reaction with ultrasonic power of 70w, keeping the temperature of the water bath between 30 ℃ and 33 ℃, adding ethyl acetate with 4 times of volume after 2 hours of reaction to stop the reaction, centrifuging at 8000r/min for 5min, and removing supernatant. Blowing the precipitate with a nitrogen blower to obtain vitamin B₁₂-a synthetic sample of OD.

(2) Vitamin B₁₂Silica gel column chromatography purification of-carbonyl-putrescine synthesis product

The above reaction sample was prepared by purification using silica gel column chromatography (silica gel purchased from Nicoti chemical industry research institute, silica gel particles 5 μm to 10 μm, column diameter 1 cm. times.5 cm) dissolved in a pre-prepared eluent (45% (v/v) n-butanol, 30% (v/v) isopropanol, 25% (v/v) deionized water) at a concentration of 15 mg/ml. Loading 1ml each time, adding 2% (v/v) ammonia water into the pre-prepared eluent to elute the reaction product to obtain vitamin B₁₂-carbonyl-putrescine. The vitamin B12-carbonyl-putrescine eluted product with a grey value ratio of about 95% as determined by Thin Layer Chromatography (TLC). The mass spectrum of the purified product has a mass-to-charge ratio of 1469.39[ H +]Corresponding to the molecular weight of vitamin B12-carbonyl-putrescine, as shown in figure 2.

(3) Vitamin B₁₂-carbonyl-putrescine-phenylalanine dipeptide-Boc (vitamin B)₁₂-OP) Synthesis

Respectively activating carboxyl of Boc-Phe-Phe-OH by using an onium phosphate coupling agent or a carbodiimide coupling agent to generate an active intermediate product, and then adding vitamin B₁₂Carbonyl-putrescine to produce more stable vitamin B₁₂-carbonyl-putrescine-phenylalanine dipeptide-Boc:

(I) condensing agent method of onium salts: HATU (2- (7-benzotriazole oxide) was used) Activating carboxyl of Boc-Phe-Phe-OH by-N, N, N ', N' -tetramethyluronium hexafluorophosphate) or HBTU (O-benzotriazol-tetramethyluronium hexafluorophosphate) to obtain active amide intermediate, and adding vitamin B₁₂OD, production of more stable vitamin B₁₂-OP. Vitamin B₁₂The synthesis of-OP involves two steps, activation of the carboxyl group and formation of the amide, as follows: 2.0ml of DMF solution was taken in a 5ml syringe and taken in a penicillin bottle, and 0.034mol of HATU or HBTU, 0.034mol of DIEA (N, N-diisopropylethylamine) and 0.025mol of Boc-phenylalanine dipeptide (Boc-Phe-Phe-OH, BACHEM from supplier) were added and capped and sonicated at 30 ℃ and 70w for 5 min. After the ultrasound is finished, 0.017mol of vitamin B is taken₁₂adding-OD into a penicillin bottle, uniformly mixing, and carrying out 70w ultrasonic oscillation reaction for 2 hours at 30 ℃. After the reaction is finished, the reaction system is transferred into a 10ml centrifuge tube, 5ml of ethyl acetate is added, 8000r/min is carried out, and centrifugation is carried out for 5 min. Removing supernatant, vacuum drying the precipitate to obtain vitamin B₁₂-OP. Wherein VB is synthesized by using HBTU as coupling agent₁₂The HPLC analysis pattern of-OP is shown in FIG. 3; synthesis of VB Using HATU as coupling agent₁₂The HPLC analysis pattern of-OP is shown in FIG. 4, from which it can be seen that VB12-OP has a better synthesis effect when HATU or HBTU is used as the coupling agent, and that the effect of HATU is higher than that of HBTU.

(II) chemical synthesis method of carbodiimide compound (carbodiimide coupling agent): 2.0mL of DMF solution was taken in a penicillin bottle by a 5mL syringe, 0.025mol DIC (N, N-diisopropylcarbodiimide), 0.025mol HOAT (1-hydroxy-7-azobenzotriazol) and 0.025mol Boc-phenylalanine dipeptide were added in this order, and after covering the bottle, sonication was carried out for 30min (70W, 30 ℃ C.). After the ultrasound is finished, 0.017mol of vitamin B is taken₁₂Adding the-carbonyl-putrescine into a penicillin bottle, uniformly mixing, and carrying out ultrasonic water bath reaction for 2 hours (70W at 30 ℃). Adding 8mL of ethyl acetate to terminate the reaction, transferring the reaction system into a centrifuge tube, and centrifuging for 5min at the rotating speed of 8000 r/min. Removing supernatant, precipitating, freeze drying to obtain vitamin B₁₂-OP. Wherein VB is synthesized by using DIC as coupling agent₁₂The HPLC analysis pattern of-OP is shown in FIG. 5, and when DIC is used as the coupling agent, it can be achievedSo as to obtain better synthetic effect.

(4) Vitamin B₁₂Reverse desalination of-OP

Using a C18 reversed phase column with a diameter-height ratio of 2.5cm × 15cm, a silica gel-based filler and a particle size of 30-60 μm (C)

Bonded phase rapid separation column, santai technologies ltd). Mixing vitamin B₁₂Dissolving the product of the-OP synthesis in 20% by volume aqueous acetonitrile, and adding 0.1% (v/v) TFA (trifluoroacetic acid) to water to prepare a solution₁₂The OP sample solution concentration was 2 mg/ml. During the desalting, the flow rate was constant at 10ml/min, the reverse phase column was equilibrated with a 20% acetonitrile solution containing 0.1% TFA, and the sample was started after 12min of equilibration. After the completion of the loading, the salt was eluted with 20% (v/v) acetonitrile (without TFA), and after 12min, the vitamin B was eluted with 50% (v/v) acetonitrile (without TFA)₁₂-OP, collecting the red eluted product under this gradient, eluting until there is no red solution. After the elution was completed, the reverse phase column was regenerated by using an 80% (v/v) acetonitrile solution containing 0.1% (v/v) TFA. And (3) carrying out vacuum rotary evaporation on the collected elution product at the temperature of 45 ℃, freezing the concentrated solution, and placing the frozen concentrated solution in a freeze dryer for 48 hours to obtain a desalted product. Vitamin B₁₂HPLC analysis patterns of the-OP synthetic product before and after desalting are shown in FIG. 6, and A, B are absorption patterns corresponding to 361nm before and after desalting respectively. As can be seen from the A, B graph, the peaks with retention times of 2.20min, 10.04min and 13.99min after desalting were significantly reduced or disappeared, indicating that the desalting process described above removed excessive HATU and its reaction products.

(5) Vitamin B₁₂Purification preparation of-OP

Rotary evaporating the desalted sample, freeze drying, dissolving in 20% (v/v) acetonitrile solution with concentration of 10mg/ml, and preparing high-purity vitamin B by semi-preparative high performance liquid chromatography₁₂-OP. A semi-preparative column using a C18 reverse phase column, height ratio of 9.4mm X250 mm, particle size of 5 μm, pore size of 110A (Agilent, ZORBAX Eclipse XDB-C18), elution gradient as shown in Table 1, elution rate of 3 ml/min. Sample (I)Eluting at room temperature in a volume of 300 μ L to collect target peak with retention time of 25.5min, and freeze drying the collected sample for 48 hr to obtain vitamin B₁₂-OP lyophilized powder. Vitamin B purified by HPLC₁₂The results of mass spectrometric detection of-OP are shown in FIG. 7, with VB appearing₁₂Mass-to-charge ratio 1885[ H +corresponding to-OP]. The HPLC purity analysis of the collected sample showed that the content of the purified product was 99% or more as shown in FIG. 8.

TABLE 1 vitamin B₁₂Elution gradient for HPLC semi-preparative of-OP synthetic products

In that¹³In the C assay, vitamin B was compared₁₂Theoretical calculation and actual calculation of chemical shift of C atom of the modified moiety substantially correspond to each other, thereby judging that vitamin B is synthesized₁₂the-OP is consistent with the expected designed structure, and the structural formula is shown as a formula I.

Example 2 vitamin B₁₂-OP self-assembly and its polypeptide drug encapsulation

Taking preparation of 1ml nano system embedded glucagon-like peptide derivative 6-KTP (Wangmang, Zhouyin, Rayanhong, etc.. Gene recombination GLP-1 derivative (6KTP) for fermentation, purification and identification [ EB/OL ]. Beijing: China scientific and technical paper on-line [2012-05-14 ]):

(1) adding 200 mul of ethanol with volume fraction of 50% and 50 mul of 6-KTP/hexafluoroisopropanol solution with concentration of 20mg/ml into an EP tube, and slightly reversing the upper part and the lower part to mix evenly;

(2) 40. mu.l of Boc-FF (Boc-Phe-Phe-OH, BACHEM)/hexafluoroisopropanol solution at a concentration of 100mg/ml and VB at a concentration of 1mg/ml₁₂-OP (VB from example 1)₁₂10 mu l of-OP)/hexafluoroisopropanol solution, mixing, adding into the EP tube of the step (1), and mixing by slightly reversing the upper part and the lower part;

(3) taking out 300 mul of mixed solution in the EP tube, dripping into another EP tube filled with 690 mul of deionized water, and slightly reversing the upper part and the lower part to mix evenly;

(4) at this time, 10 mul of PEG200 is added, and the mixture is mixed by slightly reversing the upper part and the lower part, so that the final volume fraction of PEG200 in the nano system is 1 percent, and the final volume fraction of ethanol is 10 percent;

(5) and (3) drying the nano solution in a freeze dryer for 24 hours, removing the organic solvent, adding water for redissolving and resuspending to obtain the organic phase-free nano solution.

Scanning 1ml nanometer system with Malvern laser nanometer particle size analyzer, selecting protein as dispersion and water as dispersion medium. The scanning result of the nanoparticles on the laser nanometer particle size analyzer is shown in fig. 9, the nanometer particle size is mainly distributed between 100nm and 400nm, and the average particle size is 200 nm. The morphology of the nanoparticle structure was characterized by Field Emission Scanning Electron Microscopy (FESEM) as shown in fig. 10, the nanoparticles were spherical, distributed relatively uniformly, free of other impurities, and the nanoparticle size was substantially consistent with the scanning results of the laser nanoparticle sizer. The scanning of a field emission scanning electron microscope and a laser nanometer particle size analyzer proves that the prepared nanometer particles are self-assembled into a sphere.

Example 3 encapsulation efficiency and drug Loading of nanoparticles to polypeptide drugs

The drug-loaded nano solution (the nano solution prepared in example 2) is filtered by an ultrafiltration centrifugal tube with a 30KD filter membrane, free 6-KTP cannot be trapped by the filter membrane and is then centrifuged into the filtrate from the solution, and the 6-KTP adsorbed or embedded on the nano carrier is trapped by the filter membrane. And measuring the content of 6-KTP in the filtrate by using high performance liquid chromatography.

The encapsulation efficiency EE% of the nanoparticles is [ (W1-W2)/W1] × 100%;

the drug loading DL% of the nanoparticles is [ (W1-W2)/W ] × 100%;

wherein W1: total amount of 6-KTP, W2: 6-KTP content not wrapped in the nano-carrier,

w: wrapping the weight of the nanoparticles of 6-KTP.

A chromatographic column: 300Extend-C18 analytical column (specification: 4.6 mm. times.250 mm,5 μm)

Mobile phase: gradient elution with acetonitrile and water, pH adjustment with TFA (0.1% (v/v) TFA in acetonitrile and water)

Detection wavelength: 214nm

Flow rate: 1.0ml/min

Column temperature: at room temperature

Sample introduction amount: 20 μ l

TABLE 2 elution gradient for HPLC analysis of nanoparticle ultrafiltrate

According to liquid phase analysis, the substance with the peak retention time of 20.7min is 6-KTP, the substance with the peak retention time of 25.3min is Boc-FF, the peak area of 6-KTP can be known from FIG. 11, the regression standard curve equation y of the peak area of 6-KTP to the concentration is 16114x-35.407 (the 6-KTP solution with the concentration of 25 mug/ml, 50 mug/ml, 100 mug/ml, 200 mug/ml, 500 mug/ml, 1000 mug/ml and 2000 mug/ml is prepared, the standard curve of 6-KTP is established according to the relation between the peak area and the concentration by HPLC analysis), and the entrapment rate EE percent of the nanoparticles prepared by the experiment is 96.96 +/-0.42 percent, and the drug loading DL percent is 24.25 +/-0.1 percent. The nano particles prepared by the experiment have higher encapsulation efficiency and better drug loading rate and can meet the requirements of nano drugs.

Example 4 vitamin B₁₂Mediated in vitro experiments of nanoparticles

In the experiment, colon cancer epithelial cells Caco-2 (American ATCC cell bank) are used as an in vitro experiment model. Caco-2 cells at 1X 10⁵The individual/ml concentrations were plated on 24-well Transwell plates, 0.4ml per well, and cultured continuously for 21 days to form confluent monolayers. After Caco-2 culture to form a single-layer membrane, washing the membrane twice with HBSS, and respectively adding 6-KTP and vitamin B into the upper layer₁₂Mediated Nanoparticles (FOK Nanoparticles, FOK Nano, particles prepared in example 2), vitamin B₁₂The mediated Nanoparticles and internal factors (FOK Nanoparticles + IF, FOK Nano + IF, internal factor IF cat # ab101419 from abcam) were mixed in 400. mu.l each, and 400. mu.l HBSS solution was added to the lower layer, 3 wells; wherein vitamin B₁₂The mediated nanoparticles were incubated with intrinsic factor for 0.5h at room temperature in advance. The concentration of 6-KTP in all three groups was 1 mg/ml. Placing the Transwell plate on a constant temperature incubator at 37 deg.CAfter 0.5h, 1h, 1.5h, 2h, 3h and 4h, 200 mu l of sample of the liquid in the receiving pool (lower layer) is put in an EP tube (total 54 samples) to be tested. After each sampling, a blank of 200. mu.l HBSS solution was added back.

The amount of drug in the sample was analyzed by HPLC in the same manner as in example 2. The apparent permeability coefficient (Papp) can be used to assess the transport and absorption capacity of a drug. Papp ═ dQ/dt)/(AC0, where dQ/dt is drug transport per unit time (mg/s); a is the area of the transport membrane, in which case A is 0.3cm². C0 is the initial concentration of drug (mg/ml). Papp>2*10^-6cm/s is of a well absorbed drug, and Papp<10^-6cm/s is a poorly absorbed drug. As shown in FIG. 12, it can be seen that FOK nanoparticles added with IF started to have a Papp value gradually higher than that of FOK nanoparticles after 1.5h, indicating that IF and VB on the surface of the nanoparticles₁₂After binding, the absorption and the transportation of the protein on a Caco-2 cell monolayer membrane are facilitated. And 6-KTP which is not embedded by the nano-carrier can not be absorbed and transported on a Caco-2 cell monolayer membrane basically. This example illustrates vitamin B₁₂The mediated nanoparticle can obviously improve the absorption and transportation of the nanoparticle on cells.

Example 5 vitamin B₁₂Mediated in vivo experiments with nanoparticles

In this example, a 7-8 week old male Kunming mouse was selected as a model mouse, purchased from southern medical university laboratory animal center, and has license number SCXK (Guangdong) 2011-. The experimental mice are divided into 4 groups, namely a solvent PBS group and a 6-KTP solution group, and vitamin B is not added₁₂Nanoparticle (FK Nano) group of-OPs (prepared as in example 2, except that vitamin B was not added)₁₂-OP), adding vitamin B₁₂Group of nanoparticles (FOK Nano) with OP participated in Assembly (nanoparticles prepared in example 2), administered by gavage, with a concentration of drug 6-KTP of 2mg/ml, according to a standard of 0.2ml/10g of mean body weight. The eyeball is picked at different time points for blood collection, and EDTA-Na is used₂The blood of the mice was collected by an anticoagulation tube, and 15. mu.L of DPP-IV (dipeptidyl peptidase IV) inhibitor (DPP4-010, Milipore Corp.) was previously added to the anticoagulation tube to prevent GLP-1 from being enzymatically hydrolyzed. The blood sample is taken, i.e.Centrifuging at 4 deg.C and 2000g for 15min, collecting supernatant, placing in 1.5ml EP tube, and storing in-80 deg.C refrigerator to avoid repeated freeze thawing. Using Millipore corporation

The GLP-1 kit is used for detecting blood of a mouse, and the kit has the Cat.

The results are shown in FIG. 13, from which it can be seen that the polypeptide drug 6-KTP could not be efficiently absorbed by direct oral administration without vitamin B₁₂Mediated nanoparticles and vitamin B₁₂After drug loading, the mediated nanoparticles reach the peak of blood concentration after oral administration for 4 hours, and the peak is respectively 50pM and 180 pM. Comparison with vitamin B₁₂Mediated nanoparticles via vitamin B₁₂The mediated drug released by the nanoparticles entering blood is remarkably improved, and the effective blood concentration of 40-50 pM can be achieved after 24 hours, which shows that vitamin B₁₂The mediated nano particles have a certain slow release effect.

Comparative example 1 addition of PEG before assembling of the nanosystem

(2) 40. mu.l of Boc-FF (Boc-Phe-Phe-OH, BACHEM)/hexafluoroisopropanol solution at a concentration of 100mg/ml and 10. mu.l of VB12-OP (VB 12-OP prepared in example 1)/hexafluoroisopropanol solution at a concentration of 1mg/ml were taken, mixed well, added to the EP tube of step (1), and mixed well by gently inverting the top and bottom;

(3) taking out 300 μ l of the mixed solution in the EP tube, adding into another EP tube (containing 10 μ l of PEG200) containing 700 μ l of deionized water dropwise, and mixing by slightly reversing the upper and lower parts;

(4) and (3) drying the nano solution in a freeze dryer for 24 hours, removing the organic solvent, adding water for redissolving and resuspending to obtain the organic phase-free nano solution.

Scanning 1ml nanometer system with Malvern laser nanometer particle size analyzer, selecting protein as dispersion and water as dispersion medium. The scanning result of the nano particles on a laser nano particle size analyzer shows that the nano particle size is mainly distributed between 800nm and 1100nm, the average particle size is 960nm, and the nano particles are not beneficial to intestinal absorption.

Comparative example 2 effect of different ethanol concentrations on nanosphere particle size

Fixing the concentration of Boc-FF to be 3mg/mL, the concentration of VB12-OP to be 5 mu g/mL, the concentration of polypeptide to be 1mg/mL, changing the concentration of ethanol to be 10% (v/v), 20% (v/v), 30% (v/v), 40% (v/v), 50% (v/v) and 60% (v/v), and determining the Z-Average diameter by using a laser nanometer particle sizer after preparing a nanometer solution. The particle size is basically concentrated between 100-400nm when the ethanol concentration is 10% (v/v); when the concentration of the ethanol is 20% (v/v), the nano-structures are mainly distributed in two regions, one is about 50nm, and the other is about 4 mu m; when the concentration of the ethanol is 30 percent (v/v), the particle size is larger and is mainly distributed between 2 and 5 mu m; when the concentration of the ethanol is 40% (v/v), the particle size is more below 1000nm and basically concentrated between 300nm and 700 nm; the ethanol concentration is 50 percent and is similar to that of 60 percent (v/v), and the particle size range is concentrated between 300 and 800 nm.

In the single factor test of the ethanol concentration, it can be seen from the results that the particle size of the nano solution is most suitable at the ethanol concentration of 10% (v/v).

Comparative example 3 effect of different Boc-FF concentrations on nanosphere particle size

Fixing the concentration of ethanol to be 10% (v/v), the concentration of VB12-OP to be 5 mug/mL, the concentration of polypeptide to be 1mg/mL, changing the concentration of Boc-FF to be 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL and 6mg/mL respectively, preparing a nano solution, and then determining the Z-Average diameter by adopting a laser nano particle size analyzer. As the concentration of Boc-FF increased, the Z-average particle size of the nanostructures first decreased and then increased. The average grain diameter of the nano structure is 4759nm when the concentration of Boc-FF is 1 mg/mL; the average particle size of the nano structure is 1334 at 2 mg/mL; the average grain diameter of the nano structure is 611nm when the grain size is 3 mg/mL; the average grain diameter of the nano structure is 361nm when the grain size is 4 mg/mL; the average particle size of the nano structure is 1461 at 5 mg/mL; the average particle size of the nanostructure at 6mg/mL was 2199 nm.

From the results, it was found that the particle size of the 3mg/mL or 4mg/mL nano solution was small and the distribution was relatively uniform, but when the Boc-FF concentration was low, the number of nano-structures formed per unit volume was likely to be small, and the number of nano-molecules capable of adsorbing or entrapping the protein drug was also decreased, so that the Boc-FF concentration of 4mg/mL was selected as the optimum concentration.

Comparative example 4 Effect of different VB12-OP concentrations on nanosphere particle size

The fixed ethanol concentration is 10 percent (v/v), the Boc-FF concentration is 4mg/mL, the polypeptide concentration is 1mg/mL, the VB12-OP concentration is changed to be 2.5 mu g/mL, 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 40 mu g/mL and 80 mu g/mL respectively, and the Z-Average diameter is determined by adopting a laser nanometer particle sizer after preparing the nanometer solution.

When VB12-OP is not added, the average particle size of the nano Z is obviously higher, and after VB12-OP is added, the average particle size of the nano Z is obviously reduced. In the range of 5-40 μ g/mL, the nano-particle size increases with the increase of VB12-OP concentration, wherein the average particle size is smaller and smaller than 1 μm when the VB12-OP concentration is 5 μ g/mL, 10 μ g/mL and 20 μ g/mL. The VB12-OP concentration is 2.5 mu g/mL, and the particle sizes of the nano-structure are mainly distributed in three regions, one is about 30nm, one is about 100nm, and the other is between 200 and 800 nm; the particle size of the nano structure is mainly distributed between 200 and 500nm at 5 mu g/mL; the particle size of the nano structure is mainly concentrated between 200 and 500nm at 10 mu g/mL; the particle size of the nano structure is mainly distributed in two areas at 20 mu g/mL, one is between 200 and 600nm, and the other is between 2 and 6 mu m; the grain diameter of the nano structure is mainly concentrated between 1 and 4 mu m at 40 mu g/mL; the grain diameter of the nano structure is mainly distributed in two blocks at 80 mu g/mL, one part is about 100nm, and the other part is between 200nm and 1 mu m. The nanoparticle size is most suitable when the VB12-OP concentration is 5. mu.g/mL or 10. mu.g/mL.

Comparative example 5 effect of different 6-KTP concentrations on nanosphere particle size

The fixed ethanol concentration is 10% (v/v), the Boc-FF concentration is 4mg/mL, the VB12-OP concentration is 5 mu g/mL, the 6-KTP concentrations are respectively changed to be 0.25mg/mL, 0.5mg/mL, 0.75mg/mL, 1mg/mL, 1.25mg/mL, 1.5mg/mL, 1.75mg/mL and 2mg/mL, and the Z-Average diameter is determined by a laser nanometer particle sizer after preparing a nanometer solution.

The results showed that the Z-average particle size decreased with increasing 6-KTP concentration, and increased again as the concentration increased to 1.5 mg/mL. When the concentration of 6-KTP is respectively 0.25mg/mL, 0.5mg/mL and 1.25mg/mL, the particle size of the nano structure is basically distributed at about 1 mu m; when the concentration of 6-KTP is respectively 0.75mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL, the particle size of the nano structure is basically distributed between 300nm and 800 nm; the particle size of the nano-structure is concentrated to about 1 μm when the concentration of 6-KTP is 1.75 mg/mL. From the results, it was found that the concentration of 6-KTP was most suitable at 1 mg/mL.

Examples using 1, 4-butanediamine as a linker, Boc-phenylalanine dipeptide as the linker peptide of interest, other linear diamines, and phenylalanine dipeptide compounds, and using similar concepts and principles as the present invention, considered equivalent substitutions, are included within the scope of the present invention. Use of vitamin B₁₂The linker-peptide and other phenylalanine dipeptide compounds are self-assembled together to form nanoparticles and are studied for oral drug loading without departing from the spirit of the present invention and are also included in the scope of the present invention.

Claims

1. Vitamin B loaded with medicine₁₂The preparation method of the derivative self-assembly nanometer particle is characterized by comprising the following steps:

(D) mixing the solution E with water, adding PEG200, and mixing to obtain solution F; freeze drying, adding water for re-dissolving and re-suspending to obtain vitamin B loaded with medicine₁₂Derivative self-assembled nanoparticles;

2. the method of claim 1Vitamin B loaded with medicine₁₂The preparation method of the derivative self-assembly nanometer particle is characterized in that:

the concentration of the medicine in the solution A is 20 mg/ml;

the concentration of the phenylalanine dipeptide in the solution B is 100 mg/ml;

the solution C can be self-contained with vitamin B which forms nano particles₁₂The concentration of the derivative is 1 mg/ml;

in the solution D, the concentration of the medicine is 4mg/ml, and the concentration of the organic solvent X is 40 percent by volume;

the concentration of the organic solvent X solution is 50 percent by volume;

in the solution E, the concentration of the medicine is 3.3mg/ml, the concentration of the organic solvent X is 33 percent by volume, the concentration of the phenylalanine dipeptide is 13.3mg/ml, and the vitamin B which can be automatically packaged into nano particles₁₂The concentration of the derivative is 0.03 mg/ml;

in the solution F, the concentration of the medicine is 0.1mg/ml, the concentration of the organic solvent X is 10 percent by volume, the concentration of the phenylalanine dipeptide is 4mg/ml, and the vitamin B which can be automatically packaged into nano particles₁₂The concentration of the derivative is 0.01mg/ml, and the final concentration of PEG200 is 1% by volume.

3. The drug loaded vitamin B of claim 1₁₂The preparation method of the derivative self-assembly nanometer particle is characterized in that: the medicine is polypeptide medicine and/or protein medicine.

4. The drug loaded vitamin B of claim 1₁₂The preparation method of the derivative self-assembly nanometer particle is characterized in that: the vitamin B capable of being self-assembled to form nano particles₁₂The derivative is prepared by the following steps:

(1) preparation of amide intermediate:

firstly, mixing an onium phosphate coupling agent, DMF, DIEA and Boc-phenylalanine dipeptide, and carrying out ultrasonic water bath reaction to obtain an amide intermediate productX₁；

② mixing DMF, carbodiimide couplant, HOAT and Boc-phenylalanine dipeptide, and carrying out ultrasonic water bath reaction to obtain amide intermediate product X₂；

(2) Vitamin B₁₂-preparation of OP:

amide intermediate product X obtained in the previous step (1)₁Or the amide intermediate product X obtained in the step (1)₂Adding vitamin B₁₂Performing ultrasonic water bath reaction on the vitamin B, adding ethyl acetate to stop the reaction, centrifuging, removing supernatant, taking precipitate, and drying to obtain vitamin B₁₂-OP；

Wherein, the raw materials participating in the reaction are proportioned according to the following molar ratio:

by amide intermediates X₂Obtaining vitamin B₁₂Carbonyl-putrescine, carbodiimide-based coupling agents: HOAT: boc-phenylalanine dipeptide: vitamin B₁₂-carbonyl-putrescine ═ 0.75 to 3: 0.75-3: 0.75-3: 1;

further comprises the step of mixing the vitamin B obtained in the step (2)₁₂A step of desalting the OP by a C18 reverse phase column and further purifying the desalted product by semi-preparative high performance liquid chromatography.

5. Vitamin B which can be self-assembled into nanoparticles according to claim 4₁₂A process for the preparation of derivatives, characterized in that:

the phosphate onium salt coupling agent in the step (1) is HATU or HBTU;

the carbodiimide coupling agent in the step (1) is DIC;

the conditions of the ultrasonic water bath reaction I in the first step and the second step are as follows: carrying out ultrasonic reaction at 30 ℃ and 70w for 5 min;

the conditions of the ultrasonic water bath reaction II in the step (2) are as follows: carrying out ultrasonic reaction at 30 ℃ and 70w for 2 h;

the dosage of the ethyl acetate in the step (2) is that the volume ratio of the ethyl acetate to DMF is 2-8: 1, calculating the mixture ratio;

the centrifugation conditions in the step (2) are as follows: centrifuging at 8000r/min for 5 min.

6. Vitamin B which can be self-assembled into nanoparticles according to claim 4₁₂A process for the preparation of derivatives, characterized in that:

the desalting is realized by the following steps:

(I) mixing vitamin B₁₂-OP is dissolved in aqueous acetonitrile containing TFA to obtain vitamin B₁₂-an OP solution;

(II) gradient elution with C18 reverse phase column: the reverse phase column was equilibrated with 20% (v/v) acetonitrile in water containing 0.1% (v/v) TFA, and then desalted with 20% (v/v) acetonitrile, followed by elution of vitamin B with 50% (v/v) acetonitrile₁₂-OP, collecting the red eluted product under this gradient;

(III) concentrating, freezing and drying the red elution product obtained in the step (ii) to obtain desalted vitamin B₁₂-OP；

The purification is realized by the following steps:

7. Vitamin B loaded with medicine₁₂A derivative self-assembled nanoparticle characterized by: the preparation method of any one of claims 1 to 6.

8. The drug loaded vitamin B of claim 7₁₂A derivative self-assembled nanoparticle characterized by: the vitamin B loaded with the medicine₁₂The derivative self-assembled nanoparticles have a particle size of 100 to 400nm and a spherical shape.

9. The drug loaded vitamin B of claim 7₁₂The application of the derivative self-assembly nano particles in preparing intestinal absorption medicines.