CN103202812A - Method for preparing protein nanoparticles for delivering pharmacological active substances inside human bodies - Google Patents
Method for preparing protein nanoparticles for delivering pharmacological active substances inside human bodies Download PDFInfo
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Abstract
The invention relates to a method for preparing protein nanoparticles for delivering pharmacological active substances inside human bodies, and belongs to the field of delivery of pharmacological active substances inside human bodies and clinical application of delivery of the pharmacological active substances inside the human bodies. The method has the advantages that the pharmacological active substances are wrapped in the protein nanoparticles by means of unfolding and then refolding proteins and polypeptide or automatically assembling the proteins and the polypeptide, so that the pharmacological active substances can be delivered inside the human bodies.
Description
The application is to be on 08 09th, 2010 the applying date, and application number is 201010247885.7, and invention and created name is divided an application for " a kind of method for the preparation of the protein nano grain of sending pharmacological active substance in the body ".
Technical field
The present invention relates to a kind of method for the preparation of the protein nano grain of sending pharmacological active substance in the body, belong in the body of pharmacological active substance and send and the clinical practice field.Specifically, the present invention is that a kind of albumen and polypeptide of utilizing launches with method folding or self assembly again pharmacological active substance to be wrapped into albumen, forms the method for nanoparticle.
Background technology
Intravenous administration can directly act on body fast.In order to reduce the side effect of intravenous administration, one of effective method is exactly that pharmacological active substance is wrapped in micron or the nano level particle.On the one hand, intravenous particle can the slow release pharmacological active substance and is prolonged half-life of pharmacological active substance; On the other hand, the targeting material can discharge the pharmacological active substance targeting ground in the particle.
In the preparation method of the albumen particle that prior art relates to, preparation method (U.S. Patent No. 6 as Abraxane, 749,868, U.S. Patent No. 5,560,933), protein solution and organic solvent are uniformly mixed to form emulsion, high-pressure homogenization, forming with the paclitaxel is the albumin nano granular of core.Except this preparation method more complicated, also need to remove organic solvent with high pressure and corresponding high temperature, thereby obtain nanoparticle.Dichloromethane, organic solvents such as acetonitrile have toxicity, need its residual quantity of control.In addition, the ability of these method bag medicine carrying reason active substances is limited.Simultaneously, because high pressure and shearing force in the preparation process, albumen and polypeptide also may lose biological activity.
Albumen particle is also by the carrier of numerous bibliographical informations as pharmacological active substance and diagnostic reagent.Wherein, albumin microsphere can prepare with the method for heat cross-linking and chemical crosslinking.The heat cross-linking microsphere is to prepare from emulsifying mixt (as albumin, need pharmacological active substance that bag carries and suitable oil phase) under 100～150 ℃.Microsphere washs and preserves with appropriate solvent then.Leucuta etc. have reported the preparation method [International Journal of Pharmaceutics Vol. 41:213-217 (1988)] of heat cross-linking microsphere; The preparation of chemical crosslinking microsphere, such as document [Science Vol. 213:233-235 (1981)] report, use glutaraldehyde cross-linking albumen, wash then and preserve.
And the preparation method of protein nano grain is difficult to keep the biological activity of albumen in the prior art.And the pharmacological active substance that is insoluble in water also is difficult to be wrapped in the protein nano grain, because this method itself relies on the water of emulsion crosslinked.Water solublity pharmacology active substance can be crosslinked and enters protein arrays because it is dissolved in protein-contg water, and poorly water-soluble or fat-soluble pharmacological active substance are difficult to enter in the formed protein arrays of this method.
Summary of the invention
Technical purpose of the present invention is to provide a kind of method for the preparation of the protein nano grain of sending pharmacological active substance in the body, solving some pharmacological active substance owing to its hydrophobic character causes and can't send in the body, and the problem that forms the protein active forfeiture after the nanoparticle.
In order to realize technical purpose of the present invention, technical scheme of the present invention is:
A kind of method for the preparation of the protein nano grain of sending pharmacological active substance in the body may further comprise the steps: (a) obtain protein solution with first kind of dissolution with solvents albumen; (b) in denaturant or suitable degeneration condition, pharmacological active substance is added in the protein solution described in the step (a), albumen is launched and folding or self assembly again, pharmacological active substance is wrapped into albumen, form the protein nano grain.
Protein nano grain mean diameter described in the technical solution of the present invention is 5～2000 nm, optimization be 25～500 nm, that optimum is 50～300 nm.
Protein nano grain described in the technical solution of the present invention can wrap and carry the pharmacological active substance that accounts for protein nano grain gross weight 1%～40%.
The described pharmacological active substance of technical solution of the present invention is the hydrophobicity pharmacological active substance, as antitumor drug, and cardiovascular drugs, anti-inflammatory drug, hypoglycemic medicine, medicine for central nervous system, immunosuppressive drug, and antiviral drugs.
Wherein, described hydrophobicity pharmacological active substance is paclitaxel, Docetaxel, irinotecan, 5-fluorouracil, carmustine, amycin, phenesterin, piposulfan, tamoxifen, lomustine, gamlogic acid, rubescensine A, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, piroxicam, glibenclamide, diazepam, Risperidone, Ziprasidone, tacrolimus, rapamycin, indinavir, ritonavir, Telaprevir(CAS 402957-28-2), Lopinavir, or their combination.
Wherein, preferred hydrophobicity pharmacological active substance is paclitaxel or Docetaxel.
The described pharmacological active substance of technical solution of the present invention is the hydrophilic pharmacological active substance.
Wherein, described hydrophilic pharmacological active substance is cyclophosphamide, bleomycin, daunomycin, epirubicin, methotrexate, 5-fluorouracil or its analog, platinum or its analog, vinblastine or its analog, homoharringtonine or derivatives thereof, actinomycin D, Mitomycin-C, etoposide or their combination.
Albumen described in the technical solution of the present invention is albumin, transferrins, insulin, blood vessel endothelium chalone, hemoglobin, Myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, fine layer albumen, collagen protein, gelatin, artificial polypeptide and albumen, perhaps their combination.
Wherein, preferred albumen is albumin, transferrins, insulin, blood vessel endothelium chalone or hemoglobin.
First kind of solvent of the described step of technical solution of the present invention (a) is water, normal saline, sugar, freeze drying protectant or protein stabiliser.Wherein, described freeze drying protectant is phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose or their combination.Described protein stabiliser is trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination.
Wherein, described first kind of solvent preferred water, phosphate, acetate, acetyltryptophan, sodium caprylate or normal saline.
The operative temperature of the step described in the technical solution of the present invention (a) is-20～100 ℃; Be preferably 50～85 ℃; Optimum is 55～75 ℃.
The operation pH value of the step described in the technical solution of the present invention (a) is pH3～9; Be preferably pH5～8.5; Optimum is pH6～8.
The denaturant of the step described in the technical solution of the present invention (b) or suitable degeneration condition comprise water, strong acid, highly basic, inorganic salt, organic solvent, structure developing solvent or surfactant.Wherein, described strong acid and strong base comprises hydrochloric acid, sulphuric acid, sodium hydroxide etc.Described organic solvent is methanol, ethanol, isopropyl alcohol, formalin, chloroform, acetone, hydrogen sulfide or their combination.Described structure developing solvent is water, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination.Described inorganic salt is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, sucrose, mannitol, trehalose, acetyltryptophan, sodium caprylate or their combination.
Wherein, preferred denaturant or suitable degeneration condition are water, phosphate, acetate, glycine, Tris, sodium chloride, glucose, ethanol, acetone, hydrogen sulfide, 2 mercapto ethanol, carbamide or their combination.
The pH value of the denaturant described in the technical solution of the present invention or suitable degeneration condition is pH3～9; Be preferably pH5.5～8.5.
Step described in the technical solution of the present invention (b) comprises that also the external force operation launches with auxilin.
Wherein, described external force comprises transformation temperature, change pressure, applies mechanical force or radiation.
Wherein, described change pressure is for applying the pressure of 10～100,000 psi to reaction; Preferably reaction is applied 2000～60, the pressure of 000 psi.
The method of technical scheme of the present invention further comprises: (c) dialysis of protein nano grain is removed unnecessary micromolecular compound or further concentrated.
The method of technical scheme of the present invention further comprises: the protein nano grain after (d) will dialysing is prepared into pharmaceutical preparation through dehydration.
Wherein, the step of described dehydration is lyophilization, spray drying or distilling under reduced pressure.
Be further describing technical solution of the present invention below:
Except above-mentioned total technical scheme, the present invention has further proposed a kind of for the preparation of the nanoparticle preparation method of sending the hydrophobicity pharmacological active substance in the body, described method comprises following step: (a) at-20～100 ℃, under the condition of pH3～9, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, the hydrophobicity pharmacological active substance is added the protein solution described in the step (a), thereby cause that albumen launches and folding or self assembly again, wraps into albumen to pharmacological active substance.The nanoparticle mean diameter that forms is 5～500 nm, can wrap to carry the hydrophobicity pharmacological active substance that accounts for particle gross weight about 1～40%.The hydrophobicity pharmacological active substance here can be including, but not limited to paclitaxel, Docetaxel, irinotecan, carmustine, amycin, phenesterin, piposulfan, tamoxifen, lomustine, gamlogic acid, rubescensine A, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, piroxicam, glibenclamide, diazepam, Risperidone, Ziprasidone, tacrolimus, rapamycin, indinavir, ritonavir, Telaprevir, Lopinavir, and their combination.
Albumen in the method for the technical program generally can be including, but not limited to albumin, transferrins, insulin, Endostatin, hemoglobin, Myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, fine layer albumen, collagen protein, gelatin, artificial peptides and albumen, and their combination.First kind of solvent in the method for the present embodiment can comprise water, normal saline, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate, and their combination.In addition, the denaturant in the method for the present embodiment or suitable degeneration condition can comprise water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, isopropyl alcohol, formalin, chloroform, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine and their combination.
Wherein, the average diameter of described nanoparticle is preferably 25 nm～500 nm; Optimum is 50 nm～300 nm.Described step (a) is preferably carried out at 50 ℃～85 ℃, and optimum carries out at 55 ℃～75 ℃.PH preferably carries out under 5～8.5 condition, and optimum carries out under 6～8 condition.
Described denaturant or suitable degeneration condition are water, sodium chloride, phosphate, acetate, glycine, Tris, ethanol, acetone, hydrogen sulfide, 2 mercapto ethanol, carbamide or their combination.
Wherein, the pH value of described denaturant or suitable degeneration condition is 3～9, is preferably 5.5～8.5.
Said method further comprises: (c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated.Also further comprise: the nanoparticle after (d) will dialysing is prepared into pharmaceutical preparation through dehydration.Wherein, described dehydration is lyophilizing, distilling under reduced pressure or spray drying.
Technical solution of the present invention has further proposed a kind of for the preparation of the method for sending paclitaxel protein nano grain in the body again, described method comprises following step: (a) at 55～75 ℃, under the condition of pH6～8, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, paclitaxel is added the protein solution described in the step (a), thereby cause that albumen launches and folding or self assembly again, wraps in paclitaxel in the described albumen; (c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated; (d) gained solution is carried out dehydration, make the pharmaceutical dosage form that to preserve.Here the nanoparticle mean diameter that makes is 50～300 nm, can wrap to carry the pharmacological active substance that accounts for particle gross weight about 1～40%.Denaturant in the method for the present embodiment or suitable degeneration condition can be from water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, isopropyl alcohol, formalin, chloroform, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole is selected in acetylcysteine and their combination.In addition, the albumen in the method for the present embodiment can be selected from albumin, transferrins, insulin, Endostatin, hemoglobin.
Technical solution of the present invention has further proposed a kind ofly Docetaxel is wrapped into albumen to be used for the nanoparticle preparation method of sending in the body again, this method comprises following step: (a) at 55～75 ℃, dissolve described albumen with first kind of solution under the condition of pH6～8 and obtain protein solution; (b) under denaturant or suitable degeneration condition, Docetaxel is joined the protein solution described in the step (a), thereby cause and launch and folding or self assembly again, Docetaxel is wrapped into albumen; (c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated; (d) nanoparticle after will dialysing is prepared into pharmaceutical preparation through dehydration; Here the nanoparticle mean diameter that makes is about 50～300 nm, can wrap to carry the Docetaxel that accounts for particle gross weight about 1～40%.Denaturant in the method for the present embodiment or suitable degeneration condition can be from water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, isopropyl alcohol, formalin, chloroform, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole is selected in acetylcysteine and their combination.In addition, the albumen in the method for the present embodiment can be selected from albumin, transferrins, insulin, Endostatin, hemoglobin.
Technical solution of the present invention has further proposed a kind of albumen bag medicine carrying reason active substance again for the nanoparticle of sending in the body, the method for preparing this nanoparticle comprises following step: (a) at-20～100 ℃, under the condition of pH3～9, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, pharmacological active substance is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, pharmaceutical pack is rolled in the described albumen.
Technical solution of the present invention has further proposed a kind of albumen bag again and has carried paclitaxel for the nanoparticle of sending in the body, the method for preparing this nanoparticle comprises following step: (a) at 55～75 ℃, under the condition of pH6～8, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, paclitaxel is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, pharmaceutical pack is rolled in the described albumen; (c) the nanoparticle solution of dialysis formation obtains the solution of high concentration to remove superfluous material; (d) gained solution is carried out dehydration, make the pharmaceutical formulation that to preserve.
Technical solution of the present invention has further proposed a kind of albumen bag again and has carried Docetaxel for the nanoparticle of sending in the body, the method for preparing this nanoparticle comprises following step: (a) at 55～75 ℃, under the condition of pH6～8, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, Docetaxel is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, pharmaceutical pack is rolled in the described albumen; (c) the nanoparticle solution of dialysis formation obtains the solution of high concentration to remove superfluous material; (d) gained solution is carried out dehydration, make the pharmaceutical formulation that to preserve.
Explanation to technical scheme of the present invention:
" nanoparticle " of the present invention refers to little unit, and refers in particular to as a whole in transhipment and properties.The prepared protein nano grain average particle size distribution of the present invention in 5 nm between 2000 nm.Better interval be 25 nm to 500 nm, another better interval is that 50 nm are to 300 nm.And the prepared nanoparticle of the present invention can be in conjunction with up to 40% pharmacological active substance.
Described in the present invention pharmacological active substance is wrapped into albumen, referring to pharmacological active substance can be by the expansion of albumen and fold again, enter the albumen central area.In general, pharmacological active substance is included in: when dosing a patient with, can produce any material of pharmacological reaction.Pharmacological active substance among the present invention comprises hydrophobic and hydrophilic chemical compound.The water solublity that those skilled in the art know that the hydrophobicity pharmacological active substance is bad, and what the hydrophilic pharmacological active substance can be optimized is dissolved in the water.Hydrophobic pharmacological active substance comprises following chemical compound, but is not limited only to this: antitumor drug, cardiovascular drugs, anti-inflammatory drug, hypoglycemic medicine, medicine for central nervous system, immunosuppressive drug, and antiviral drugs.
The hydrophobicity pharmacological active substance that the present invention relates to can comprise, but not only be confined to this: paclitaxel, Docetaxel, irinotecan, carmustine, amycin, phenesterin, piposulfan, tamoxifen, lomustine, gamlogic acid, rubescensine A, podophyllotoxin, atorvastatin, simvastatin, fenofibrate, nifedipine, ibuprofen, indomethacin, piroxicam, glibenclamide, diazepam, Risperidone, Ziprasidone, tacrolimus, rapamycin, indinavir, ritonavir, Telaprevir, Lopinavir, and their combination.
That more optimizes says that the lyophobic dust with pharmacologically active comprises: antitumor pharmacology active substance such as paclitaxel, Docetaxel, irinotecan, card chlorine mustard, amycin, phenesterine, piposulfan, tamoxifen, lomustine, gamlogic acid, oridonin, podophyllotoxin and their derivant, and their combination.
In a scope of more optimizing, the hydrophobicity pharmacological active substance comprises paclitaxel and Docetaxel.Above-mentioned pharmacological active substance comprises crystal form and amorphous form, and its crystal form comprises the form that has water of crystallization and do not have water of crystallization.
Pharmacological active substance involved in the present invention also comprises hydroaropic substance.
Hydroaropic substance can comprise following chemical compound, but not only be confined to this: cyclophosphamide, bleomycin, daunomycin, epirubicin, methotrexate, 5-fluorouracil and analog thereof, platinum and analog thereof, vinblastine and analog thereof, homoharringtonine and derivant thereof, actinomycin D, Mitomycin-C, etoposide and their combination.
The amount that those skilled in the art can understand the pharmacological active substance that uses among the present invention can change according to the variation of the amount of albumen, and the variation according to the amount of nanoparticle simultaneously changes.Simultaneously, the veteran can recognize the pharmacological active substance that uses among the present invention, can be pure material, or mixture, and these all do not deviate from scope of the present invention.
Among the present invention, can select different albumen to remove to form the interested nanoparticle of those skilled in the art.Related albumen comprises all can launch and in conjunction with albumen or the polypeptide of pharmacological active substance among the present invention.The example of suitable albumen comprises as follows, but not only is confined to this: albumin, transferrins, insulin, blood vessel endothelium chalone, hemoglobin, myosin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, lamin, collagen protein, gelatin, man-made protein and their combination.
A scope of more optimizing, be suitable for albumen of the present invention and comprise as follows: albumin, transferrins, insulin, blood vessel endothelium chalone, hemoglobin and their compositions.The amount that those skilled in the art can know employed albumen in the inventive method is along with the variation of the amount of the amount of active substance and nanoparticle and change [Annalytical Biochemistry Vol. 72:248-254 (1976)].
Step in the inventive method (a) is for obtaining protein solution with first kind of described albumen of dissolution with solvents.The protein solution here refers to and comprises albumen and can proteolytic solvent in the solution, after albumen launches, can fold or self assembly again.Used hereinly foldingly again refer to an albumen of separating folding or degeneration and can fold the process that returns to suitable three dimensional structure again.Self assembly used herein refers to folding again protein binding to the process that forms nanoparticle together.The folding process again that those skilled in the art know that albumen can be finished by a lot of conditions.The first kind of solution that uses in protein solution is exemplified below, but not only is confined to this: water, normal saline, sugar; freeze drying protectant and protein stabiliser, in more accurate scope, solvent comprises water, sodium chloride solution; phosphate solution, acetum, glycine solution, tris solution; aqueous hydrogen peroxide solution, glutathion aqueous solution, glucose solution; aqueous trehalose, mannitol solution, sucrose solution; acetyltryptophan solution, sodium caprylate solution, and their mixture.
An accurate more scope, the solvent in the protein solution comprises water, phosphate, acetate and sodium chloride solution.The concentration of the solvent among the present invention in the employed protein solution all is feasible as long as suitable soluble protein and albumen are folding again.In general, in the protein solution content range of solvent from 0.001 M to 1.6 M.The scope of optimizing, from 0.03 M to 1.5 M.The scope of You Huaing again, from 0.05 M to 0.8 M.A more optimal scope, from 0.1 M to 0.3 M.The amount that those skilled in the art can understand proteolytic solvent can change according to the amount of albumen and the variation of protein solution concentration.
Experiment shows that the response parameter in the step among the present invention (a) is very important for forming nanoparticle.In general, obtain an ideal results, the step among the present invention (a) must be reacted between-20 ℃ to 100 ℃ scopes.A more accurate scope is from 50 ℃ to 85 ℃.An accurate more scope is from 55 ℃ to 75 ℃.Test verifiedly, obtain a more satisfactory result, the pH of step among the present invention (a) must be between 3 to 9, and a more accurate scope is from 5 to 8.5, are from 6 to 8 an accurate more scope.Those skilled in the art can know that step (a) needs a period of time so that albumen dissolves fully.In general, the length of time depends on employed kinds of proteins, and the use amount of albumen is used solvent types, the content of solvent, the concentration of solvent and other some factors.In general, those skilled in the art can fully recognize, each step of course of reaction and course of reaction all needs the sufficient time, give one example, and course of reaction needed do not wait by 8 hours in 5 minutes.
Second step of the present invention is that step (b) is included under denaturant or the suitable degeneration condition, and pharmacological active substance is joined through in the described protein solution of step (a), and albumen is launched and folding or self assembly again.Denaturant used herein or suitable degeneration condition refer to can induced protein or their three-dimensional of polypeptide change or the solution of two-dimensional structure.In general, the denaturant of mentioning here or suitable degeneration condition can the induced protein gentleness degeneration.Those skilled in the art can fully recognize gentle degeneration refer to the albumen solution folding/degeneration after, under certain conditions (as using again folding solution) be folded into suitable structure again.Denaturant or suitable degeneration condition can provide an environment to destroy the disulfide bond of albumen, form hydrogen bond, so that water can disturb the hydrophobic interaction of active site of protein.Those skilled in the art can recognize fully that many solution can serve as denaturant or suitable degeneration condition.Denaturant described here or suitable degeneration condition are drawn together water, strong acid, highly basic, inorganic salt, organic solvent, structure developing solvent and surfactant.Suitable denaturant or suitable degeneration condition are exemplified below, but not only are confined to this: water, sodium hydroxide, hydrochloric acid, sulphuric acid, methanol solution, alcoholic solution, isopropyl alcohol, formalin, chloroform, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine, carbamide, perchloric acid, tri-n-butyl phosphine, mercaptomethyl propionyl proline, performic acid, penicillamine, glutathion, methimazole, acetylcysteine, sodium chloride solution, phosphate solution, acetate solution, glycine solution, tris solution, hydrogen peroxide, glutathion, glucose, sucrose, mannitol, trehalose, acetyltryptophan, sodium caprylate and their mixture.A more accurate scope, denaturant or suitable degeneration condition comprise water, phosphate, acetate, glycine, Tris, ethanol, acetone, hydrogen sulfide, 2 mercapto ethanol, carbamide.Experiment showed, pH when denaturant or suitable degeneration condition between 3～9, the result of acquisition is gratifying.An accurate more pH scope is from 5.5 to 8.5.
In addition, except denaturant, the albumen solution is folding among the present invention can also use impressed pressure.In general, impressed pressure comprises and can cause the folding strength of albumen solution.Applied force comprises: temperature, pressure changes, mechanical force, radiation.Applied force comprises the pressure of from 10 to 100,000 psi, one more suitably scope be from 2000 to 60000 psi.
The present invention may need a step (c) that unnecessary micromolecular compound or further concentrated is removed in the nanoparticle dialysis in addition.In general, this step comprises the method that micromolecule can be separated arbitrarily from nanoparticle, and those skilled in the art can recognize fully that the method for separation comprises arbitrarily can purifying protein or the method for polypeptide.These methods can comprise salt precipitation, dialysis, chromatography and their combination, the selection that these methods can be suitable.A meticulousr scope, dialysis is feasible.
The present invention may also need the nanoparticle after a step (d) will be dialysed to be prepared into pharmaceutical preparation through dehydration.In general, the method for protection comprises to nanoparticle dewaters with convenient storage and transportation, so that obtains suitable dosage form.The method of protection involved in the present invention comprises: centrifugal, and drying under reduced pressure, lyophilizing, spray drying.
Those skilled in the art will appreciate that scope of the present invention and marrow change.Separate folding material and change, many pharmacological active substancies are spendable simultaneously, and many albumen and synthetic polypeptide also can be used as carrier.The present invention will obtain clear and definite more in the following embodiments and clearly describe.
Beneficial effect of the present invention is:
At first, the nanoparticle that forms through method provided by the invention can reach 90% envelop rate, surpasses prior art, has formed a kind of efficient method; Secondly, method provided by the present invention can obtain to be up to 40% drug loading, namely contains 40% pharmacological active substance in the nanoparticle.Because higher drug loading is arranged, when treatment, can obtain littler medication volume, shorter administration time, more convenient to patient.Higher drug loading has reduced the use amount of albumen when sending pharmacological active substance, has improved the cost efficiency of product; At last, the low medicine carrying ability that prior art provides can not satisfy the high dose administration, because the administration of high dose needs very big administration volume.Yet the nanoparticle with high medicine carrying ability of this experiment invention can not be subjected to the restriction of administration volume.
Another beneficial effect of nanoparticle by the present invention preparation is that nanoparticle can specificly be sent pharmacological active substance.Nanoparticle by the present invention's preparation can some organs of targeting and system.Such as, when using albumin as carrier, by changing the size of nanoparticle, targeting liver, lung, spleen or lymphsystem etc.When with transferrins or insulin as carrier the time, their receptor by the affinity of carrier and tumor cell surface, can specificly be transported to tumor tissues with pharmacological active substance at the cell surface high expressed.When carrier was the blood vessel endothelium chalone, its receptor was distributed in vascular cell, owing to there is the cause of a large amount of blood vessels in the tumor tissues, what nanoparticle can be a large amount of accumulates in tumor tissues.In a word, different protein carriers can different tissue or the organs of targeting.Therefore we can say, the invention provides a kind of efficient method and carry pharmacological active substance to the different parts of health.
Description of drawings
Fig. 1 is the influence (15% dispensing, medicine/albumen) of nanoparticle size among the present invention of different pH
Fig. 2 is the particle size distribution figure of albumin-paclitaxel nano grain among the present invention
Fig. 3 is the transmission electron microscope image (drug loading 10.59%) of albumin-paclitaxel nano grain among the present invention
Fig. 4 is the high-resolution TEM photo of paclitaxel-albumin nano granular
Wherein, (a) albumin-paclitaxel nano grain; (b) albumin nano granular of the zero load of Fang Daing; (c) the free paclitaxel that amplifies; (d) albumin of Fang Daing-paclitaxel nano grain
Fig. 5 is the X-ray powder diffraction image of albumin-paclitaxel nano grain
Wherein, (a) paclitaxel; (b) Kong Bai albumin nano granular; (c) paclitaxel-albumin nano granular (drug loading 12.9%); (d) physical mixture of albumin and paclitaxel (12.9%)
Fig. 6 is the redispersion of albumin-paclitaxel nano grain and medicine Abraxane
Wherein, (a) 2 mg/mL albumin-paclitaxel nano grain solution; (b) 2 mg/mL Abraxane solution; (c) 20 mg/mL albumin-paclitaxel nano grain solution; (d) 20 mg/mL Abraxane solution; (e) 50 mg/mL albumin-paclitaxel nano grain solution; (f) 50 mg/mL Abraxane solution.
The specific embodiment
Below all be based on representative embodiment of the present invention, but following embodiment can be not in office face restriction protection scope of the present invention where.
The preparation of embodiment 1. paclitaxels-albumin nano granular
100 mgHSA are dissolved in the phosphate buffer that contains 0.5 mg/mL EDTA and 0.05 M mercaptoethanol of 10 mL pH6,55 ℃ of reactions continue two hours, finish the back with 5% trichloroacetic acid precipitation and wash albumen, add paclitaxel (dissolve with ethanol) solution of 1.6 mL10 mg/mL in precipitation, mixed 2 minutes, the phosphate buffer that adds 0.08 M of 50 mL stirs dissolving mixt.The suspension that obtains is transparent, and the mean diameter of medicine carrying particle is 80～200 nm, (BIC 90plus Particle Size Analyzer).With the envelop rate of anti-phase C18 post mensuration paclitaxel, mobile phase is acetonitrile: water (60:40), detect wavelength 227 nm.HPLC the analysis showed that the paclitaxel envelop rate of this experiment reaches more than 90%.
Trichloroacetic acid can also replace with other denaturants in the experiment, such as water, and strong acid (hydrochloric acid and sulphuric acid), highly basic (sodium hydroxide), hydrogen peroxide, glutathion etc.Found that 5% trichloroacetic acid can reach narrower particle size distribution (50～300 nm).
The preparation of embodiment 2. paclitaxels-albumin nano granular
100 mg HSA are dissolved in the TRIS buffer of 50 mL pH7.4, and 37 ℃ of water-baths add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, add the paclitaxel (dissolve with ethanol) of 2 mL, 10 mg/mL.After 30 minutes, sample was dialysed gained sample lyophilizing 48 hours 24 hours with the TRIS buffer of pH7.4.Bulk sample behind obtained freeze-drying water or normal saline easily redissolves and is original solution, and the nanoparticle particle diameter remains unchanged.Particle diameter mainly is distributed in 80～200 nm (BIC 90plus Particle Size Analyzer) after the lyophilizing, and HPLC the analysis showed that the paclitaxel envelop rate of this experiment reaches more than 90%.
The preparation of embodiment 3. paclitaxels-albumin nano granular
100 mg paclitaxels are dissolved in the buffer of 10 mL pH4.8, and ice-water bath 30 minutes drips the acetone of 0 ℃ of pre-cooling of 7.5 mL, continues ice bath 1 hour.Centrifugal, collecting precipitation adds the paclitaxel (acetone solution) of 1 mL, 10 mg/mL in the precipitation, and ultrasonic mixing adds 50 mL normal saline, and magnetic agitation forms suspension.Carry out granularmetric analysis with BIC 90plus Particle Size Analyzer, the result is 150～220 nm.The sample of lyophilizing can redissolve, and it is 8.34% that HPLC analyzes drug loading.
Additional experiment shows glycine, and mannitol, lactose and trehalose all can be as freeze drying protectants, and does the particle diameter minimum that freeze drying protectant obtains with lactose.
In preparation process, we have investigated different buffer (water, normal saline, phosphate buffer, acetate buffer, glycine, Tris, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate and glucose solution etc.) and the different particle diameter of pH and the influence of drug loading, the result shows that pH is better 8.0.But pH can not surpass 8.5 again, because pharmacological active substance can decompose when pH surpasses 8.5.
The preparation of embodiment 4. paclitaxels-transferrins nanoparticle
100 mg transferrinss are dissolved in the TRIS buffer of 50 mL pH7.4, and 75 ℃ of stirrings add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, slowly add the paclitaxel (dissolve with ethanol) of 1 mL, 10 mg/mL.Record particle size distribution at 154.4 nm(BIC 90plus Particle Size Analyzer).
In preparation process, we have investigated the influence of different temperatures to particle diameter and drug loading, and the result shows that temperature all can form nanoparticle between 0 ℃ to 100 ℃, and is better at the nanoparticle of 55～75 ℃ of formation.
The preparation of embodiment 5. Docetaxels-transferrins nanoparticle
100 mg transferrinss are dissolved in the TRIS buffer of 50 mL pH7.4, and 65 ℃ of stirrings add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, add the Docetaxel (dissolve with ethanol) of 5 mL, 10 mg/mL.Recording mean diameter is 177.1 nm(BIC 90plus Particle Size Analyzer).
If 2 mercapto ethanol and other inorganic salts are used, can obtain better particle diameter result, such as water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, sucrose, mannitol, trehalose, acetyltryptophan and sodium caprylate etc.When these inorganic salts mixed as denaturant with 2 mercapto ethanol, particle diameter distributed narrower, wherein, Tris, the distribution of particles of acetyltryptophan and sodium caprylate is the narrowest, is approximately 80～130 nm.
The preparation of embodiment 6. gamlogic acids-albumin nano granular
100 mg HSA are dissolved in the 10 mL pure water.55 ℃ of stirrings, gained solution and isopyknic 5% trichloroacetic acid solution mix and are centrifugal, remove supernatant.Add the alcoholic solution that contains gamlogic acid and mix, add the TRIS of 50 mL pH8.0 then, stir and dissolve fully until albumen precipitation.The nanoparticle particle diameter of gained is 110 nm(BIC 90plus Particle Size Analyzer).
The preparation of embodiment 7. paclitaxels-hemoglobin nanoparticle
Under-20 ℃ of conditions, in the haemoglobin aqueous solution of 10 mL3%, slowly add the acetone soln that 300 mL contain the HCl of 3 mL, 2 M.Solution is powerful to stir centrifugal 15 minutes then 15 minutes.After treating that remaining acetone evaporated is fallen, collecting precipitation is dissolved in the cold deionized water, with deionized water dialysis 5 hours, uses 0.0016 M NaHCO then under 2 ℃ of conditions
3Dialysed 30 hours, and filtered and obtain globin solution.
Get the above-mentioned globin solution of 7 mL and add 28 mL water, obtain the protein solution of 1 mg/mL.Under 2～8 ℃ of conditions, add the alcoholic solution that 0.7 mL contains 10 mg/mL paclitaxels, stir until forming light blue solution.The gained average particle size is 308.1 nm(BIC 90plus Particle Size Analyzer).
The influence of the nanoparticle particle diameter of embodiment 8. pH is investigated
The present invention has investigated the phosphate buffer of different pH and they to the influence of nanoparticle particle diameter.All add 15% paclitaxel (10 mg/mL) in all protein solution groups, particle diameter is measured under constant temperature, and the result as shown in Figure 1.
(dynamic light scaterring DLS) analyzes the dynamic light scattering of embodiment 9. paclitaxels-albumin nano granular
Paclitaxel-albumin nano granular according to method preparation of the present invention is carried out granularmetric analysis, and used instrument is BIC 90plus Particle Size Analyzer.The result as shown in Figure 4, mean diameter is 121 nm, and particle size distribution is in narrower scope.
(Transmission Electron Microscopy TEM) characterizes the transmission electron microscope of embodiment 10. paclitaxels-albumin nano granular
Preparing drug loading according to the present invention is that paclitaxel-albumin nano granular of 10.59% carries out the analysis of transmission electron microscope.Used instrument is EM 2100 type 200KV high-resolution transmission electron microscopes (Japan).The result shows that nanoparticle is spherical in shape as shown in Figure 2.
The stability study of paclitaxel-albumin nano granular that embodiment 11. redissolves
The paclitaxel that lyophilizing obtains-albumin block normal saline, it is 5 mg/mL that 5% glucose and calf serum are dissolved into paclitaxel concentration respectively, the solution of 5 mg/mL and 2 mg/mL.After 25 ℃ and 37 ℃ were placed 12 hours, the mean diameter of nanoparticle did not change (DLS, BIC 90plus Particle Size Analyzer) respectively, and precipitation produces.The result is as shown in table 1.
The stability study of table 1 paclitaxel-albumin nano granular
| Diameter (nm) | 0 h | 3 h | 6 h | 12 h | 18 h | 24 h | 36 h |
| NS, 25℃ | 113.8 | 111.5 | 108.1 | 108.0 | 108.9 | 106.2 | 108.8 |
| GS, 25℃ | 113.7 | 123.3 | 122.1 | 121.1 | 123.1 | 116.5 | 125.3 |
| CS, 37℃ | 119.7 | 151.8 | 144.6 | 147.1 | 160.4 | 156.8 | 184.9 |
The research of embodiment 12. paclitaxels-transferrins nanoparticle
In the process of preparation paclitaxel-transferrins nanoparticle, find that except the TRIS buffer normal saline also can be used for dissolving transferrins.In addition, be 3～9 at pH, particularly in 6～8 scopes, can obtain the nanoparticle of high drug load and high stability.And, sucrose, glucose, glycine and trehalose can be used as lyophilizing or drying under reduced pressure protective agent.
The stability study of paclitaxel-transferrins nanoparticle that embodiment 13. redissolves
The paclitaxel that lyophilizing obtains--it is 5 mg/mL that transferrins block normal saline, 5% glucose and calf serum are dissolved into paclitaxel concentration respectively, the solution of 5 mg/mL and 2 mg/mL.After 25 ℃ and 37 ℃ were placed 12 hours, the mean diameter of nanoparticle did not change (DLS, BIC 90plus Particle Size Analyzer) respectively, and precipitation produces.The result is as shown in table 2.
The stability study of table 2 paclitaxel-transferrins nanoparticle
| Diameter (nm) | 0 h | 3 h | 6 h | 12 h | 18 h | 24 h | 36 h |
| NS, 25℃ | 143.2 | 132.7 | 132.9 | 133.4 | 134.0 | 127.3 | 139.6 |
| GS, 25℃ | 168.4 | 152.3 | 146.5 | 144.0 | 147.5 | 144.7 | 145.6 |
| CS, 37℃ | 118.8 | 139.3 | 152.7 | 165.6 | 171.5 | 177.6 | 228.8 |
Embodiment 14. usefulness transmission electron microscopes (Transmission Electron Microscopy, TEM) sign that paclitaxel-albumin nano granular is carried out
With 2～3 paclitaxel-albumin nano granular drips of solution (200 order) on the copper mesh that is coated with the carbon supporting film according to the present invention's preparation, blot excessive solution with filter paper after 2 minutes, then copper mesh is placed on air drying, analyzes with EM-2100 200 KV type high-resolution transmission electron microscopes (Japan).
The result is as shown in Figure 4: a) paclitaxel-albumin nano granular is spherical in shape; B) Kong Bai albumin nano granular presents irregular shape, and mean diameter is about 100 nm; C) You Li paclitaxel presents the higher electron density in place in the centre, is surrounded by club shaped structure on every side; D) paclitaxel-albumin nano granular presents nucleocapsid structure.
(x-ray powder diffraction XRD) characterizes the X-ray powder diffraction of embodiment 15. paclitaxels-albumin nano granular
Will crystallization when paclitaxel surpasses 1 mg/mL in aqueous solution.Therefore, the paclitaxel of amorphous state is very beneficial for injection.In order to detect among the present invention the solid forms of paclitaxel in paclitaxel-albumin nano granular, we have used X-ray powder diffraction to characterize.Four samples have been prepared altogether: a) paclitaxel; B) albumin nano granular; C) paclitaxel albumin nano granular (drug loading is 12.9%); D) paclitaxel and albuminous physical mixture (12.9%).The Zeta angular measurement of each sample decide scope from 5 the degree to 50 the degree (ARL, X'TRA, Applied Research Laboratories, Switzerland).
The result is as shown in Figure 5: figure a) has shown the characteristic peak of paclitaxel crystal; At 2 θ angles be 15 degree in 45 degree scopes, figure b) demonstrated albuminous dizzy type peak; Figure c) show at 2 θ angles be 15 the degree in 45 degree scopes, also shown albuminous dizzy type peak, illustrate that paclitaxel is to exist with unbodied state in nanoparticle; With the paclitaxel and albuminous physical mixture of nanoparticle same ratio (12.9%) in, figure d) shown the paclitaxel of crystal state and the albuminous existence of amorphous state.Therefore, we can obtain conclusion: paclitaxel exists with amorphous state in the paclitaxel-albumin nanometer of the present invention's preparation.
The redispersion research of embodiment 16. paclitaxels-albumin nano granular
Press following protein concentration water dissolution according to the sample after the paclitaxel-albumin nano granular lyophilizing of the present invention's preparation and business-like pharmacological active substance Abraxane: (a, b) 2 mg/mL; (c, d) 20 mg/mL; (e, f) 50 mg/mL.The photo of these samples all can obtain the colloid solution of stable transparent paclitaxel-albumin nano granular as shown in Figure 6 in three samples.
Embodiment 17. irinotecans-Preparation of insulin nanoparticles
100 mg insulins are dissolved in 10 mL water or the normal saline, and 65 ℃ of stirrings finish the back with methanol extraction and wash albumen, add the irinotecan solution of 1.6 mL10 mg/mL in precipitation, mixed 2 minutes, the sodium chloride solution that adds 50 mL stirs dissolving mixt.The diameter of the medicine carrying particle that obtains is 2000 nm to the maximum, and minimum is 5 nm, and the main scope that distributes is at 50～300 nm, and the part nanoparticle is distributed in 2000 nm (BIC 90plus Particle Size Analyzer).With the envelop rate that anti-phase C18 post is measured, mobile phase is acetonitrile: water (60:40), detect wavelength 227 nm.HPLC the analysis showed that the irinotecan medicine carrying of this experiment is 2%.
Experiment finds except water or physiological saline solution insulin, and some protein stabilisers and freeze drying protectant also can be used for dissolving insulin.Such as glycine, glutathion, acetyltryptophan, sodium caprylate and mannitol etc.In addition, be 0 ℃～100 ℃ in reaction temperature, particularly in 55～75 ℃ of scopes, can obtain the nanoparticle of high drug load and high stability.The protein nano grain that obtains can obtain the dryness powder with spray-dired mode.
The preparation of embodiment 18. 5-fluorouracil-blood vessel endothelium chalone nanoparticle
100 mg blood vessel endothelium chalones be dissolved in 10 mL pH 9 the solution of 5% glucose that contains in, 75 ℃ of water-baths add the glutathion of 25 mL, 0.5 mg/mL, react 10 minutes, add the 5-fluorouracil of 1.6 mL10 mg/mL, reaction continues two hours.Sample was dialysed 24 hours with the TRIS buffer of pH7.4, and the nanoparticle particle diameter remained unchanged after the gained sample carried out spray drying.Particle diameter mainly is distributed in 25～500 nm (BIC 90plus Particle Size Analyzer), and drug loading reaches 40%.
Wherein, it is any in the freeze drying protectant that 5% glucose can change into, as phosphate, and acetate, glycine, Tris.Glutathion can be any in the structure developing solvent, as 2 mercapto ethanol, and dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination.The result shows that the solution of solubilising protein is to the not influence of size of final particle.In denaturant or the suitable degeneration condition, the effect of 2 mercapto ethanol is best, and the nanoparticle narrow distribution that obtains is between 50～300 nm.
The preparation of embodiment 19. 5-fluorouracil-paclitaxel-blood vessel endothelium chalone nanoparticle
100 mg blood vessel endothelium chalones be dissolved in 10 mL pH7.4 the solution of 5% trehalose that contains in, 25 ℃ of water-baths, add the mercaptoethanol of 25 mL, 0.5 mg/mL and the carbamide of 6 M, reacted 10 minutes, the 5-fluorouracil and the 0.8 mL10 mg/mL paclitaxel that add 0.8 mL10 mg/mL, continue to stir, it is light blue that solution is.Particle diameter mainly is distributed in 25～500 nm (BIC 90plus Particle Size Analyzer).
Wherein, it is any in the protein stabiliser that 5% glucose can change into, as mannitol, and sucrose, acetyltryptophan, sodium caprylate.The result shows that the solution of solubilising protein is to the not influence of size of final particle, and the nanoparticle narrow distribution that obtains is between 50～300 nm.
The preparation of embodiment 20. carmustines-Myoglobin nanoparticle
100 mg Myoglobin are dissolved in the phosphate buffer that contains 0.5 mg/mL sodium caprylate and 0.05 M acetyltryptophan of 10 mL pH 3, ice bath 30 minutes, acetone 7.5 mL of 0 ℃ of pre-cooling of dropping.Continued ice bath 1 hour, centrifugal, collecting precipitation, add the carmustine solution of 1.6 mL, 10 mg/mL in the precipitation, ultrasonic mixing adds 50 mL Tris buffer, magnetic agitation, the suspension that obtains is transparent, and solution obtains the particle that mean diameter is 80～200 nm (BIC 90plus Particle Size Analyzer) through distilling under reduced pressure
Wherein degeneration condition acetone can be replaced and become other and can make protein-denatured organic solvent, as methanol, and ethanol, isopropyl alcohol, formalin, chloroform, hydrogen sulfide or their combination.The result shows that if the degeneration condition is with an organic solvent, and then the effect of acetone is best, and particle diameter is distributed between 50～300 nm.
The preparation of embodiment 21. amycin-lysozyme nanoparticle
100 mg lysozyme are dissolved in the TRIS buffer of 50 mL pH 6, and 55 ℃ of stirrings feed hydrogen sulfide gas, and reaction continues 10 minutes, adds the amycin of 5 mL, 10 mg/mL.Record particle size distribution at 347.1 nm(BIC 90plus Particle Size Analyzer).
The Tris buffer can change any in the protein stabiliser into, as trehalose, and mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination.
The preparation of embodiment 22. phenesterins-immunoglobulin nanoparticle
Present embodiment is identical with embodiment 5, and only changing Docetaxel is phenesterin, and transferrins is immunoglobulin.Mean diameter is approximately 250 nm, drug loading 9.8%.
The preparation of embodiment 23. piposulfans-α-2-macroglobulin nanoparticle
Present embodiment is identical with embodiment 5, and only changing Docetaxel is piposulfan, and transferrins is α-2-macroglobulin, and reaction temperature is 65 ℃.Particle size distribution 25 nm～500 nm, drug loading 11.2%.
The preparation of embodiment 24. tamoxifens-fibronectin nanoparticle
Present embodiment is identical with embodiment 5, and only changing Docetaxel is tamoxifen, and transferrins is fibronectin.45 ℃ of reaction temperatures, particle size distribution 50 nm～200 nm, drug loading 9.8%.
The preparation of embodiment 25. lomustines-fibre layer protein nano grain
Present embodiment is identical with embodiment 5, and only changing Docetaxel is lomustine, and transferrins is fine layer albumen.Reaction temperature is 75 ℃, particle size distribution between 100 nm～200 nm, drug loading 35%.
The preparation of embodiment 26. rubescensine A-collagen protein nanoparticle
Present embodiment is identical with embodiment 5, and only changing Docetaxel is rubescensine A, and transferrins is collagen protein.The reaction pH be 8.0, particle size distribution between 300 nm～600 nm, drug loading 22%.
The preparation of embodiment 27. podophyllotoxins-gelatin nanoparticle
Present embodiment is identical with embodiment 5, and only changing Docetaxel is podophyllotoxin, and transferrins is gelatin.The reaction temperature be 70 ℃, particle size distribution between 50 nm～300 nm, drug loading 20%.
The preparation of embodiment 28. atorvastatins-albumin nano granular
100 mgHSA are dissolved in the 10 mL normal saline, contain 0.5 mg/mL sodium caprylate and 0.05 M acetyltryptophan, 55 ℃ of water-baths.Add 350 μ L 2 mercapto ethanols, reaction continues 10 min, adds the atorvastatin of 2 mL, 10 mg/mL, continues to stir to be light blue up to solution, the nanoparticle particle diameter of gained is 50～300 nm(BIC 90plus Particle Size Analyzer), drug loading is 24%.
The preparation of embodiment 29. simvastatins-transferrins nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is simvastatin, and albumin is transferrins.The gained nano particle diameter is 50～300 nm, and drug loading is 5%.
The preparation of embodiment 30. luxuriant and rich with fragrance nobert-hemoglobin nanoparticles
Present embodiment is identical with embodiment 28, and only changing atorvastatin is luxuriant and rich with fragrance nobert, and albumin is hemoglobin.The gained nano particle diameter is 30～300 nm, and drug loading is 11%.
The preparation of embodiment 31. nifedipines-blood vessel endothelium chalone nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is nifedipine, and albumin is the blood vessel endothelium chalone.The gained nano particle diameter is 20～250 nm, and drug loading is 9.7%.
The preparation of embodiment 32. ibuprofen-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is ibuprofen.The gained nano particle diameter is 20～250 nm, and drug loading is 8.7%.
The preparation of embodiment 33. indomethacins-collagen protein nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is indomethacin, and albumin is collagen protein.The particle diameter of gained nano-particle is 30～500 nm, and drug loading is 8.9%.
The preparation of embodiment 34. piroxicams-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is piroxicam.The particle diameter of gained nano-particle is 25～500 nm, and drug loading is 8.0%.
The preparation of embodiment 35. glibenclamides-Myoglobin nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is glibenclamide, and albumin is Myoglobin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 7.8%.
The preparation of embodiment 36. diazepam-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is diazepam.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 8.8%.
The preparation of embodiment 37. Risperidones-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is Risperidone.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 9.8%.
The preparation of embodiment 38. Ziprasidones-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is Ziprasidone, and albumin is immunoglobulin, and the particle diameter of gained nano-particle is 50～300 nm, and drug loading is 7.3%.
The preparation of embodiment 39. tacrolimuss-lysozyme nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is tacrolimus, and albumin is lysozyme.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 12.8%.
The preparation of embodiment 40. rapamycins-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is rapamycin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 9.8%.
The preparation of embodiment 41. indinavirs-transferrins nanoparticle
Present embodiment is identical with embodiment 28, and only changing atorvastatin is indinavir, and albumin is transferrins.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 13.5%.
Embodiment 42. ritonavirs-Preparation of insulin nanoparticles
Present embodiment is identical with embodiment 28, and only changing atorvastatin is ritonavir, and albumin is insulin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 9.8%.
The preparation of embodiment 43. Lopinavirs-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is Lopinavir.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 11.3%.
The preparation of embodiment 44. cyclophosphamide-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is cyclophosphamide.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 11.9%.
The preparation of embodiment 45. bleomycin-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is bleomycin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 12.8%.
The preparation of embodiment 46. daunomycin-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is daunomycin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 13.8%.
The preparation of embodiment 47. epirubicins-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is epirubicin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 7.3%.
The preparation of embodiment 48. methotrexates-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is the methylamine pterin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 6.8%.
The preparation of embodiment 49. 5-fluorouracil-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is fluorouracil.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 11.1%.
The preparation of embodiment 50. cisplatin-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is cisplatin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 12.8%.
The preparation of embodiment 51. vinblastine-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is vinblastine.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 6.3%.
The preparation of embodiment 52. actinomycin D-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is actinomycin D.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 5.8%.
The preparation of embodiment 53. ametycins-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is ametycin.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 4.8%.
The preparation of embodiment 54. etoposides-albumin nano granular
Present embodiment is identical with embodiment 28, and only changing atorvastatin is etoposide.The particle diameter of gained nano-particle is 50～300 nm, and drug loading is 14.9%.
Claims (23)
1. method for the preparation of the protein nano grain of sending pharmacological active substance in the body may further comprise the steps:
(a) obtain protein solution with first kind of dissolution with solvents albumen; Described albumen is albumin;
(b) under denaturant or suitable degeneration condition, pharmacological active substance is added in the protein solution described in the step (a), albumen is launched and folding or self assembly again, pharmacological active substance is wrapped into albumen, form the protein nano grain; Described pharmacological active substance is Docetaxel;
Wherein, the denaturant in the described step (b) or suitable degeneration condition are water, strong acid, highly basic, inorganic salt, organic solvent, structure developing solvent or surfactant;
Wherein, described organic solvent is methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone, hydrogen sulfide or their combination.
2. method according to claim 1, the mean diameter that it is characterized in that described protein nano grain is 25 nm～500 nm.
3. method according to claim 1, the mean diameter that it is characterized in that described protein nano grain is 50 nm～300 nm.
4. method according to claim 1 is characterized in that described pharmacological active substance shared weight ratio in nanoparticle is 1%～40%.
5. method according to claim 1 is characterized in that first kind of solvent of described step (a) is water, normal saline, sugar, freeze drying protectant or protein stabiliser.
6. method according to claim 5 is characterized in that described freeze drying protectant is phosphate, acetate, glycine, Tris, glucose or their combination.
7. method according to claim 5 is characterized in that described protein stabiliser is trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination.
8. method according to claim 1 is characterized in that the operative temperature of described step (a) is-20～100 ℃.
9. method according to claim 1, the operative temperature that it is characterized in that described step (a) is 55～75 ℃.
10. method according to claim 1, the operation pH value that it is characterized in that described step (a) is pH3～9.
11. method according to claim 1 is characterized in that described inorganic salt is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, sucrose, mannitol, trehalose, acetyltryptophan, sodium caprylate or their combination.
12. method according to claim 1 is characterized in that described structure developing solvent is water, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine, trichloroacetic acid or their combination.
13. method according to claim 1, its described denaturant or suitable degeneration condition are water, phosphate, acetate, glycine, Tris, sodium chloride, glucose, ethanol, acetone, hydrogen sulfide, 2 mercapto ethanol, carbamide or their combination.
14. method according to claim 1 is characterized in that described method further comprises: (c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated.
15. method according to claim 14 is characterized in that described method further comprises: the nanoparticle after (d) will dialysing is prepared into pharmaceutical preparation through dehydration.
16. method according to claim 15 is characterized in that described dehydration is lyophilizing, distilling under reduced pressure or spray drying.
17. the method for the preparation of the protein nano grain of sending lyophobic dust in the body may further comprise the steps:
(a) at-20～100 ℃, under the condition of pH3～9, obtain protein solution with first kind of described albumen of dissolution with solvents; (b) under denaturant or suitable degeneration condition, pharmacological active substance is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, pharmaceutical pack is rolled in the described albumen;
Described diameter of nano particles 25 nm～500 nm, described protein nano grain contains the hydrophobicity pharmacological active substance of 1%～40% weight ratio;
Described hydrophobicity pharmacological active substance is Docetaxel;
Described albumen is albumin;
Described first kind of solvent is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination;
Described denaturant or suitable degeneration condition are water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, isopropyl alcohol, formalin, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine, trichloroacetic acid or their combination.
18. method according to claim 17 is characterized in that described method further comprises:
(c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated.
19. method according to claim 18 is characterized in that described method further comprises:
(d) nanoparticle after will dialysing is prepared into pharmaceutical preparation through dehydration.
20. method according to claim 19 is characterized in that described dehydration is lyophilizing, distilling under reduced pressure or spray drying.
21. one kind for the preparation of the method for sending Docetaxel protein nano grain in the body, described method comprises following step:
(a) at 55～75 ℃, under the condition of pH6～8, obtain protein solution with first kind of described albumen of dissolution with solvents;
(b) under denaturant or suitable degeneration condition, Docetaxel is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, Docetaxel is wrapped in the described albumen;
(c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated;
(d) nanoparticle after will dialysing is prepared into pharmaceutical preparation through dehydration;
Described diameter of nano particles is 50 nm～300 nm, and described protein nano grain contains the Docetaxel of 1%～40% weight ratio;
Described denaturant or suitable degeneration condition are water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination;
Described first kind of solvent is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination;
Described albumen is albumin.
22. a protein nano grain that includes pharmacological active substance is used for drug disposition and sends; It is characterized in that the preparation process of described nanoparticle by the following method:
(a) at-20～100 ℃, under the condition of pH3～9, obtain protein solution with first kind of described albumen of dissolution with solvents;
(b) under denaturant or suitable degeneration condition, pharmacological active substance is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, pharmaceutical pack is rolled in the described albumen;
Described diameter of nano particles is 5 nm～2000 nm, and described protein nano grain contains the hydrophobicity pharmacological active substance of 1%～40% weight ratio;
Described pharmacological active substance is Docetaxel;
Described albumen is albumin;
Described first kind of solvent is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination;
Described denaturant or suitable degeneration condition are water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination.
23. a protein nano grain that includes Docetaxel is used for drug disposition and sends; It is characterized in that the preparation process of described nanoparticle by the following method:
(a) at 55～75 ℃, under the condition of pH6～8, obtain protein solution with first kind of described albumen of dissolution with solvents;
(b) under denaturant or suitable degeneration condition, Docetaxel is joined in the described protein solution of step (a), albumen is launched and folding or self assembly again, Docetaxel is wrapped in the described albumen;
(c) the nanoparticle dialysis is removed unnecessary micromolecular compound or further concentrated;
(d) nanoparticle after will dialysing is prepared into pharmaceutical preparation through dehydration;
Wherein said diameter of nano particles is 50 nm～300 nm, and described protein nano grain contains the Docetaxel of 1%～40% weight ratio;
Described denaturant or suitable degeneration condition are water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone, hydrogen sulfide, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination;
Described albumen is albumin;
Described first kind of solvent is water, sodium chloride, phosphate, acetate, glycine, Tris, hydrogen peroxide, glutathion, glucose, trehalose, mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515244A (en) * | 1996-10-01 | 2004-07-28 | �Ϻ���ͨ��ѧ | Protein-stable pharmacological active matter, its preparation and application method |
| US20080199911A1 (en) * | 2003-05-29 | 2008-08-21 | Academia Sinica | Vp1 of foot-and-mouth disease virus |
| WO2008124165A2 (en) * | 2007-04-09 | 2008-10-16 | Chimeros, Inc. | Self-assembling nanoparticle drug delivery system |
| CN101298610A (en) * | 2008-06-06 | 2008-11-05 | 浙江大学 | Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide |
| CN101333243A (en) * | 2003-05-14 | 2008-12-31 | 多曼蒂斯有限公司 | Process for recovering polypeptides that unfold reversibly from a polypeptide repertoire |
| CN101346131A (en) * | 2005-12-27 | 2009-01-14 | Lts罗曼治疗方法有限公司 | Protein-based delivery system for overcoming resistance in tumour cells |
| CN101385857A (en) * | 2008-10-21 | 2009-03-18 | 中国药科大学 | Novel nano preparation with stable protein and preparation method and use thereof |
| CN101495148A (en) * | 2005-10-21 | 2009-07-29 | 万能药生物有限公司 | Pharmaceutical composition comprising at least one anticancer drug and at least one polymer |
-
2010
- 2010-08-09 CN CN201310101755.6A patent/CN103202812B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1515244A (en) * | 1996-10-01 | 2004-07-28 | �Ϻ���ͨ��ѧ | Protein-stable pharmacological active matter, its preparation and application method |
| CN101333243A (en) * | 2003-05-14 | 2008-12-31 | 多曼蒂斯有限公司 | Process for recovering polypeptides that unfold reversibly from a polypeptide repertoire |
| US20080199911A1 (en) * | 2003-05-29 | 2008-08-21 | Academia Sinica | Vp1 of foot-and-mouth disease virus |
| CN101495148A (en) * | 2005-10-21 | 2009-07-29 | 万能药生物有限公司 | Pharmaceutical composition comprising at least one anticancer drug and at least one polymer |
| CN101346131A (en) * | 2005-12-27 | 2009-01-14 | Lts罗曼治疗方法有限公司 | Protein-based delivery system for overcoming resistance in tumour cells |
| WO2008124165A2 (en) * | 2007-04-09 | 2008-10-16 | Chimeros, Inc. | Self-assembling nanoparticle drug delivery system |
| CN101298610A (en) * | 2008-06-06 | 2008-11-05 | 浙江大学 | Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide |
| CN101385857A (en) * | 2008-10-21 | 2009-03-18 | 中国药科大学 | Novel nano preparation with stable protein and preparation method and use thereof |
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