CN102133190A - Transferrin nanoparticles and preparation method and application thereof - Google Patents
Transferrin nanoparticles and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to transferrin nanoparticles and a preparation method and application thereof. The transferrin nanoparticles can carry medicines which target tumors and brain, improve oral bioavailability of orally indigestible medicines, have the characteristics of high medicine loading rate, simple preparation process, low cost, high biocompatibility and the like, and have a clear application prospect.
Description
Technical field
The present invention relates to a kind of transferrins nanoparticle and its production and use, belong to medical technical field.
Background technology
Nanometer formulation for example liposome, solid-state lipid nanoparticle, nano structured lipid carrier etc. is the research focus of current field of medicaments, yet these nano-carriers all exist drug loading low, and the intravenous injection targeting is poor, the weak shortcoming of the short Absorption of oral administration.
Transferrins (Transferrin, Tf are called siderophillin, transferrin again) is a kind of important betaglobulin, is the transporter of ferrum in the vertebrates body.Research to the vertebrates transferrins proves, the molecular weight of transferrins about 80000Da, single peptide chain, iron content, sugar account for 6%.Different types of transferrins has different physics, chemistry and immunological characteristic, but two ferric ion binding sites are all arranged.In difference research,, divide the saturated transferrins of common transferrins or ferrum, single ferrum transferrins, apotransferrin by its iron content number.By its configuration, divide plain edition transferrins and heterogeneous transferrins.The single peptide chain that human transferrin is made up of the spherical structure territory that lays respectively at N end and C end of 2 structural similarities.Transferrins contains 679 amino acid residues, has 38 Cys, forms 19 pairs of disulfide bond.
Transferrins has polymorphism, mainly is divided into 3 classes: serum transferrin, ovum (clearly) transferrins, breast (clearly) transferrins.As amphoteric compound, except that the isoelectric point, IP (PI) of Lactotransferrin is 8.7, other transferrins isoelectric point, IPs are about 5.6～5.8, all are acidic proteins.
Transferrins combines with two kinds of dissimilar TfRs at least, i.e. TfR 1 (TfR1) and TfR 2 (TfR2).TfR is wide expression in vivo, and the expression of pronormoblast is the highest, reaches 80%; In intestinal epithelial cell, brain cell, brain capillary endothelial cell, the proliferative cell (as tumor cell, activated lymphocyte, the inductive fibroblast of serum) great expression is arranged also.Therefore transferrins can be used for cancer target administration, brain target administration and the oral administration of Chinese medicine, biomacromolecule, chemicals, treatment metal ion, diagnosis radiosiotope, nutrient etc.
Because transferrins has only part (about 30%) saturated by iron ion, so with other metal ions higher affinity is arranged.Have at least 30 kinds of other metal ions also can be, comprise in this metal ion that treatment is with metal ion such as bismuth (Bi), ruthenium Ru in conjunction with transferrins), titanium (Ti) waits and diagnosis usefulness radiosiotope gallium (Ga), indium (In) etc.Proved that transferrins can carry these metal ion targeting in tumor cell.
Owing to exist a large amount of TfRs at tumor cell and blood brain barrier surface, this provides hope for transferrins as pharmaceutical carrier.Transferrins and chemicals or gene combine, and the distributivity in tissue is good, have prolonged the half-life of medicine in endochylema, also can reach controlled-release function simultaneously.Studies show that of recent years, transferrins has all been obtained effect preferably in treatment tumor and brain targeting.
Transferrins can be partly to the Degradation of antitrypsin and chymase, and stability better in gastrointestinal tract.The molecular weight of transferrins self is relatively large in addition, the drug molecule that volume is little can be hidden enzyme in the external environment with it as carrier destruction.The transferrins mediated endocytosis is one of transport process of the tool characteristics of biological cell.If drug molecule and transferrins are coupled together, under the mediation of transferrins, medicine-transferrins conjugate combines with TfR.Then can be absorbed by the intestinal epithelial cell that the TfR height is expressed.
Because the transferrins good biocompatibility is a kind of very promising nano carrier material.Transferrins does not also contain the report of the transferrins nanoparticle of medicine at present generally by covalent bond and medicine combination now.
Summary of the invention
The purpose of this invention is to provide a kind of transferrins nanoparticle and preparation method thereof.Another object of the present invention provides the application that described transferrins nanoparticle is carried medicine in vivo.
At the foregoing invention purpose, the invention provides following technical scheme:
A kind of transferrins nanoparticle is characterized in that being made by medicine and transferrins, and the mass ratio of medicine and transferrins is 500～1: 1000, and the average diameter of described nanoparticle is no more than 1000 nanometers, and the diameter range of preferred nanoparticle is 20～500 nanometers.
Described transferrins nanoparticle, it is characterized in that described transferrins is made up of in the transferrins of serum transferrin, ovotransferrin, lactoferrin, breast (clearly) transferrins, melanocyte transferrins, common transferrins, the saturated transferrins of ferrum, single ferrum transferrins, apotransferrin, plain edition transferrins, heterogeneous transferrins, crosslinking Treatment one or more, the preferably transferrins of serum transferrin, ovotransferrin, breast (clearly) transferrins, crosslinking Treatment, the more preferably transferrins of crosslinking Treatment.
The transferrins of described crosslinking Treatment is characterized in that passing through disulfide bond crosslinking between the part transferrins.The transferrins of crosslinking Treatment is manually to be dispersed in transferrins in the aqueous medium or to contain in the aqueous medium of finite concentration organic solvent, by high pressure homogenize, ultrasonic, grind, in the methods such as breast is even, shearing, collision, heating one or more handle, by disulfide bond crosslinking, obtain the transferrins of crosslinking Treatment between the part transferrins.
Described transferrins nanoparticle, its preparation method comprises: with a kind of mixture that contains medicine and dispersive therein organic facies and contain the aqueous medium composition of transferrins, by high pressure homogenize, ultrasonic, grind, breast is even, in the shearing, collision method one or more are handled, the processing time long enough is to produce transferrins nanoparticle, separated and collected transferrins nanoparticle.Wherein said organic facies is by methanol, dehydrated alcohol, the ethanol that contains certain water gaging, normal propyl alcohol, isopropyl alcohol, n-butyl alcohol, isobutanol, the tert-butyl alcohol, dimethyl sulfoxide, N, dinethylformamide, N, one or more compositions in N-dimethyl acetylamide, methyl acetate, ethyl acetate, isopropyl acetate, acetone, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), toluene, oxolane, 1,4 one dioxane, dimethoxy-ethane, dichloromethane, dichloroethanes, chloroform, tetrachloromethane, the ether etc.
Described transferrins nanoparticle, its preparation method comprises: with medicine and transferrins mixed grinding, the processing time long enough is to produce transferrins nanoparticle, separated and collected transferrins nanoparticle.
Described transferrins nanoparticle, its preparation method comprises: be dispersed in transferrins in the aqueous medium or contain in the aqueous medium of finite concentration organic solvent, take to add dehydrant, in the adjusting pH method one or more are handled, form the transferrins aggregate, pass through high pressure homogenize, ultrasonic, grind, breast is even, shear, collision, in the heating means one or more are cured processing, the blank transferrins nanoparticle of separated and collected, blank transferrins nanoparticle is dispersed in the solution that is dissolved with medicine, by stirring, evaporating solvent, ultrasonic, heating, grind, in the cutting method one or more are handled, make medicine be adsorbed in the blank transferrins nanoparticle separated and collected transferrins nanoparticle.
Described transferrins nanoparticle, its preparation method comprises:: be dispersed in transferrins and medicine in the aqueous medium or contain in the aqueous medium of finite concentration organic solvent, take to add dehydrant, regulate in the pH method one or more and handle, form the aggregate of transferrins and medicine, by high pressure homogenize, ultrasonic, grind, breast is even, in the shearing, collision, heating means one or more are cured processing, separated and collected transferrins nanoparticle.
Described transferrins nanoparticle, it is characterized in that described medicine is selected from Chinese medicine, natural drug, chemicals and biotech drug, preferably poorly water soluble drugs and biomacromolecule, more preferably tripterygium glycosides, Herba Epimedii total flavones, Cordyceps polysaccharide, breviscapine, ursolic acid, Nobiletin, curcumin, paclitaxel, amycin, insulin.
Described transferrins nanoparticle is carried the application of medicine in vivo.
The application of described transferrins nanoparticle in the preparation oral drugs.
Beneficial effect of the present invention mainly is:
1, described transferrins nanoparticle drug loading height is generally 10～20%, the highest can reaching more than 30%.
2, transferrins nanoparticle preparation technology is simple, does not need complicated chemical reaction to modify, and has avoided toxic organic solvent and catalyst residue, and preparation cost is low, and clear and definite application prospect is arranged.
3, adopt the transferrins of crosslinking Treatment to prepare the transferrins nanoparticle, because the part transferrins is crosslinked mutually, thereby process treatment time is shorter, helps the stable of medicine, and entrapment efficiency is also higher simultaneously.
4, the transferrins nanoparticle can carry drug targeting in tumor and brain, reduces untoward reaction, increases the drug level of diseased region, thereby strengthens drug effect.
5, the transferrins nanoparticle can improve the oral administration biaavailability that oral difficulty absorbs the drug.
The specific embodiment
Below in conjunction with example the present invention is described in further detail, but scope of the present invention is not subjected to any restriction of these examples.
Embodiment 1
(Paclitaxel) is dissolved in the 3.5ml dichloromethane with the 30mg paclitaxel, add 27.0ml serum transferrin solution (1%, w/v), mixture homogenate 5 minutes under the slow-speed of revolution, to form rough emulsion, then it is transferred to (Avestin) in the high pressure homogenization device, high pressure homogenization circulates 5 times under 9000-18000 pound/inch (psi), even matter liquid evaporates under 40 ℃ of decompressions, remove dichloromethane rapidly, obtain translucent dispersion liquid, the average diameter of paclitaxel transferrins nanoparticle is 185nm.
Embodiment 2
Take by weighing ovotransferrin and amycin, mix, add an amount of pH value and be 6.0 solution according to mass ratio 9: 1, ultrasonic to dissolving.25 ℃ of waters bath with thermostatic control, add proper amount of acetone under the electromagnetic agitation, spray drying, the an amount of washing with alcohol of powder is flung to ethanol, with 5% mannitol solution ultra-sonic dispersion, lyophilization, after gained cake piece adds sterilized water or normal saline, measure particle diameter, the average diameter of amycin transferrins nanoparticle is 218nm.
Embodiment 3
With GaCl3 titration apotransferrin or single ferrum transferrins, prepare the gallium transferrins.The gallium transferrins is dissolved in the deionized water, regulates pH to about 7.0, slowly add the dehydrated alcohol of ormal weight, constant speed stirs, and thermal denaturation more than 80 ℃, spray drying are promptly.After gained cake piece adds sterilized water or normal saline, measure particle diameter, the average diameter of gallium transferrins nanoparticle is 336nm.
Embodiment 4
The 100mg serum transferrin is soluble in water, regulate pH to about 7.0; The 15mg insulin is dissolved in 0.1% the hydrochloric acid, regulates pH to about 7.0; Mix transferrins and insulin solutions, slowly add the sodium chloride of ormal weight, stir, form the aggregate of transferrins and insulin, high pressure homogenization promptly obtains insulin transferrins nanoparticle, and mean diameter is 252nm.
Embodiment 5
100mg ursolic acid and 800mg lactoferrin are placed nano-milled machine, grind, add aqueous dispersion after the grinding, centrifugal, get upper strata liquid, be ursolic acid transferrins nanoparticle, measure particle diameter, average diameter is 97nm.
Embodiment 6
The 35mg paclitaxel is dissolved in the 3.5ml dichloromethane, the serum transferrin solution (1% that adds the 27.0ml crosslinking Treatment, w/v), mixture homogenate 5 minutes under the slow-speed of revolution, to form rough emulsion, then it is transferred to (Avestin) in the high pressure homogenization device, high pressure homogenization circulates 3 times under 8000-15000 pound/inch (psi), even matter liquid evaporates under 40 ℃ of decompressions, remove dichloromethane rapidly, obtain translucent dispersion liquid, the average diameter of paclitaxel transferrins nanoparticle is 173nm.
Embodiment 7
100mg melanocyte transferrins and 20mg Cordyceps polysaccharide is soluble in water, 95% ethanol that slowly adds ormal weight, constant speed stirs, and forms the aggregate of melanocyte transferrins and Cordyceps polysaccharide, 180 ℃ of inlet temperature, 50 ℃ of leaving air temps, spray drying promptly obtains Cordyceps polysaccharide transferrins nanoparticle, after the adding aqueous dispersion, measure particle diameter, mean diameter is 365nm.
Embodiment 8
The 1g serum transferrin is soluble in water, regulate pH, slowly add 95% ethanol of ormal weight, form the aggregate of serum transferrin, 180 ℃ of inlet temperature, 50 ℃ of leaving air temps, spray drying promptly obtains blank transferrins nanoparticle.100mg transferrins nanoparticle is added in 50% alcoholic solution of 16mg Herba Epimedii total flavones, stirred 4 hours, slowly reclaim ethanol, spray drying promptly obtains Herba Epimedii total flavones transferrins nanoparticle, after the adding aqueous dispersion, measure particle diameter, mean diameter is 268nm, and in icariin, the envelop rate of Herba Epimedii total flavones is 86%.
Embodiment 9
The serum transferrin of 90mg tripterygium glycosides and 900mg crosslinking Treatment is placed nano-milled machine, adding water grinds, centrifugal, get upper strata liquid, be tripterygium glycosides transferrins nanoparticle, measure particle diameter, average diameter is 136nm, in tripterine, the envelop rate of tripterygium glycosides is 97.3%.
Embodiment 10
The 20mg Nobiletin is dissolved in the dehydrated alcohol of the dichloromethane of 4.5ml and 2ml, (concentration of serum transferrin and ovotransferrin is 0.3% to the mixed solution of adding 30.0ml serum transferrin and ovotransferrin, w/v), mixture homogenate 5 minutes under the slow-speed of revolution, to form rough emulsion, then it is transferred to (Avestin) in the high pressure homogenization device, high pressure homogenization circulates 5 times under 9000-18000 pound/inch (psi), even matter liquid evaporates under 45 ℃ of decompressions, remove organic solvent rapidly, obtain translucent dispersion liquid, the average diameter of Nobiletin transferrins nanoparticle is 162nm.
Embodiment 11
The 1g serum transferrin is soluble in water, slowly add the ethanol and the sodium chloride of ormal weight, form the aggregate of serum transferrin, 160 ℃ of inlet temperature, 50 ℃ of leaving air temps, spray drying promptly obtains blank transferrins nanoparticle.100mg transferrins nanoparticle is added in the alcoholic solution of 15mg curcumin, stirred 2 hours, reclaim ethanol, spray drying promptly obtains curcumin transferrins nanoparticle, add aqueous dispersion after, measure particle diameter, mean diameter is 337nm, and the envelop rate of curcumin is 93%.
Embodiment 12
1g breviscapine and 10g ovotransferrin are placed nano-milled machine, add an amount of 50% ethanol grinding, centrifugal, get upper strata liquid, be breviscapine transferrins nanoparticle, measure particle diameter, average diameter is 165nm, and in breviscapine, the envelop rate of breviscapine is 95.2%.
Embodiment 13
(Paclitaxel) is dissolved in the 35ml dichloromethane with the 300mg paclitaxel, add 270ml serum transferrin solution (1%, w/v), mixture homogenate 5 minutes under the slow-speed of revolution, to form rough emulsion, then it is transferred to (Avestin) in the high pressure homogenization device, high pressure homogenization circulates 5 times under 9000-18000 pound/inch (psi), even matter liquid evaporates under 40 ℃ of decompressions, removes dichloromethane rapidly, obtains translucent dispersion liquid, the mean diameter of paclitaxel transferrins nanoparticle is 163nm, add an amount of mannitol as the lyophilizing proppant, lyophilization 48 hours promptly gets injection paclitaxel transferrins nanoparticle.Gained cake piece is easy to reconstruct original dispersion liquid after adding sterilized water, measures particle diameter average out to 178nm.
[acute toxicity test]:
Experimental animal: Kunming mouse, body weight 18～22g, male and female half and half are provided by Nanjing General Hospital, Nanjing Military Area Command, PLA's animal center, raise with full-valence pellet feed, freely drink water.
Trial drug: injection paclitaxel transferrins nanoparticle, specification: 30mg/ bottle.Face with preceding usefulness 5% glucose injection and be mixed with desired concn.Paclitaxel injection, Beijing consonance pharmaceutical factory, specification: 30mg/5ml faces with preceding usefulness 5% glucose injection and is mixed with desired concn.
Test method: described in the non-CLINICAL PHARMACOKINETIS STUDY ON technological guidance's principle of chemicals, arrange design experiment.
Conclusion (of pressure testing): injection paclitaxel transferrins nanoparticle and paclitaxel injection acute toxicity test in mice result show: twice administration in a day, 1. injection paclitaxel transferrins nanoparticle is through the LD of intravenous administration
50Be 128.39mg/kg.Begin to occur movable and minimizings of ingesting of mice next day after the administration, and perpendicular mao, symptom such as lose weight, and begin that dead mouse is arranged.Beginning in the 6th day after the administration, it is normal that survival mice is recovered gradually.2. paclitaxel injection is through the LD of intravenous administration
50Be 72.53mg/kg.Begin to occur the movable minimizing of mice next day after the administration, perpendicular hair, and the symptoms such as minimizing of ingesting, and begin that dead mouse is arranged.Administration the 10th day, high dose group still has dead mouse.All the other are respectively organized survival mice and recover gradually normally, and to the administration the 15th day, survival mice was no longer dead.Dissect dead mice, it is obviously unusual that naked eyes do not see that each internal organs occurs.
[to the inhibitory action of mice-transplanted tumor EAC]:
Experimental animal: ICR kind white mice, 18-22g, male and female half and half are provided by animal housing of China Medicine University.Feedstuff: pellet, supply with by animal housing of China Medicine University; Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
Dosage is provided with: blank group (normal saline), and paclitaxel injection: 10mg/kg, injection paclitaxel transferrins nanoparticle: 10mg/kg, 5mg/kg, 2.5mg/kg be totally 5 dosage groups.
Test method: get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation EAC solid type, 10 every group, male and female half and half.Inoculate iv administration after 24 hours, every other day once, administration is 4 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
Result of the test: compare with the blank group, the high, medium and low dosage of injection paclitaxel transferrins nanoparticle (10,5mg/kg) group can suppress the tumor growth (P＜0.01) of sarcoma EAC significantly, and high, middle dosage group inhibitory action is organized than paclitaxel injection (10mg/kg).Injection paclitaxel transferrins nanoparticle is little than paclitaxel injection to the body weight influence of test mice simultaneously.Specifically see Table 1.
Table 1 injection paclitaxel transferrins nanoparticle is to the inhibitory action of mice-transplanted tumor EAC
*P＜0.05
*Compare with the blank group P＜0.01
Embodiment 14
The 200mg Nobiletin is dissolved in the dehydrated alcohol of the dichloromethane of 50ml and 15ml, add 300ml serum transferrin solution (0.5%, w/v), mixture homogenate 5 minutes under the slow-speed of revolution, to form rough emulsion, then it is transferred to (Avestin) in the high pressure homogenization device, high pressure homogenization circulates 5 times under 9000-18000 pound/inch (psi), even matter liquid evaporates under 40 ℃ of decompressions, remove organic solvent rapidly, obtain translucent dispersion liquid, the average diameter of Nobiletin transferrins nanoparticle is 155nm.
[test of brain targeting]:
Experimental animal: Kunming mouse, body weight 18～22g, male and female half and half are provided by Nanjing General Hospital, Nanjing Military Area Command, PLA's animal center, raise with full-valence pellet feed, freely drink water.
Dosage is provided with: Nobiletin solution (containing 20% ethanol): 10mg Nobiletin/kg; Nobiletin transferrins nanoparticle: 10mg Nobiletin/kg.
Test method: get 110 of above-mentioned specification mices, be divided into 2 groups at random, 55 every group, difference tail vein injection Nobiletin solution and Nobiletin transferrins nanoparticle.Respectively at after the administration 10,30,60,90,120,180,240,360,420,480,600min plucks and puts to death mice after eyeball is got blood, separates the internal organs such as brain, liver,spleen,kidney of mice, handles sample, measure and data analysis.
Result of the test: Cmax and the AUC0-t of Nobiletin transferrins nanoparticle group Nobiletin in cerebral tissue is respectively 8.52 ± 1.67 times and 15.68 ± 3.22 times of Nobiletin solution.Illustrate that Nobiletin transferrins nanoparticle has brain targeting preferably.
Embodiment 15
Take by weighing ovotransferrin and amycin, mix, add an amount of pH value and be 6.0 solution according to mass ratio 9: 1, ultrasonic to dissolving.25 ℃ of waters bath with thermostatic control add proper amount of acetone under the electromagnetic agitation, spray drying, and an amount of washing with alcohol of powder is flung to ethanol, with 5% mannitol solution ultra-sonic dispersion, lyophilization, promptly gets injection amycin transferrins nanoparticle.
[cancer target test]:
Experimental animal: nude mice, 18-21g, male and female half and half are provided by China Medicine University's animal center.Feedstuff: pellet, supply with by China Medicine University's animal center; Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
Dosage is provided with: adriamycin vial: 12mg amycin/kg; Injection amycin transferrins nanoparticle: 12mg amycin/kg.
Test method: give nude mice subcutaneous vaccination human breast cancer cell strain MCF-7 according to the transplanted tumor organon, transplanting the 3rd week of back rises, select to transplant 20 of successful model mouses, be divided into 2 groups at random according to tumor size and body weight, tail vein injection gives adriamycin vial and injection amycin transferrins nanoparticle respectively, puts to death tumor-bearing mice after 24 hours, and separation tumor piece, claim tumor heavy, handle sample, measure and data analysis.
Result of the test: amycin content is 6.14 ± 1.53 times of adriamycin vial group in the injection amycin transferrins nanoparticle group nude mice tumor piece, illustrates that amycin transferrins nanoparticle has tumor-targeting preferably.
Embodiment 16
The 100mg serum transferrin is soluble in water, regulate pH to about 7.0; The 10mg insulin is dissolved in 0.1% the hydrochloric acid, regulates pH to about 7.0; Mix transferrins and insulin solutions, slowly add the sodium chloride of ormal weight, stir, form the aggregate of transferrins and insulin, ultrasonic, promptly obtain insulin transferrins nanoparticle, mean diameter is 218nm.
[oral blood sugar lowering test]:
Test method: diabetes rat is divided into 3 groups at random, 6 every group, fasting 12h before the administration, first group is the blank group, orally give insulin normal saline solution (dosage is 20IU/Kg); Second group of positive matched group, i.e. subcutaneous injection insulin normal saline solution (dosage is 2IU/Kg); The 3rd group of orally give insulin transferrins nanoparticle (dosage is 20IU/Kg).After administration 0,1,2,4,6,8,12,16,24h gets blood 0.2mL in the angular oculi vein clump, press the blood glucose kit method and measure the blood glucose mass fraction, and calculate the rate of change of its blood glucose mass fraction with respect to zero time, draw change of blood sugar percentage rate and time relation curve.Calculate bioavailability according to area under the administration group blood glucose time graph with respect to the subcutaneous injection insulin.Computational methods as shown in the formula:
Wherein, F is the relative bioavailability of oral formulations with respect to the subcutaneous injection insulin solutions; Aoral and Asc are respectively area under the blood glucose time graph of oral insulin and subcutaneous injection insulin solutions; Doral, Dsc are respectively oral and hypodermic insulin dose.
Result of the test: the oral insulin bioavailability of blank group is 0, and the oral insulin bioavailability of insulin transferrins nanoparticle is 19.8 ± 3.6%.
Embodiment 17
Artificial aweto polysaccharide (purity is 98%, Changxing, Zhejiang pharmaceutical factory) reacts with tyramine, and then reacts with Fluorescein isothiocyanate (FITC), obtains fluorescently-labeled Cordyceps polysaccharide.
100mg serum transferrin and the fluorescently-labeled Cordyceps polysaccharide of 20mg is soluble in water, 95% ethanol that slowly adds ormal weight, constant speed stirs, and forms the aggregate of transferrins and Cordyceps polysaccharide, 180 ℃ of inlet temperature, 50 ℃ of leaving air temps, spray drying promptly obtains Cordyceps polysaccharide transferrins nanoparticle, after the adding aqueous dispersion, measure particle diameter, mean diameter is 370nm.
[Caco-2 cell model absorption test]:
Cell culture: on transwell, planting density is (100,000 cells/cm with the Caco-2 cell seeding
2), culture medium is the DMEM solution that contains 10% calf serum, cell is at 37 ℃ CO
2Cultivate in the incubator, changed culture fluid every one day, cell monolayer was differentiated to form in about 19～21 days, promptly can be used for test.
Test method: 37 ℃ of HBSS that use pH7.4 down measure cell monolayer flushing 3 times to stride membrane resistance, discard and stride the membrane resistance value less than 500ohms * cm
2Cell.Cell siphons away incubation medium hatch 1h in buffer after, and a series of transmembrane transport takes place subsequently for villous surface (AP) fluorescently-labeled Cordyceps polysaccharide of adding or fluorescently-labeled Cordyceps polysaccharide transferrins nanoparticle at cellular layer.Liquid two parts of samplings when beginning and 4h finish at the bottom of the AP side, the liquid 400ul that in 30min, takes a sample at the bottom of the BL side, and replenish with liquid of the blank end of volume and keep the BL Side Volume constant, measure fluorescence intensity, analysis.
Result of the test: the absorption infiltration coefficient of fluorescently-labeled Cordyceps polysaccharide transferrins nanoparticle is 5.32 * 10
-6Cm/s, the absorption infiltration coefficient of fluorescently-labeled Cordyceps polysaccharide is 1.49 * 10
-8Cm/s illustrates that the transferrins nanoparticle has significant oral short Absorption to Cordyceps polysaccharide.
Embodiment 18
Ursolic acid transferrins nanoparticle, Herba Epimedii total flavones transferrins nanoparticle, tripterygium glycosides transferrins nanoparticle, Nobiletin transferrins nanoparticle, curcumin transferrins nanoparticle, the breviscapine transferrins nanoparticle of getting preparation in the implementation column 5,8,9,10,11,12 carry out rat in the unidirectional intestinal perfusion model of body absorption test.
[rat is in the unidirectional intestinal perfusion model of body absorption test]:
Test method: anesthetized animal, open the abdominal cavity, insert the bile conduit near the duodenum place, respectively in duodenum, jejunum, ileum and colon two ends intubate, the gauze that experiment Shi Yong etc. oozes the normal saline dipping is covered in the intestinal tissue surface to preserve moisture, change perfusate with waiting after oozing the normal saline flushing intestinal contents, with a constant speed pump perfusion enteric cavity.Collect perfusate in the outlet every 30min, collect a bile sample before the perfusion, a every the 30min collection subsequently, the length of small intestinal is measured in the perfusion back, measures substrate concentration in the detection outlet.
Result of the test: compare with ursolic acid, Herba Epimedii total flavones, tripterygium glycosides, Nobiletin, curcumin, breviscapine raw material, the apparent infiltration coefficient of medicine in the transferrins nanoparticle in corresponding intestinal be significantly increased (P＜0.01), and all have preferably at four intestinal segments to absorb (apparent infiltration coefficient is all greater than 1), illustrate that the transferrins nanoparticle absorbs the drug to difficulty and has significant oral short Absorption.
Claims (10)
1. transferrins nanoparticle, it is characterized in that making by medicine and transferrins, the mass ratio of medicine and transferrins is 500～1: 1000, and the average diameter of described nanoparticle is no more than 1000 nanometers, and the diameter range of preferred nanoparticle is 20～500 nanometers.
2. transferrins nanoparticle according to claim 1, it is characterized in that described transferrins is by serum transferrin, ovotransferrin, lactoferrin, breast (clearly) transferrins, the melanocyte transferrins, common transferrins, the saturated transferrins of ferrum, single ferrum transferrins, apotransferrin, the plain edition transferrins, the heterogeneous transferrins, one or more compositions in the transferrins of crosslinking Treatment, serum transferrin preferably, ovotransferrin, breast (clearly) transferrins, the transferrins of crosslinking Treatment, the more preferably transferrins of crosslinking Treatment.
3. the transferrins of crosslinking Treatment according to claim 2 is characterized in that passing through disulfide bond crosslinking between the part transferrins.
4. transferrins nanoparticle according to claim 1, its preparation method comprises: with a kind of mixture that contains medicine and dispersive therein organic facies and contain the aqueous medium composition of transferrins, by high pressure homogenize, ultrasonic, grind, breast is even, in the shearing, collision, heating means one or more are handled, the processing time long enough is to produce transferrins nanoparticle, separated and collected transferrins nanoparticle.
5. transferrins nanoparticle according to claim 1, its preparation method comprises: with medicine and transferrins mixed grinding, the processing time long enough is to produce transferrins nanoparticle, separated and collected transferrins nanoparticle.
6. transferrins nanoparticle according to claim 1, its preparation method comprises: be dispersed in transferrins in the aqueous medium or contain in the aqueous medium of finite concentration organic solvent, take to add dehydrant, in the adjusting pH method one or more are handled, form the transferrins aggregate, pass through high pressure homogenize, ultrasonic, grind, breast is even, shear, collision, in the heating means one or more are cured processing, the blank transferrins nanoparticle of separated and collected, blank transferrins nanoparticle is dispersed in the solution that is dissolved with medicine, by stirring, evaporating solvent, ultrasonic, heating, grind, in the cutting method one or more are handled, make medicine be adsorbed in the blank transferrins nanoparticle separated and collected transferrins nanoparticle.
7. transferrins nanoparticle according to claim 1, its preparation method comprises: be dispersed in transferrins and medicine in the aqueous medium or contain in the aqueous medium of finite concentration organic solvent, take to add dehydrant, regulate in the pH method one or more and handle, form the aggregate of transferrins and medicine, by high pressure homogenize, ultrasonic, grind, breast is even, in the shearing, collision, heating means one or more are cured processing, separated and collected transferrins nanoparticle.
8. transferrins nanoparticle according to claim 1, it is characterized in that described medicine is selected from Chinese medicine, natural drug, chemicals and biotech drug, preferably poorly water soluble drugs and biomacromolecule, more preferably tripterygium glycosides, Herba Epimedii total flavones, Cordyceps polysaccharide, breviscapine, ursolic acid, Nobiletin, curcumin, paclitaxel, amycin, insulin.
9. the described transferrins nanoparticle of claim 1 application of carrying medicine in vivo.
10. the application of the described transferrins nanoparticle of claim 1 in the preparation oral drugs.
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| CN102327230A (en) * | 2011-09-30 | 2012-01-25 | 中国药科大学 | Protein nanometer granules wrapped with taxane medicaments and preparation method for nanometer granules |
| CN102357076A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Preparation method of protein nanoparticles coating insoluble drug |
| CN102357077A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Protein nanometer particle for wrapping slightly soluble medicines and preparation method thereof |
| CN104256048A (en) * | 2014-09-30 | 2015-01-07 | 华南理工大学 | Preparation method of high-load curcumin soybean protein nano product |
| CN105343005A (en) * | 2015-11-06 | 2016-02-24 | 中国药科大学 | Novel traditional Chinese medicinal nanoparticle oral absorption enhancing technology |
| CN109908964A (en) * | 2019-04-02 | 2019-06-21 | 南京大学 | Iron-sulfur cluster-protein gel compound and the preparation method and application thereof |
| US10660941B2 (en) | 2016-02-19 | 2020-05-26 | Indian Institute Of Technology, Bombay | Orally administrable pharmaceutical preparation containing protein |
| CN112094861A (en) * | 2020-10-26 | 2020-12-18 | 南京林业大学 | Expression and purification method of green alga plant ferritin and application thereof |
| CN112274495A (en) * | 2020-10-31 | 2021-01-29 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN114668771A (en) * | 2022-03-18 | 2022-06-28 | 南京林业大学 | Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together |
| CN115025054A (en) * | 2022-06-09 | 2022-09-09 | 四川普锐特药业有限公司 | Preparation method of nano composition with lactoferrin as carrier |
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| CN102357076A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Preparation method of protein nanoparticles coating insoluble drug |
| CN102357077A (en) * | 2011-09-30 | 2012-02-22 | 中国药科大学 | Protein nanometer particle for wrapping slightly soluble medicines and preparation method thereof |
| CN102327230B (en) * | 2011-09-30 | 2014-05-21 | 中国药科大学 | Protein nanometer granules wrapped with taxane medicaments and preparation method for nanometer granules |
| CN102357076B (en) * | 2011-09-30 | 2014-06-11 | 中国药科大学 | Preparation method of protein nanoparticles coating insoluble drug |
| CN102357077B (en) * | 2011-09-30 | 2014-06-11 | 中国药科大学 | Protein nanometer particle for wrapping slightly soluble medicines and preparation method thereof |
| CN102327230A (en) * | 2011-09-30 | 2012-01-25 | 中国药科大学 | Protein nanometer granules wrapped with taxane medicaments and preparation method for nanometer granules |
| CN104256048A (en) * | 2014-09-30 | 2015-01-07 | 华南理工大学 | Preparation method of high-load curcumin soybean protein nano product |
| CN105343005A (en) * | 2015-11-06 | 2016-02-24 | 中国药科大学 | Novel traditional Chinese medicinal nanoparticle oral absorption enhancing technology |
| US10660941B2 (en) | 2016-02-19 | 2020-05-26 | Indian Institute Of Technology, Bombay | Orally administrable pharmaceutical preparation containing protein |
| CN109908964A (en) * | 2019-04-02 | 2019-06-21 | 南京大学 | Iron-sulfur cluster-protein gel compound and the preparation method and application thereof |
| CN112094861A (en) * | 2020-10-26 | 2020-12-18 | 南京林业大学 | Expression and purification method of green alga plant ferritin and application thereof |
| CN112274495A (en) * | 2020-10-31 | 2021-01-29 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN112274495B (en) * | 2020-10-31 | 2022-05-03 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN114668771A (en) * | 2022-03-18 | 2022-06-28 | 南京林业大学 | Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together |
| CN114668771B (en) * | 2022-03-18 | 2022-12-06 | 南京林业大学 | Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together |
| CN115025054A (en) * | 2022-06-09 | 2022-09-09 | 四川普锐特药业有限公司 | Preparation method of nano composition with lactoferrin as carrier |
| CN115025054B (en) * | 2022-06-09 | 2023-12-22 | 四川普锐特药业有限公司 | Preparation method of nano composition taking lactoferrin as carrier |
| CN116735864A (en) * | 2023-08-15 | 2023-09-12 | 迦进生物医药(上海)有限公司 | Kit for evaluating blood safety of TfR1 antibody |
| CN116735864B (en) * | 2023-08-15 | 2023-11-10 | 迦进生物医药(上海)有限公司 | Kit for evaluating blood safety of TfR1 antibody |
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