CN102133190A - Transferrin nanoparticles and preparation method and application thereof - Google Patents

Abstract

The invention relates to a transferrin nanoparticle, a preparation method and application thereof. The transferrin nanoparticles provided by the invention can carry drugs targeted to tumors and brains, improve the oral bioavailability of oral ingestible drugs, and have high drug loading capacity, simple preparation process, low cost, good biocompatibility, etc. Features, clear application prospects.

Description

A kind of transferrin nanoparticle and its preparation method and application

technical field

The invention relates to a transferrin nanoparticle, a preparation method and application thereof, and belongs to the technical field of medicine.

Background technique

Nano-preparations such as liposomes, solid lipid nanoparticles, and nano-structured lipid carriers are currently research hotspots in the field of medicine. However, these nano-carriers have low drug loading, poor targeting of intravenous injections, and absorption-promoting effects of oral administration. Weakness.

Transferrin (Tf, also known as transferrin, transferrin) is an important β-globulin, which is the transporter of iron in vertebrates. Studies on transferrin in vertebrates have proved that the molecular weight of transferrin is about 80,000 Da, a single peptide chain, and iron and sugar account for about 6%. Different types of transferrin have different physical, chemical and immune properties, but all have two binding sites for ferric ions. In different studies, according to the number of iron, it is divided into ordinary transferrin or iron-saturated transferrin, single iron transferrin, and apotransferrin. According to its configuration, it is divided into ordinary transferrin and isoform transferrin. Human transferrin is a single peptide chain composed of two structurally similar globular domains located at the N-terminus and C-terminus. Transferrin contains 679 amino acid residues, a total of 38 Cys, forming 19 pairs of disulfide bonds.

Transferrin has polymorphisms and is mainly divided into three types: serum transferrin, egg (clear) transferrin, and milk (clear) transferrin. As an amphoteric substance, except for the isoelectric point (PI) of lactotransferrin which is 8.7, other transferrins have an isoelectric point of about 5.6-5.8, and they are all acidic proteins.

Transferrin binds to at least two different types of transferrin receptors, transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2). Transferrin receptors are widely expressed in vivo, and the expression of primitive erythrocytes is the highest, reaching 80%; intestinal epithelial cells, brain cells, brain capillary endothelial cells, proliferating cells (such as tumor cells, activated lymphocytes, serum-induced fibroblasts) cells) are also highly expressed. Therefore, transferrin can be used for tumor-targeted drug delivery, brain-targeted drug delivery, and oral drug delivery of traditional Chinese medicines, biomacromolecules, chemical drugs, metal ions for treatment, radioactive isotopes for diagnosis, nutritional agents, and the like.

Since transferrin is only partially (about 30%) saturated with iron ions, it has a higher affinity with other metal ions. At least 30 other metal ions can also bind transferrin, including therapeutic metal ions such as bismuth (Bi), ruthenium (Ru), titanium (Ti), etc., and diagnostic radioactive isotopes gallium (Ga), indium, etc. (In) etc. Transferrin has been shown to carry these metal ions to target tumor cells.

Since there are a large number of transferrin receptors on the surface of tumor cells and the blood-brain barrier, this provides hope for transferrin as a drug carrier. Transferrin is combined with chemical drugs or genes, which has good distribution in tissues, prolongs the half-life of drugs in the cytoplasm, and can also achieve controlled release. Studies in recent years have shown that transferrin has achieved good results in the treatment of tumors and brain targeting.

Transferrin can partially resist the degradation of trypsin and chymotrypsin, and has good stability in the gastrointestinal tract. In addition, the molecular weight of transferrin itself is relatively large, and small drug molecules can use it as a carrier to avoid the destruction of enzymes in the external environment. Transferrin-mediated endocytosis is one of the most characteristic transport processes in biological cells. If a drug molecule is linked to transferrin, the drug-transferrin conjugate binds to the transferrin receptor under the mediation of transferrin. It can be absorbed by intestinal epithelial cells with high expression of transferrin receptor.

Due to its good biocompatibility, transferrin is a very promising nanocarrier material. At present, transferrin is generally combined with drugs through covalent bonds, and there is no report on transferrin nanoparticles containing drugs.

Contents of the invention

The object of the present invention is to provide a transferrin nanoparticle and a preparation method thereof. Another object of the present invention is to provide the application of the transferrin nanoparticle for drug delivery in vivo.

For the purpose of the above invention, the present invention provides the following technical solutions:

A transferrin nanoparticle is characterized in that it is made of medicine and transferrin, the mass ratio of medicine to transferrin is 500～1:1000, and the average diameter of the nanoparticle is no more than 1000 nanometers, preferably nanometer The particle diameter ranges from 20 to 500 nanometers.

Described transferrin nanoparticle is characterized in that described transferrin is made of serum transferrin, ovotransferrin, lactoferrin, whey (serum) transferrin, melanin transferrin, common transferrin One or more components of protein, iron-saturated transferrin, monoiron transferrin, apo-transferrin, common transferrin, isoform transferrin, and cross-linked transferrin, preferably It is serum transferrin, ovotransferrin, whey (serum) transferrin, cross-linked transferrin, more preferably cross-linked transferrin.

The cross-linked transferrin is characterized in that part of the transferrin is cross-linked through disulfide bonds. The cross-linked transferrin is artificially dispersed in the aqueous medium or the aqueous medium containing a certain concentration of organic solvent, through high-pressure homogenization, ultrasonic, grinding, creaming, shearing, collision, heating and other methods. One or more of them are processed, and some transferrins are cross-linked through disulfide bonds to obtain cross-linked transferrins.

The preparation method of the transferrin nanoparticle comprises: a mixture of an organic phase containing a drug dispersed therein and an aqueous medium containing transferrin is subjected to high-pressure homogenization, ultrasonication, grinding, and emulsification. One or more of methods of processing, shearing, and collision are processed, and the processing time is long enough to produce transferrin nanoparticles, and the transferrin nanoparticles are separated and collected. Wherein the organic phase is composed of methanol, absolute ethanol, ethanol containing a certain amount of water, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, dimethylsulfoxide, N,N- Dimethylformamide, N,N-dimethylacetamide, methyl acetate, ethyl acetate, isopropyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, toluene, tetrahydrofuran, 1, 4-one or more of dioxane, dimethoxyethane, dichloromethane, dichloroethane, chloroform, tetrachloromethane, ether, etc.

The preparation method of the transferrin nanoparticle includes: mixing and grinding the drug and the transferrin, treating for a long enough time to produce the transferrin nanoparticle, and separating and collecting the transferrin nanoparticle.

The preparation method of the transferrin nanoparticle includes: dispersing the transferrin in an aqueous medium or an aqueous medium containing a certain concentration of an organic solvent, and adopting one or more methods of adding a dehydrating agent and adjusting pH be processed to form transferrin aggregates, solidify by one or more of high-pressure homogenization, ultrasonication, grinding, homogenization, shearing, collision, and heating methods, separate and collect blank transferrin nanoparticles, and The blank transferrin nanoparticles are dispersed in the solution containing the drug, and are treated by one or more of stirring, solvent evaporation, ultrasound, heating, grinding, and shearing methods, so that the drug is adsorbed on the blank transferrin nanoparticles. , separate and collect transferrin nanoparticles.

The preparation method of the transferrin nanoparticle includes: dispersing the transferrin and the drug in an aqueous medium or an aqueous medium containing a certain concentration of an organic solvent, and taking one of the methods of adding a dehydrating agent and adjusting the pH or multiple treatments to form aggregates of transferrin and drugs, solidify by one or more of high-pressure homogenization, ultrasonication, grinding, homogenization, shearing, collision, and heating methods, and separate and collect transferrin protein nanoparticles.

The transferrin nanoparticle is characterized in that the medicine is selected from traditional Chinese medicine, natural medicine, chemical medicine and biotechnology medicine, preferably water insoluble medicine and biomacromolecule, more preferably tripterygium glycoside, Epimedium total flavonoids, Cordyceps polysaccharide, breviscapine, ursolic acid, nobiletin, curcumin, paclitaxel, doxorubicin, insulin.

The application of the transferrin nanoparticle in the delivery of medicines in vivo.

The application of the transferrin nanoparticle in the preparation of oral medicine.

The beneficial effects of the present invention are mainly:

1. The drug-loading capacity of the transferrin nanoparticles is high, generally 10-20%, and the highest can reach more than 30%.

2. The preparation process of transferrin nanoparticles is simple, does not require complex chemical reaction modification, avoids toxic organic solvents and catalyst residues, has low preparation cost, and has a clear application prospect.

3. The cross-linked transferrin is used to prepare transferrin nanoparticles. Since part of the transferrin has been cross-linked with each other, the processing time is shorter, which is beneficial to the stability of the drug, and the drug encapsulation efficiency is also higher.

4. Transferrin nanoparticles can carry drugs to target tumors and brains, reduce adverse reactions, increase drug concentrations in lesion sites, and enhance drug efficacy.

5. Transferrin nanoparticles can improve the oral bioavailability of orally ingestible drugs.

Detailed ways

The present invention will be described in further detail below in conjunction with examples, but the scope of the present invention is not limited by these examples.

Example 1

30mg paclitaxel (Paclitaxel) was dissolved in 3.5ml methylene chloride, 27.0ml serum transferrin solution (1%, w/v) was added, the mixture was homogenized at low speed for 5 minutes to form a coarse emulsion, and then It is transferred to a high-pressure homogenizer (Avestin), and the high-pressure homogenizer is circulated 5 times at 9000-18000 pounds per inch (psi), and the homogenate solution is evaporated under reduced pressure at 40 ° C, and dichloromethane is removed rapidly to obtain half Transparent dispersion, the average diameter of paclitaxel-transferrin nanoparticles is 185nm.

Example 2

Weigh ovotransferrin and doxorubicin, mix according to the mass ratio of 9:1, add an appropriate amount of solution with a pH value of 6.0, and sonicate until dissolved. 25°C constant temperature water bath, add appropriate amount of acetone under electromagnetic stirring, spray dry, wash the powder with appropriate amount of ethanol, evaporate the ethanol, disperse ultrasonically with 5% mannitol solution, freeze-dry, add sterile water or normal saline to the obtained cake, and measure Particle size, the average diameter of doxorubicin-transferrin nanoparticles is 218nm.

Example 3

Gallium transferrin was prepared by titrating apotransferrin or monoferrotransferrin with GaCl3. Dissolve gallium transferrin in deionized water, adjust the pH to about 7.0, slowly add a specified amount of absolute ethanol, stir at a constant speed, heat denaturation above 80°C, and spray dry. After adding sterile water or physiological saline to the obtained cake, the particle diameter is measured, and the average diameter of the gallium transferrin nanoparticles is 336nm.

Example 4

Dissolve 100mg of serum transferrin in water, adjust the pH to about 7.0; dissolve 15mg of insulin in 0.1% hydrochloric acid, adjust the pH to about 7.0; mix transferrin and insulin solution, slowly add the specified amount of sodium chloride , stirred to form aggregates of transferrin and insulin, and homogenized under high pressure to obtain insulin transferrin nanoparticles with an average particle size of 252nm.

Example 5

Put 100mg ursolic acid and 800mg lactoferrin in a nano grinder, grind, add water to disperse after grinding, centrifuge, take the upper layer, which is ursolic acid transferrin nanoparticles, measure the particle size, the average diameter is 97nm.

Example 6

35 mg of paclitaxel was dissolved in 3.5 ml of dichloromethane, and 27.0 ml of cross-linked serum transferrin solution (1%, w/v) was added, and the mixture was homogenized at low speed for 5 minutes to form a coarse emulsion, and then It was transferred to a high-pressure homogenizer (Avestin), and the high-pressure homogenizer was circulated 3 times at 8000-15000 pounds per inch (psi), and the homogenate was evaporated under reduced pressure at 40°C to remove methylene chloride rapidly to obtain Translucent dispersion, the average diameter of paclitaxel-transferrin nanoparticles is 173nm.

Example 7

Dissolve 100mg of melanotransferrin and 20mg of Cordyceps polysaccharide in water, slowly add the prescribed amount of 95% ethanol, and stir at a constant speed to form aggregates of melanotransferrin and Cordyceps polysaccharide. The temperature was 50° C., spray drying, and the Cordyceps polysaccharide-transferrin nanoparticles were obtained. After adding water to disperse, the particle size was measured, and the average particle size was 365 nm.

Example 8

Dissolve 1g of serum transferrin in water, adjust the pH, slowly add a specified amount of 95% ethanol to form aggregates of serum transferrin, spray-dry at an inlet temperature of 180°C and an outlet temperature of 50°C, and obtain a blank Transferrin nanoparticles. Add 100 mg of transferrin nanoparticles into 16 mg of total flavonoids of Epimedium in 50% ethanol solution, stir for 4 hours, slowly recover ethanol, spray dry, and obtain transferrin nanoparticles of total flavonoids of Epimedium, add water to disperse , Determination of particle size, the average particle size is 268nm, based on icariin, the encapsulation efficiency of total flavonoids of Epimedium is 86%.

Example 9

Put 90mg tripterygium glycosides and 900mg cross-linked serum transferrin in a nano grinder, add water to grind, centrifuge, take the upper layer, which is tripterygium glycosides transferrin nanoparticles, measure the particle size, average diameter It is 136nm, and the encapsulation efficiency of tripterygium glycosides is 97.3% based on tripterine.

Example 10

20mg nobiletin is dissolved in the dichloromethane of 4.5ml and the dehydrated alcohol of 2ml, add the mixed solution of 30.0ml serum transferrin and ovotransferrin (the concentration of serum transferrin and ovotransferrin is 0.3%, w/v), the mixture was homogenized at low speed for 5 minutes to form a coarse emulsion, which was then transferred to a high-pressure homogenizer (Avestin), and the high-pressure homogenization was performed at 9000-18000 psi ( psi) was circulated 5 times, the homogeneous liquid was evaporated under reduced pressure at 45° C., and the organic solvent was removed rapidly to obtain a translucent dispersion. The average diameter of nobiletin-transferrin nanoparticles was 162 nm.

Example 11

Dissolve 1g of serum transferrin in water, slowly add specified amount of ethanol and sodium chloride to form aggregates of serum transferrin, spray dry at 160°C at the air inlet temperature, and 50°C at the outlet air temperature, and obtain blank transferrin ferritin nanoparticles. Add 100mg transferrin nanoparticles into the ethanol solution of 15mg curcumin, stir for 2 hours, reclaim ethanol, spray dry, promptly obtain curcumin transferrin nanoparticles, after adding water to disperse, measure the particle size, the average particle size is 337nm, The encapsulation rate of curcumin is 93%.

Example 12

Put 1g breviscapine and 10g ovotransferrin in a nano grinder, add an appropriate amount of 50% ethanol to grind, centrifuge, take the upper layer, which is breviscapine transferrin nanoparticles, measure the particle size, the average diameter is 165nm, based on scutellarin, the encapsulation efficiency of scutellarin is 95.2%.

Example 13

Dissolve 300 mg of paclitaxel (Paclitaxel) in 35 ml of dichloromethane, add 270 ml of serum transferrin solution (1%, w/v), and homogenize the mixture at low speed for 5 minutes to form a crude emulsion, which is then transferred In the high-pressure homogenizer (Avestin), the high-pressure homogenizer is circulated 5 times at 9,000-18,000 pounds per inch (psi), and the homogenate is evaporated under reduced pressure at 40°C to quickly remove dichloromethane to obtain a translucent dispersion The average particle diameter of paclitaxel-transferrin nanoparticles was 163nm, and an appropriate amount of mannitol was added as a freeze-dried proppant, and freeze-dried for 48 hours to obtain paclitaxel-transferrin nanoparticles for injection. The obtained cake is easy to reconstitute the original dispersion after adding sterile water, and the average particle diameter is 178nm.

【Acute Toxicity Test】:

Experimental animals: Kunming mice, weighing 18-22 g, half male and half male, provided by the Animal Center of Nanjing General Hospital of Nanjing Military Command, fed with full-price pellet feed and free to drink water.

Test drug: paclitaxel transferrin nanoparticles for injection, specification: 30mg/bottle. Prepare the required concentration with 5% glucose injection before use. Paclitaxel injection, Beijing Concord Pharmaceutical Factory, specification: 30mg/5ml, prepared to the required concentration with 5% glucose injection before use.

Experimental method: Arrange and design experiments as described in the Technical Guidelines for Nonclinical Pharmacokinetic Research of Chemical Drugs.

Test conclusion: The results of the acute toxicity test of paclitaxel-transferrin nanoparticles for injection and paclitaxel injection in mice showed that the LD ₅₀ of paclitaxel-transferrin nanoparticles for injection administered intravenously was 128.39 mg after two administrations in one day. /kg. The next day after the administration, the mice began to exhibit symptoms such as decreased activity and food intake, piloerection, and weight loss, and some mice began to die. From day 6 after administration, the surviving mice gradually returned to normal. ②The LD ₅₀ of paclitaxel injection administered by intravenous injection is 72.53mg/kg. The next day after the administration, symptoms such as decreased activity, piloerection, and decreased food intake began to appear in mice, and mice began to die. On the 10th day of administration, mice in the high-dose group still died. The surviving mice in other groups gradually returned to normal, and on the 15th day after administration, the surviving mice no longer died. The dead mice were dissected, and no obvious abnormalities were found in the organs.

【Inhibitory effect on mouse transplanted tumor EAC】:

Experimental animals: ICR white mice, 18-22g, half male and half male, provided by the animal room of China Pharmaceutical University. Feed: pellet feed, supplied by the animal room of China Pharmaceutical University; feeding conditions: air-conditioned room, temperature 18-24°C, relative humidity 70%.

Dose setting: blank control group (normal saline), paclitaxel injection: 10 mg/kg, paclitaxel transferrin nanoparticles for injection: 10 mg/kg, 5 mg/kg, 2.5 mg/kg, a total of 5 dosage groups.

Test method: 50 mice of the above-mentioned specifications were inoculated with EAC solid type according to the transplanted tumor research method, weighed the mice 24 hours after inoculation, and randomly divided them into 5 groups, 10 mice in each group, half male and half male. 24 hours after inoculation, iv administration was given once every other day, for a total of 4 administrations. The mice were weighed on the second day after drug withdrawal, the tumor-bearing mice were sacrificed, the tumor mass was separated, and the tumor weight was weighed. The obtained data were statistically processed. (t test).

Test results: Compared with the blank control group, the high, medium and low dose (10, 5mg/kg) groups of paclitaxel transferrin nanoparticles for injection can significantly inhibit the tumor growth of sarcoma EAC (P<0.01), and the high, medium and low doses (P<0.01) of sarcoma EAC The inhibitory effect of the middle dose group was better than that of the paclitaxel injection (10mg/kg) group. At the same time, paclitaxel injection with transferrin nanoparticles has a smaller effect on the body weight of the test mice than paclitaxel injection. See Table 1 for details.

Table 1 Inhibitory effect of paclitaxel-transferrin nanoparticles for injection on EAC of transplanted tumor in mice

^* P＜0.05 ^** P＜0.01 compared with blank control group

Example 14

200mg nobiletin was dissolved in 50ml of dichloromethane and 15ml of absolute ethanol, 300ml of serum transferrin solution (0.5%, w/v) was added, and the mixture was homogenized at low speed for 5 minutes to form crude The emulsion is then transferred to a high-pressure homogenizer (Avestin), and the high-pressure homogenizer is circulated 5 times at 9,000-18,000 pounds per inch (psi), and the homogenate is evaporated under reduced pressure at 40°C to quickly remove the organic solvent to obtain a translucent dispersion, and the average diameter of nobiletin-transferrin nanoparticles is 155nm.

【Brain targeting test】:

Experimental animals: Kunming mice, weighing 18-22 g, half male and half male, provided by the Animal Center of Nanjing General Hospital of Nanjing Military Command, fed with full-price pellet feed and free to drink water.

Dosage setting: nobiletin solution (containing 20% ethanol): 10 mg nobiletin/kg; nobiletin-transferrin nanoparticles: 10 mg nobiletin/kg.

Test method: 110 mice of the above-mentioned specifications were randomly divided into 2 groups, 55 mice in each group, and nobiletin solution and nobiletin-transferrin nanoparticles were injected into the tail vein respectively. At 10, 30, 60, 90, 120, 180, 240, 360, 420, 480, and 600 minutes after administration, the mice were sacrificed after taking blood from the eyeballs, and the brain, liver, spleen, kidney and other organs of the mice were separated. Processing samples, assays and data analysis.

Test results: The Cmax and AUC0-t of nobiletin in the nobiletin-transferrin nanoparticle group in the brain tissue were 8.52±1.67 times and 15.68±3.22 times that of the nobiletin solution, respectively. It shows that nobiletin-transferrin nanoparticles have better brain targeting.

Example 15

Weigh ovotransferrin and doxorubicin, mix according to the mass ratio of 9:1, add an appropriate amount of solution with a pH value of 6.0, and sonicate until dissolved. In a constant temperature water bath at 25°C, add an appropriate amount of acetone under electromagnetic stirring, spray dry, wash the powder with an appropriate amount of ethanol, evaporate the ethanol, disperse ultrasonically with 5% mannitol solution, and freeze-dry to obtain doxorubicin-transferrin nanoparticles for injection.

【Tumor targeting test】:

Experimental animals: nude mice, 18-21g, half male and half male, provided by the Animal Center of China Pharmaceutical University. Feed: Pellet feed, supplied by Animal Center of China Pharmaceutical University; Breeding conditions: Air-conditioned room, temperature 18-24°C, relative humidity 70%.

Dosage setting: doxorubicin hydrochloride for injection: 12mg doxorubicin/kg; doxorubicin transferrin nanoparticles for injection: 12mg doxorubicin/kg.

Test method: Inoculate nude mice with human breast cancer cell line MCF-7 subcutaneously according to the transplanted tumor research method. From the 3rd week after transplantation, 20 model mice with successful transplantation were selected and randomly divided into 2 groups according to tumor size and body weight. Doxorubicin hydrochloride for injection and doxorubicin-transferrin nanoparticles for injection were injected into the tail vein respectively, and the tumor-bearing mice were sacrificed 24 hours later, and the tumor mass was separated, and the tumor weight was weighed, and the samples were processed, measured and analyzed.

Test results: the content of doxorubicin in the nude mouse tumors of the doxorubicin-transferrin nanoparticle group was 6.14 ± 1.53 times that of the doxorubicin hydrochloride group, indicating that the doxorubicin-transferrin nanoparticle has a better Tumor targeting.

Example 16

Dissolve 100mg of serum transferrin in water, adjust the pH to about 7.0; dissolve 10mg of insulin in 0.1% hydrochloric acid, adjust the pH to about 7.0; mix the transferrin and insulin solution, slowly add the specified amount of sodium chloride , stirred to form aggregates of transferrin and insulin, and ultrasonicated to obtain insulin transferrin nanoparticles with an average particle size of 218nm.

【Oral hypoglycemic test】:

Test method: Diabetic rats were randomly divided into 3 groups, 6 in each group, fasted for 12 hours before administration, the first group was a blank control group, and insulin saline solution was orally administered (dose was 20IU/Kg); the second group was Positive control group, that is, subcutaneous injection of insulin saline solution (dose of 2IU/Kg); the third group orally administered insulin transferrin nanoparticles (dose of 20IU/Kg). At 0, 1, 2, 4, 6, 8, 12, 16, and 24 hours after administration, 0.2 mL of blood was collected from the canthus venous plexus, and the blood glucose mass fraction was measured according to the method of the blood glucose kit, and its relative blood glucose mass fraction at zero time was calculated The rate of change of blood sugar, draw the relationship curve of the percentage change of blood sugar and time. The bioavailability relative to subcutaneously injected insulin was calculated according to the area under the blood glucose time curve of the administration group. The calculation method is as follows:

$\mathrm{<span\; class="notranslate">\; f</span>}<span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; Aoral</span>}}{\mathrm{<span\; class="notranslate">\; Asc</span>}}<span\; class="notranslate">\; \&times;</span>\frac{\mathrm{<span\; class="notranslate">\; Dsc</span>}}{\mathrm{<span\; class="notranslate">\; Doral</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$

Among them, F is the relative bioavailability of oral preparations relative to subcutaneous insulin solution; Aoral and Asc are the areas under the blood glucose time curve of oral insulin and subcutaneous insulin solution, respectively; Doral, Dsc are the oral and subcutaneous insulin doses, respectively.

Test results: the oral bioavailability of insulin in the blank control group was 0, and the oral bioavailability of insulin in the insulin transferrin nanoparticles was 19.8±3.6%.

Example 17

Artificial Cordyceps polysaccharide (purity: 98%, Zhejiang Changxing Pharmaceutical Factory) was reacted with tyramine, and then reacted with fluorescein isothiocyanate (FITC) to obtain fluorescently labeled Cordyceps polysaccharide.

Dissolve 100mg of serum transferrin and 20mg of fluorescently labeled Cordyceps polysaccharide in water, slowly add a specified amount of 95% ethanol, and stir at a constant speed to form aggregates of transferrin and Cordyceps polysaccharide. The temperature was 50° C., spray drying, and the Cordyceps polysaccharide-transferrin nanoparticles were obtained. After adding water to disperse, the particle size was measured, and the average particle size was 370 nm.

【Caco-2 cell model absorption test】:

Cell culture: Caco-2 cells were planted on the transwell at a planting density of (100,000 cells/cm ² ), the medium was DMEM solution containing 10% calf serum, and the cells were cultured in a CO ₂ incubator at 37°C. The culture medium was changed every day, and the monolayer cells were differentiated and formed in about 19-21 days, and then they could be used for the experiment.

Test method: wash monolayer cells with HBSS of pH 7.4 at 37°C for 3 times, measure transmembrane resistance, and discard cells with transmembrane resistance value less than 500ohms×cm ² . After the cells were incubated in the buffer for 1 h, the incubation medium was sucked away, and fluorescently labeled Cordyceps polysaccharide or fluorescently labeled Cordyceps polysaccharide transferrin nanoparticles were added to the villi surface (AP) of the cell layer, followed by a series of transmembrane transport. The AP side bottom liquid was sampled twice at the beginning and the end of 4h, and the BL side bottom liquid was sampled 400ul every 30 minutes, and the same volume of blank bottom liquid was added to keep the volume of the BL side constant, and the fluorescence intensity was measured for analysis.

Test results: the absorption and permeability coefficient of fluorescently labeled Cordyceps polysaccharide transferrin nanoparticles is 5.32×10 ^-6 cm/s, and the absorption and permeability coefficient of fluorescently labeled Cordyceps polysaccharide is 1.49×10 ^-8 cm/s, indicating that transferrin Nanoparticles have significant oral absorption-promoting effect on Cordyceps polysaccharide.

Example 18

Get the ursolic acid transferrin nanoparticles prepared in Example 5, 8, 9, 10, 11, 12, Epimedium total flavonoid transferrin nanoparticles, tripterygium polyglycoside transferrin nanoparticles, nobietin Transferrin nanoparticles, curcumin transferrin nanoparticles, and breviscapine transferrin nanoparticles were subjected to the in vivo one-way intestinal perfusion model absorption test in rats.

[Rat in vivo one-way intestinal perfusion model absorption test]:

Test method: anesthetize the animal, open the abdominal cavity, insert a bile catheter near the duodenum, intubate the two ends of the duodenum, jejunum, ileum and colon respectively, cover the intestinal tissue with gauze soaked in isotonic saline The surface is moistened, the intestinal contents are washed with isotonic saline, and the perfusion fluid is changed, and the intestinal cavity is perfused with a constant speed pump. The perfusate in the outlet tube was collected every 30 minutes. A bile sample was collected before perfusion, and then every 30 minutes. After perfusion, the length of the small intestine was measured and the concentration of the analyte in the outlet tube was detected.

Test results: Compared with ursolic acid, total flavonoids of Epimedium, tripterygium glycosides, nobiletin, curcumin, and breviscapine raw materials, the apparent permeability coefficient of the drugs in the transferrin nanoparticles in the corresponding intestinal tract All were significantly improved (P<0.01), and all had good absorption in four intestinal segments (the apparent permeability coefficients were all greater than 1), indicating that transferrin nanoparticles had a significant oral absorption-promoting effect on difficult-to-absorb drugs.

Claims (10)

1. A transferrin nanoparticle is characterized in that being made of medicine and transferrin, the mass ratio of medicine and transferrin is 500～1: 1000, and the average diameter of described nanoparticle is no more than 1000 nanometers, preferably The diameter of the nanoparticles ranges from 20 to 500 nm. 2. transferrin nanoparticle according to claim 1, is characterized in that described transferrin is by serum transferrin, ovotransferrin, lactoferrin, milk (clear) transferrin, melanin transferrin One of ferritin, ordinary transferrin, iron-saturated transferrin, monoiron transferrin, apo-transferrin, ordinary transferrin, isoform transferrin, and cross-linked transferrin or multiple compositions, preferably serum transferrin, ovotransferrin, whey (serum) transferrin, cross-linked transferrin, more preferably cross-linked transferrin. 3. The cross-linked transferrin according to claim 2, characterized in that part of the transferrin is cross-linked by disulfide bonds. 4. transferrin nanoparticle according to claim 1, its preparation method comprises: with a kind of mixture that contains medicine and the organic phase that is dispersed therein and the aqueous medium that contains transferrin forms, by high-pressure homogenization, One or more of ultrasonication, grinding, homogenization, shearing, collision, and heating are processed, and the processing time is long enough to produce transferrin nanoparticles, and the transferrin nanoparticles are separated and collected. 5 . The transferrin nanoparticle according to claim 1 , its preparation method comprising: mixing and grinding the medicine and the transferrin, treating for a long enough time to produce the transferrin nanoparticle, and separating and collecting the transferrin nanoparticle. 6. transferrin nanoparticle according to claim 1, its preparation method comprises: transferrin is dispersed in the aqueous medium or in the aqueous medium containing certain concentration organic solvent, takes to add dehydrating agent, regulates in the pH method One or more of them are processed to form transferrin aggregates, which are solidified by one or more of high-pressure homogenization, ultrasonication, grinding, homogenization, shearing, collision, and heating methods, and the blank transferrin is separated and collected. Ferritin nanoparticles, the blank transferrin nanoparticles are dispersed in the solution of the drug, and treated by one or more of stirring, evaporating solvent, ultrasonic, heating, grinding, and shearing methods, so that the drug is adsorbed on the From the blank transferrin nanoparticles, separate and collect the transferrin nanoparticles. 7. transferrin nanoparticle according to claim 1, its preparation method comprises: transferrin and medicine are dispersed in aqueous medium or in the aqueous medium containing certain concentration organic solvent, take and add dehydrating agent, regulate pH One or more of the methods are processed to form aggregates of transferrin and drugs, which are solidified by one or more of high-pressure homogenization, ultrasonication, grinding, homogenization, shearing, collision, and heating. , to separate and collect transferrin nanoparticles. 8. transferrin nanoparticle according to claim 1, is characterized in that described medicine is selected from Chinese medicine, natural medicine, chemical medicine and biotechnology medicine, is preferably water insoluble medicine and biomacromolecule, more preferably Tripterygium glycosides, total flavonoids of Epimedium, Cordyceps polysaccharide, breviscapine, ursolic acid, nobiletin, curcumin, paclitaxel, doxorubicin, insulin. 9. The application of the transferrin nanoparticle according to claim 1 in the delivery of medicine in vivo. 10. The application of the transferrin nanoparticle according to claim 1 in the preparation of oral medicine.