CN103202813B - A kind of method of protein nano grain for the preparation of sending pharmacological active substance in body - Google Patents
A kind of method of protein nano grain for the preparation of sending pharmacological active substance in body Download PDFInfo
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Abstract
The present invention relates to a kind of method of protein nano grain for the preparation of sending pharmacological active substance in body, sending and clinical practice field in the body belonging to pharmacological active substance.The present invention utilizes the method for albumen and polypeptide expansion and refolding or self assembly, pharmacological active substance is wrapped into protein nano grain, for sending in body.
Description
The application is the applying date is on 08 09th, 2010, and application number is 201010247885.7, and invention and created name is the divisional application of " a kind of method of protein nano grain for the preparation of sending pharmacological active substance in body ".
Technical field
The present invention relates to a kind of method of protein nano grain for the preparation of sending pharmacological active substance in body, sending and clinical practice field in the body belonging to pharmacological active substance.Specifically, the present invention is that a kind of albumen and polypeptide of utilizing launches, with the method for refolding or self assembly, pharmacological active substance is wrapped into albumen, forms the method for nanoparticle.
Background technology
Intravenous administration fast direct ground connection can act on body.In order to reduce the side effect of intravenous administration, pharmacological active substance wraps in micron or nano level particle by one of effective method exactly.On the one hand, intravenous particle can slow release pharmacological active substance extend half-life of pharmacological active substance; On the other hand, the pharmacological active substance targeting in particle can discharge by targeting material.
In the preparation method of the albumen particle related in prior art, as the preparation method (U.S. Patent No. 6 of Abraxane, 749,868, U.S. Patent No. 5,560,933), protein solution and organic solvent are uniformly mixed to form emulsion, high-pressure homogenization, being formed with 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, thus obtain nanoparticle.Dichloromethane, the organic solvents such as acetonitrile have toxicity, need to control its residual quantity.In addition, the ability that these method bags carry pharmacological active substance is limited.Meanwhile, due to the high pressure in preparation process and shearing force, proteins and peptides also may lose biological activity.
Albumen particle is also by the carrier of numerous bibliographical information as pharmacological active substance and diagnostic reagent.Wherein, albumin microsphere can be prepared by the method for heat cross-linking and chemical crosslinking.Heat cross-linking microsphere is preparation from emulsifying mixt (as albumin, need wrap the pharmacological active substance and suitable oil phase that carry) at 100 ~ 150 DEG C.Then microsphere is preserved with suitable solvent wash.Leucuta etc. report the preparation method [International Journal of Pharmaceutics Vol. 41:213-217 (1988)] of heat cross-linking microsphere; The preparation of chemical crosslinking microsphere, as document [Science Vol. 213:233-235 (1981)] report, use glutaraldehyde cross-linking albumen, then wash and preserve.
And the preparation method of protein nano grain is difficult to the biological activity keeping albumen in prior art.And the pharmacological active substance being insoluble in water is also difficult to be typically entrapped within protein nano grain, because this method itself relies on the aqueous phase of emulsion to be cross-linked.Water solublity pharmacological active substance can be crosslinked because it is dissolved in protein-contg aqueous phase and enter protein arrays, and poorly water-soluble or fat-soluble pharmacological active substance are difficult to enter in the protein arrays that this method formed.
Summary of the invention
Technical purpose of the present invention is to provide a kind of method of protein nano grain for the preparation of sending pharmacological active substance in body, cause to send in body due to its hydrophobic character to solve some pharmacological active substance, and form the problem of the protein active forfeiture after nanoparticle.
In order to realize technical purpose of the present invention, technical scheme of the present invention is:
For the preparation of the method for protein nano grain of sending pharmacological active substance in body, comprise the following steps: (a) obtains protein solution with the first dissolution with solvents albumen; B pharmacological active substance adds in the protein solution described in step (a) by () in denaturant or applicable Denaturing, albumen is launched and refolding or self assembly, pharmacological active substance is wrapped into albumen, form protein nano grain.
Protein nano grain mean diameter described in 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 technical solution of the present invention can wrap the pharmacological active substance carrying and account for protein nano grain gross weight 1% ~ 40%.
Pharmacological active substance described in technical solution of the present invention is 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.
Pharmacological active substance described in technical solution of the present invention is 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 technical solution of the present invention is albumin, transferrins, insulin, endostatin research, hemoglobin, Myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, fine layer albumen, collagen protein, gelatin, artificial polypeptide and albumen, or their combination.
Wherein, preferred albumen is albumin, transferrins, insulin, endostatin research or hemoglobin.
The first solvent of step (a) described in technical solution of the present invention 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 the first solvent preferred water, phosphate, acetate, acetyltryptophan, sodium caprylate or normal saline.
The operative temperature of the step (a) described in technical solution of the present invention is-20 ~ 100 DEG C; Be preferably 50 ~ 85 DEG C; Optimum is 55 ~ 75 DEG C.
The operation pH value of the step (a) described in technical solution of the present invention is pH3 ~ 9; Be preferably pH5 ~ 8.5; Optimum is pH6 ~ 8.
The denaturant of the step (b) described in technical solution of the present invention or applicable Denaturing 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 applicable Denaturing 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 technical solution of the present invention or applicable Denaturing is pH3 ~ 9; Be preferably pH5.5 ~ 8.5.
Step (b) described in technical solution of the present invention also comprises external force operation and launches with auxilin.
Wherein, described external force comprises transformation temperature, change pressure, applying mechanical force or radiation.
Wherein, described change pressure is the pressure to reaction applying 10 ~ 100,000 psi; Preferably to the pressure of reaction applying 2000 ~ 60,000 psi.
The method of technical scheme of the present invention comprises further: (c) is by the protein nano grain dialysis unnecessary micromolecular compound of removing or concentrate further.
The method of technical scheme of the present invention comprises further: the protein nano grain after dialysis is prepared into pharmaceutical preparation through dehydration by (d).
Wherein, the step of described dehydration is lyophilization, spraying dry or distilling under reduced pressure.
Here is further describing technical solution of the present invention:
Except above-mentioned total technical scheme, present invention further proposes a kind of nanoparticle preparation method for the preparation of sending hydrophobicity pharmacological active substance in body, described method comprises following step: (a) is at-20 ~ 100 DEG C, under the condition of pH3 ~ 9, obtain protein solution with albumen described in the first dissolution with solvents; B hydrophobicity pharmacological active substance, under denaturant or applicable Denaturing, is added the protein solution described in step (a) by (), thus cause albumen to launch and refolding or self assembly, and pharmacological active substance is wrapped into albumen.The nanoparticle mean diameter formed is 5 ~ 500 nm, can wrap the hydrophobicity pharmacological active substance carrying and account for particle gross weight about 1 ~ 40%.Here hydrophobicity pharmacological active substance can 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 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.The first 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 applicable Denaturing 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 DEG C ~ 85 DEG C, and optimum carries out at 55 DEG C ~ 75 DEG C.PH preferably carries out under the condition of 5 ~ 8.5, and optimum carries out under the condition of 6 ~ 8.
Described denaturant or applicable Denaturing 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 applicable Denaturing is 3 ~ 9, is preferably 5.5 ~ 8.5.
Said method comprises further: (c) is by the nanoparticle dialysis unnecessary micromolecular compound of removing or concentrate further.Also comprise further: the nanoparticle after dialysis is prepared into pharmaceutical preparation through dehydration by (d).Wherein, described dehydration is lyophilizing, distilling under reduced pressure or spraying dry.
Technical solution of the present invention further provides a kind of method for the preparation of sending paclitaxel protein nano grain in body again, described method comprises following step: (a) is at 55 ~ 75 DEG C, under the condition of pH6 ~ 8, obtain protein solution with albumen described in the first dissolution with solvents; B paclitaxel, under denaturant or applicable Denaturing, is added the protein solution described in step (a) by (), thus cause albumen to launch and refolding or self assembly, and paclitaxel is wrapped in described albumen; C () is by the nanoparticle dialysis unnecessary micromolecular compound of removing or concentrate further; D () carries out dehydration to gained solution, the obtained pharmaceutical dosage form that can preserve.Here obtained nanoparticle mean diameter is 50 ~ 300 nm, can wrap the pharmacological active substance carrying and account for particle gross weight about 1 ~ 40%.Denaturant in the method for the present embodiment or applicable Denaturing can 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, selects 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 further provides a kind of nanoparticle preparation method Docetaxel being wrapped into albumen and be used for sending in body again, this method comprises following step: (a), at 55 ~ 75 DEG C, obtains protein solution with albumen described in the first solubilize under the condition of pH6 ~ 8; B Docetaxel, under denaturant or applicable Denaturing, is joined the protein solution described in step (a) by (), thus cause expansion and refolding or self assembly, and Docetaxel is wrapped into albumen; C () is by the nanoparticle dialysis unnecessary micromolecular compound of removing or concentrate further; D nanoparticle after dialysis is prepared into pharmaceutical preparation through dehydration by (); Here obtained nanoparticle mean diameter is about 50 ~ 300 nm, can wrap the Docetaxel carrying and account for particle gross weight about 1 ~ 40%.Denaturant in the method for the present embodiment or applicable Denaturing can 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, selects 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 further provides a kind of albumen bag again and carries the nanoparticle of pharmacological active substance for sending in body, the method preparing this nanoparticle comprises following step: (a) is at-20 ~ 100 DEG C, under the condition of pH3 ~ 9, obtain protein solution with albumen described in the first dissolution with solvents; B pharmacological active substance, under denaturant or applicable Denaturing, joins in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmaceutical pack is rolled in described albumen.
Technical solution of the present invention further provides a kind of albumen bag again and carries the nanoparticle of paclitaxel for sending in body, the method preparing this nanoparticle comprises following step: (a) is at 55 ~ 75 DEG C, under the condition of pH6 ~ 8, obtain protein solution with albumen described in the first dissolution with solvents; B paclitaxel, under denaturant or applicable Denaturing, joins in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmaceutical pack is rolled in described albumen; C nanoparticle solution that () dialysis is formed, to remove superfluous material, obtains the solution of high concentration; D () carries out dehydration to gained solution, the obtained pharmaceutical formulation that can preserve.
Technical solution of the present invention further provides a kind of albumen bag again and carries the nanoparticle of Docetaxel for sending in body, the method preparing this nanoparticle comprises following step: (a) is at 55 ~ 75 DEG C, under the condition of pH6 ~ 8, obtain protein solution with albumen described in the first dissolution with solvents; B Docetaxel, under denaturant or applicable Denaturing, joins in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmaceutical pack is rolled in described albumen; C nanoparticle solution that () dialysis is formed, to remove superfluous material, obtains the solution of high concentration; D () carries out dehydration to gained solution, the obtained pharmaceutical formulation that can 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.Protein nano grain average particle size distribution prepared by the present invention is between 5 nm to 2000 nm.Better interval be 25 nm to 500 nm, another better interval is that 50 nm are to 300 nm.And, the nanoparticle prepared by the present invention can in conjunction with up to 40% pharmacological active substance.
Described in the present invention, pharmacological active substance is wrapped into albumen, refer to pharmacological active substance and by the expansion of albumen and refolding, albumen central area can be entered.In general, pharmacological active substance is included in: when dosing a patient with, and can produce any material of pharmacological reaction.Pharmacological active substance in the present invention comprises hydrophobic and hydrophilic compound.Those skilled in the art know that the water solublity of hydrophobicity pharmacological active substance is bad, what hydrophilic pharmacological active substance can be optimized is dissolved in water.Hydrophobic pharmacological active substance comprises following 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 be not only 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, the lyophobic dust with pharmacologically active comprises: antitumor pharmacological active substance 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 more optimized, hydrophobicity pharmacological active substance comprises paclitaxel and Docetaxel.Above-mentioned pharmacological active substance comprises crystal form and amorphous form, and its crystal form comprises with water of crystallization and the form without water of crystallization.
Pharmacological active substance involved in the present invention also comprises hydroaropic substance.
Hydroaropic substance can comprise following compound, but be not only confined to this: cyclophosphamide, bleomycin, daunomycin, epirubicin, methotrexate, 5-fluorouracil and analog, platinum and analog thereof, vinblastine and analog, 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 used in the present invention can change according to the change of the amount of albumen, changes according to the change of the amount of nanoparticle simultaneously.Meanwhile, veteran can recognize the pharmacological active substance used in the present invention, can be pure material, or mixture, and these all do not deviate from scope of the present invention.
In the present invention, different albumen can be selected to go to form the nanoparticle interested to those skilled in the art.Albumen involved in the present invention comprises all can to launch and in conjunction with the albumen of pharmacological active substance or polypeptide.The example of suitable albumen comprises as follows, but is not only confined to this: albumin, transferrins, insulin, endostatin research, hemoglobin, myosin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, lamin, collagen protein, gelatin, man-made protein and their combination.
In the scope that is more optimized, be suitable for albumen of the present invention and comprise as follows: albumin, transferrins, insulin, endostatin research, hemoglobin and their compositions.Those skilled in the art can know that the amount of the albumen used in the inventive method changes [Annalytical Biochemistry Vol. 72:248-254 (1976)] along with the change of the amount of active substance and the amount of nanoparticle.
Step (a) in the inventive method is for obtaining protein solution with albumen described in the first dissolution with solvents.Here protein solution refers to solution and comprises albumen and can proteolytic solvent, after albumen launches, and can refolding or self assembly.The albumen that refolding used herein refers to a unfolding or degeneration refolding can return to the process of suitable three dimensional structure.Self assembly used herein refers to the protein binding of refolding to the process forming nanoparticle together.Those skilled in the art know that the refolding process of albumen can be completed by a lot of condition.The first solution used in protein solution is exemplified below, but is not only 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.
A more accurate scope, the solvent in protein solution comprises water, phosphate, acetate and sodium chloride solution.As long as the concentration of the solvent in the protein solution used in the present invention is applicable to soluble protein and albumen refolding, is all feasible.In general, in protein solution the content range of solvent from 0.001 M to 1.6 M.The scope optimized, from 0.03 M to 1.5 M.The scope optimized again, from 0.05 M to 0.8 M.A more optimal scope, from 0.1 M to 0.3 M.Those skilled in the art can understand that the amount for proteolytic solvent can change according to the change of the amount of albumen and protein solution concentration.
Experiment shows, the response parameter in the step (a) in the present invention, for formation nanoparticle, is very important.In general, obtain a desirable result, the step (a) in the present invention must be reacted between-20 DEG C to 100 DEG C scopes.A more accurate scope is from 50 DEG C to 85 DEG C.A more accurate scope is from 55 DEG C to 75 DEG C.Test verified, obtain a more satisfactory result, in the present invention, the pH of step (a) must between 3 to 9, and a more accurate scope is from 5 to 8.5, is from 6 to 8 a more accurate scope.Those skilled in the art can know that step (a) needs a period of time to dissolve fully to make albumen.In general, the length of time depends on the kind of used albumen, the use amount of albumen, uses the kind of solvent, 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 needs the sufficient time, gives one example, and course of reaction does not need 5 minutes to 8 hours not etc.
Pharmacological active substance joins in the protein solution described in step (a) under being included in denaturant or applicable Denaturing by second step of the present invention and step (b), and albumen is launched and refolding or self assembly.Denaturant used herein or applicable Denaturing refer to energy induced protein or polypeptide changes their three-dimensional or the solution of two-dimensional structure.In general, the denaturant mentioned here or applicable Denaturing can the degeneration of induced protein gentleness.Those skilled in the art fully can recognize that gentle degeneration refers to after albumen unfolding/degeneration, (as used refolding solution) refolding can become suitable structure under certain conditions.Denaturant or applicable Denaturing can provide an environment to destroy the disulfide bond of albumen, form hydrogen bond, with the hydrophobic interaction making water can disturb active site of protein.Those skilled in the art fully can recognize that much solution can serve as denaturant or applicable Denaturing.Denaturant described here or applicable Denaturing draw together water, strong acid, highly basic, inorganic salt, organic solvent, structure developing solvent and surfactant.Suitable denaturant or suitable Denaturing are exemplified below, but be not only 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 applicable Denaturing comprise water, phosphate, acetate, glycine, Tris, ethanol, acetone, hydrogen sulfide, 2 mercapto ethanol, carbamide.Experiment proves, when the pH of denaturant or applicable Denaturing is between 3 ~ 9, the result of acquisition is gratifying.A more accurate pH scope is from 5.5 to 8.5.
In addition, except denaturant, in the present invention, albumen unfolding can also use impressed pressure.In general, impressed pressure comprises the strength that can cause albumen unfolding.Applied force comprises: temperature, and pressure changes, mechanical force, radiation.Applied force comprises from 10 to 100, the pressure of 000 psi, one more suitably scope be from 2000 to 60000 psi.
The present invention may need a step (c) by the nanoparticle dialysis unnecessary micromolecular compound of removing in addition or concentrate further.In general, this step comprises method micromolecule can separated from nanoparticle arbitrarily, and those skilled in the art fully can recognize that the method for separation comprises arbitrarily can the method for purifying protein or 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 a step (d) that the nanoparticle after dialysis is prepared into pharmaceutical preparation through dehydration.In general, the method for protection comprises to nanoparticle dehydration to facilitate storage and transport, so that obtains suitable dosage form.The method of protection involved in the present invention comprises: centrifugal, drying under reduced pressure, lyophilizing, spraying dry.
Those skilled in the art will appreciate that scope of the present invention and marrow are variations.The material of unfolding is change, and many pharmacological active substancies are spendable simultaneously, and many albumen and the polypeptide of synthesis also can be used as carrier.The present invention will obtain definitely and clearly describing in the following embodiments.
Beneficial effect of the present invention is:
First, the nanoparticle formed through method provided by the invention can reach the envelop rate of 90%, exceedes prior art, defines a kind of efficient method; Secondly, method provided by the present invention can obtain the drug loading being up to 40%, namely contains the pharmacological active substance of 40% in nanoparticle.Owing to there being higher drug loading, when treating, less medication volume can be obtained, shorter administration time, more convenient to patient.Higher drug loading reduces the use amount of albumen when sending pharmacological active substance, improves the cost efficiency of product; Finally, the lower Drug loading capacity that prior art provides can not meet high dose administration, because the administration of high dose needs very large administration volume.But the nanoparticle with high Drug loading capacity of this experiment invention can not by the restriction of administration volume.
Another beneficial effect of the nanoparticle prepared by the present invention is, nanoparticle specificly can send pharmacological active substance.The nanoparticle prepared by the present invention can some organs of targeting and system.Such as, when with albumin as carrier time, by changing the size of nanoparticle, targeting liver, lung, spleen or lymphsystem etc.When with transferrins or insulin as carrier time, pharmacological active substance, at cell surface high expressed, by the affinity of carrier and tumor cell surface, specificly can be transported to tumor tissues by their receptor.When carrier is endostatin research time, its receptor is distributed in vascular cell, and owing to there is the cause of a large amount of blood vessels in tumor tissues, what nanoparticle can be a large amount of accumulates in tumor tissues.In a word, different protein carriers can the different tissue of targeting or organ.Therefore can say, the invention provides a kind of efficient method to carry pharmacological active substance to the different parts of health.
Accompanying drawing explanation
Fig. 1 is the impact (15% dispensing, medicine/albumen) of different pH on nanoparticle size in the present invention
Fig. 2 is the grain size distribution of ABI-007 nanoparticle in the present invention
Fig. 3 is the transmission electron microscope image (drug loading 10.59%) of ABI-007 nanoparticle in the present invention
Fig. 4 is the high-resolution TEM photo of paclitaxel-albumin nano granular
Wherein, (a) ABI-007 nanoparticle; The albumin nano granular of b zero load that () amplifies; C free paclitaxel that () amplifies; D ABI-007 nanoparticle that () amplifies
Fig. 5 is the X-ray powder diffraction image of ABI-007 nanoparticle
Wherein, (a) paclitaxel; B albumin nano granular that () is blank; (c) paclitaxel-albumin nano granular (drug loading 12.9%); The physical mixture (12.9%) of (d) albumin and paclitaxel
Fig. 6 is the redispersion of ABI-007 nanoparticle and medicine Abraxane
Wherein, (a) 2 mg/mL ABI-007 nanoparticle solution; (b) 2 mg/mL Abraxane solution; (c) 20 mg/mL ABI-007 nanoparticle solution; (d) 20 mg/mL Abraxane solution; (e) 50 mg/mL ABI-007 nanoparticle solution; (f) 50 mg/mL Abraxane solution.
Detailed description of the invention
Below be all based on representative embodiment of the present invention, but following embodiment can not in office where face limit the scope of the invention.
the preparation of embodiment 1. paclitaxels-albumin nano granular
100 mgHSA are dissolved in the phosphate buffer containing 0.5 mg/mL EDTA and 0.05 M mercaptoethanol of 10 mL pH6,55 DEG C of reactions continue two hours, terminate after with 5% trichloroacetic acid precipitation and wash albumen, add paclitaxel (dissolve with ethanol) solution of 1.6 mL10 mg/mL in precipitation, mix 2 minutes, the phosphate buffer adding 0.08 M of 50 mL stirs, dissolving mixt.The suspension obtained is transparent, and the mean diameter of medicine carrying particle is 80 ~ 200 nm, (BIC 90plus Particle Size Analyzer).Measure the envelop rate of paclitaxel with anti-phase C18 post, mobile phase is acetonitrile: water (60:40), determined wavelength 227 nm.HPLC analyzes and shows that the paclitaxel envelop rate of this experiment reaches more than 90%.
In experiment, trichloroacetic acid can also replace with other denaturants, such as water, strong acid (hydrochloric acid and sulphuric acid), highly basic (sodium hydroxide), hydrogen peroxide, glutathion etc.Found that, the trichloroacetic acid of 5% 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 pH7.4,37 DEG C of water-baths, add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, adds the paclitaxel (dissolve with ethanol) of 2 mL 10 mg/mL.After 30 minutes, the TRIS buffer of sample pH7.4 is dialysed 24 hours, gained sample lyophilizing 48 hours.It is original solution that bulk sample after obtained freeze-drying can redissolve with water or normal saline easily, and nanoparticle particle diameter remains unchanged.After lyophilizing, particle diameter is mainly distributed in 80 ~ 200 nm (BIC 90plus Particle Size Analyzer), and HPLC analyzes and shows 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 7.5 mL, 0 DEG C of pre-cooling, continue ice bath 1 hour.Centrifugal, collecting precipitation, in precipitation, add the paclitaxel (acetone solution) of 1 mL 10 mg/mL, ultrasonic mixing, adds 50 mL normal saline, magnetic agitation, forms suspension.Carry out granularmetric analysis with BIC 90plus Particle Size Analyzer, result is 150 ~ 220 nm.The sample of lyophilizing can redissolve, and it is 8.34% that HPLC analyzes drug loading.
Appended experimental shows glycine, mannitol, and lactose and trehalose all can as freeze drying protectants, and it is minimum to do with lactose the particle diameter that freeze drying protectant obtains.
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 different pH on the impact of particle diameter and drug loading, result shows that pH is better 8.0.But pH again can not more than 8.5, because pharmacological active substance can decompose when pH is more than 8.5.
the preparation of embodiment 4. paclitaxels-transferrins nanoparticle
100 mg transferrinss are dissolved in the TRIS buffer of 50 mL pH7.4,75 DEG C of stirrings, add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, slowly adds 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 impact of different temperatures on particle diameter and drug loading, and result shows that temperature all can form nanoparticle between 0 DEG C to 100 DEG C, better at 55 ~ 75 DEG C of nanoparticles formed.
the preparation of embodiment 5. Docetaxels-transferrins nanoparticle
100 mg transferrinss are dissolved in the TRIS buffer of 50 mL pH7.4,65 DEG C of stirrings, add 350 μ L 2 mercapto ethanols, and reaction continues 10 minutes, adds 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 with the use of, better particle size results can be obtained, 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 mix as denaturant with 2 mercapto ethanol, particle diameter distribution is narrower, and 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 10 mL pure water.55 DEG C of stirrings, gained solution and isopyknic 5% solution of trichloroacetic acid mixing and centrifugal, removing supernatant.The alcoholic solution added containing gamlogic acid mixes, and then adds the TRIS of 50 mL pH8.0, stirs until albumen precipitation dissolves completely.The nanoparticle particle diameter of gained is 110 nm(BIC 90plus Particle Size Analyzer).
the preparation of embodiment 7. paclitaxels-further grain
Under-20 DEG C 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 strong stirring 15 minutes, then centrifugal 15 minutes.After treating that remaining acetone evaporated is fallen, collecting precipitation, is dissolved in cold deionized water, dialyses 5 hours under 2 DEG C of conditions with deionized water, then uses 0.0016 M NaHCO
3dialyse 30 hours, filter 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 DEG C of conditions, add the alcoholic solution of 0.7 mL containing 10 mg/mL paclitaxels, stir until form light blue solution.Gained average particle size is 308.1 nm(BIC 90plus Particle Size Analyzer).
embodiment 8. pH investigates the impact of nanoparticle particle diameter
The present invention has investigated the phosphate buffer of different pH and they are on the impact of nanoparticle particle diameter.All add the paclitaxel (10 mg/mL) of 15% in all protein solution groups, particle diameter measures under constant temperature, and result as shown in Figure 1.
the dynamic light scattering (dynamic light scaterring, DLS) of embodiment 9. paclitaxels-albumin nano granular is analyzed
Carry out granularmetric analysis to the paclitaxel-albumin nano granular prepared according to method of the present invention, instrument is BIC 90plus Particle Size Analyzer.As shown in Figure 4, mean diameter is 121 nm to result, and particle size distribution is in narrower scope.
the transmission electron microscope (Transmission Electron Microscopy, TEM) of embodiment 10. paclitaxels-albumin nano granular characterizes
According to the present invention prepare drug loading be 10.59% paclitaxel-albumin nano granular carry out the analysis of transmission electron microscope.Instrument is EM 2100 type 200KV high resolution TEM (Japan).As shown in Figure 2, display nanoparticle is spherical in shape for result.
the stability study of paclitaxel-albumin nano granular that embodiment 11. is redissolved
The paclitaxel that lyophilizing obtains-albumin block normal saline, the glucose of 5% and calf serum are dissolved into the solution that paclitaxel concentration is 5 mg/mL, 5 mg/mL and 2 mg/mL respectively.Respectively after 25 DEG C and 37 DEG C place 12 hours, the mean diameter of nanoparticle does not change (DLS, BIC 90plus Particle Size Analyzer), and does not precipitate generation.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 preparing paclitaxel-transferrins nanoparticle, find except TRIS buffer, normal saline also can be used for dissolving transferrins.In addition, be 3 ~ 9 at pH, particularly can obtain the nanoparticle of high drug load and high stability in 6 ~ 8 scopes.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. is redissolved
The paclitaxel that lyophilizing obtains--transferrins block normal saline, the glucose of 5% and calf serum are dissolved into the solution that paclitaxel concentration is 5 mg/mL, 5 mg/mL and 2 mg/mL respectively.Respectively after 25 DEG C and 37 DEG C place 12 hours, the mean diameter of nanoparticle does not change (DLS, BIC 90plus Particle Size Analyzer), and does not precipitate generation.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 |
the sign that embodiment 14. is carried out paclitaxel-albumin nano granular with transmission electron microscope (Transmission Electron Microscopy, TEM)
2 ~ 3 paclitaxel prepared according to the present invention-albumin nano granular solution are dropped on the copper mesh being coated with carbon supporting film (200 order), unnecessary solution is blotted with filter paper after 2 minutes, then copper mesh is placed on air drying, analyzes with EM-2100 200 KV type high resolution TEM (Japan).
Result is as shown in Figure 4: a) paclitaxel-albumin nano granular is spherical in shape; B) blank albumin nano granular presents irregular shape, and mean diameter is at about 100 nm; C) free paclitaxel presents the higher electron density in place in centre, around surrounded by club shaped structure; D) paclitaxel-albumin nano granular presents nucleocapsid structure.
the X-ray powder diffraction (x-ray powder diffraction, XRD) of embodiment 15. paclitaxels-albumin nano granular characterizes
Paclitaxel in aqueous will crystallization more than during 1 mg/mL.Therefore, the paclitaxel of amorphous state is very beneficial for injection.In order to detect the solid forms of paclitaxel in paclitaxel-albumin nano granular in the present invention, we used X-ray powder diffraction to characterize.Four samples are 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 angle measurement range of each sample is from 5 degree to 50 degree (ARL, X'TRA, Applied Research Laboratories, Switzerland).
Result is as shown in Figure 5: figure a) shows the characteristic peak of paclitaxel crystal; At 2 θ angles be 15 degree within the scope of 45 degree, figure b) show albuminous halo type peak; Figure c) show at 2 θ angles be 15 degree within the scope of 45 degree, also show albuminous halo type peak, illustrate that paclitaxel exists with unbodied state in nanoparticle; With the paclitaxel and albuminous physical mixture of nanoparticle same ratio (12.9%), scheme d) to show the paclitaxel of crystal state and the albuminous existence of amorphous state.Therefore, we can obtain conclusion: in paclitaxel-albumin nano prepared by the present invention, paclitaxel exists with amorphous state.
the redispersion research of embodiment 16. paclitaxels-albumin nano granular
Sample after the paclitaxel prepared according to the present invention-albumin nano granular lyophilizing and business-like pharmacological active substance Abraxane are by following protein concentration water dissolution: (a, b) 2 mg/mL; (c, d) 20 mg/mL; (e, f) 50 mg/mL.The photo of these samples as shown in Figure 6, all can obtain the colloid solution of stable transparent paclitaxel-albumin nano granular in three samples.
the preparation of embodiment 17. irinotecans-insulin nanoparticles
100 mg insulins are dissolved in 10 mL water or normal saline, and 65 DEG C of stirrings, terminate rear methanol extraction and wash albumen, add the irinotecan solution of 1.6 mL10 mg/mL in precipitation, mix 2 minutes, the sodium chloride solution adding 50 mL stirs, dissolving mixt.The largest diameter of the medicine carrying particle obtained is 2000 nm, and minimum is 5 nm, and the scope of main distribution is at 50 ~ 300 nm, and part nanoparticle is distributed in 2000 nm (BIC 90plus Particle Size Analyzer).With the envelop rate that anti-phase C18 post measures, mobile phase is acetonitrile: water (60:40), determined wavelength 227 nm.HPLC analyzes and shows 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 DEG C ~ 100 DEG C in reaction temperature, particularly can obtain the nanoparticle of high drug load and high stability within the scope of 55 ~ 75 DEG C.The protein nano grain obtained can obtain dryness powder by spray-dired mode.
the preparation of embodiment 18. 5-fluorouracil-endostatin research nanoparticle
100 mg endostatin researchs be dissolved in 10 mL pH 9 5% glucose contained solution in, 75 DEG C 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 continue two hours.The TRIS buffer of sample pH7.4 is dialysed 24 hours, and after gained sample carries out spraying dry, nanoparticle particle diameter remains unchanged.Particle diameter is mainly distributed in 25 ~ 500 nm (BIC 90plus Particle Size Analyzer), and drug loading reaches 40%.
Wherein, it is any one in freeze drying protectant that the glucose of 5% can change into, as phosphate, and acetate, glycine, Tris.Glutathion can be any one in 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.Result shows, and the size of solution on final particle of solubilising protein does not affect.In denaturant or applicable Denaturing, the effect of 2 mercapto ethanol is best, and the nanoparticle narrow distribution obtained, between 50 ~ 300 nm.
the preparation of embodiment 19. 5-fluorouracil-paclitaxel-endostatin research nanoparticle
100 mg endostatin researchs be dissolved in 10 mL pH7.4 5% trehalose contained solution in, 25 DEG C of water-baths, add the mercaptoethanol of 25 mL 0.5 mg/mL and the carbamide of 6 M, react 10 minutes, add 5-fluorouracil and the 0.8 mL10 mg/mL paclitaxel of 0.8 mL10 mg/mL, Keep agitation, solution is light blue.Particle diameter is mainly distributed in 25 ~ 500 nm (BIC 90plus Particle Size Analyzer).
Wherein, it is any one in protein stabiliser that the glucose of 5% can change into, as mannitol, and sucrose, acetyltryptophan, sodium caprylate.Result shows, and the size of solution on final particle of solubilising protein does not affect, and the nanoparticle narrow distribution obtained, between 50 ~ 300 nm.
the preparation of embodiment 20. carmustines-Myoglobin nanoparticle
100 mg Myoglobin are dissolved in the phosphate buffer containing 0.5 mg/mL sodium caprylate and 0.05 M acetyltryptophan of 10 mL pH 3, and ice bath 30 minutes drips acetone 7.5 mL of 0 DEG C of pre-cooling.Continue ice bath 1 hour, centrifugal, collecting precipitation, in precipitation, add the carmustine solution of 1.6 mL 10 mg/mL, ultrasonic mixing, adds 50 mL Tris buffer, magnetic agitation, the suspension obtained is transparent, and solution obtains through distilling under reduced pressure the particle (BIC 90plus Particle Size Analyzer) that mean diameter is 80 ~ 200 nm
Wherein Denaturing 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.Result shows, if Denaturing with an organic solvent, 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 DEG C of stirrings, pass into 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).
Tris buffer can change any one in protein stabiliser into, as trehalose, and mannitol, sucrose, acetyltryptophan, sodium caprylate or their combination.
the preparation of embodiment 22. phenesterins-immunoglobulin nanoparticle
The 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. piposulfan-α-2-macroglobulin nanoparticle
The present embodiment is identical with embodiment 5, and only changing Docetaxel is piposulfan, and transferrins is α-2-macroglobulin, and reaction temperature is 65 DEG C.Particle size distribution 25 nm ~ 500 nm, drug loading 11.2%.
the preparation of embodiment 24. tamoxifens-fibronectin nanoparticle
The present embodiment is identical with embodiment 5, and only changing Docetaxel is tamoxifen, and transferrins is fibronectin.Reaction temperature 45 DEG C, particle size distribution 50 nm ~ 200 nm, drug loading 9.8%.
the preparation of embodiment 25. lomustine-fine layer protein nano grain
The present embodiment is identical with embodiment 5, and only changing Docetaxel is lomustine, and transferrins is fine layer albumen.Reaction temperature is 75 DEG C, particle size distribution between 100 nm ~ 200 nm, drug loading 35%.
the preparation of embodiment 26. rubescensine A-collagen protein nanoparticle
The present embodiment is identical with embodiment 5, and only changing Docetaxel is rubescensine A, and transferrins is collagen protein.Reaction pH be 8.0, particle size distribution between 300 nm ~ 600 nm, drug loading 22%.
the preparation of embodiment 27. podophyllotoxins-gelatin nanparticles
The present embodiment is identical with embodiment 5, and only changing Docetaxel is podophyllotoxin, and transferrins is gelatin.Reaction temperature be 70 DEG C, 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 10 mL normal saline, containing 0.5 mg/mL sodium caprylate and 0.05 M acetyltryptophan, and 55 DEG C 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 until solution is light blue, 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
The present embodiment is identical with embodiment 28, and only changing atorvastatin is simvastatin, and albumin is transferrins.Gained nano particle diameter is 50 ~ 300 nm, and drug loading is 5%.
the preparation of embodiment 30. Fenofibrates-further grain
The present embodiment is identical with embodiment 28, and only changing atorvastatin is Fenofibrate, and albumin is hemoglobin.Gained nano particle diameter is 30 ~ 300 nm, and drug loading is 11%.
the preparation of embodiment 31. nifedipines-endostatin research nanoparticle
The present embodiment is identical with embodiment 28, and only changing atorvastatin is nifedipine, and albumin is endostatin research.Gained nano particle diameter is 20 ~ 250 nm, and drug loading is 9.7%.
the preparation of embodiment 32. ibuprofen-albumin nano granular
The present embodiment is identical with embodiment 28, and only changing atorvastatin is ibuprofen.Gained nano particle diameter is 20 ~ 250 nm, and drug loading is 8.7%.
the preparation of embodiment 33. indomethacins-collagen protein nanoparticle
The 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
The 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
The 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
The 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
The 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
The 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
The 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
The 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
The 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%.
the preparation of embodiment 42. ritonavirs-insulin nanoparticles
The 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
The 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
The 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
The 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
The 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
The 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
The present embodiment is identical with embodiment 28, and only changing atorvastatin is MTX.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
The 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
The 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
The 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
The 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
The 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
The 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 (18)
1., for the preparation of the method for protein nano grain of sending pharmacological active substance in body, comprise the following steps:
A () obtains protein solution with the first dissolution with solvents albumen; Described albumen is albumin, transferrins, insulin, endostatin research, hemoglobin, Myoglobin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, fine layer albumen, collagen protein, artificial polypeptide and albumen or their combination;
The operative temperature of step (a) is 50 ~ 85 DEG C;
The operation pH value of step (a) is pH5 ~ 8.5;
B pharmacological active substance, under denaturant or applicable Denaturing, adds in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmacological active substance is wrapped into albumen, form protein nano grain;
Described pharmacological active substance is hydrophobicity pharmacological active substance, 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, Lopinavir, or their combination, when the albumen described in step (a) is albumin, above-mentioned pharmacological active substance does not comprise paclitaxel or Docetaxel,
Or described pharmacological active substance is hydrophilic pharmacological active substance; 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;
Wherein, the denaturant in described step (b) or applicable Denaturing are strong acid, highly basic, organic solvent, structure developing solvent or surfactant;
Wherein, described organic solvent is methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone or their combination.
2. method according to claim 1, is characterized in that the mean diameter of described protein nano grain is 25 nm ~ 500 nm.
3. method according to claim 1, is characterized in that the mean diameter of described protein nano grain is 50 nm ~ 300 nm.
4. method according to claim 1, is characterized in that the weight ratio shared by described pharmacological active substance is in nanoparticle is 1% ~ 40%.
5. method according to claim 1, is characterized in that the first solvent of described step (a) is water, normal saline, phosphate buffer, acetum, glycine solution, TRIS buffer, hydrogen peroxide, glutathion aqueous solution, glucose solution, aqueous trehalose, mannitol solution, sucrose solution, acetyltryptophan solution, sodium caprylate solution or their combination.
6. method according to claim 5, is characterized in that the first described solvent is phosphate buffer, acetum, glycine solution, TRIS buffer, glucose solution or their combination.
7. method according to claim 5, is characterized in that the first described solvent is aqueous trehalose, mannitol solution, sucrose solution, acetyltryptophan solution, sodium caprylate solution or their combination.
8. method according to claim 1, is characterized in that the operative temperature of described step (a) is 55 ~ 75 DEG C.
9. method according to claim 1, is characterized in that described structure developing solvent is 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine, trichloroacetic acid or their combination.
10. method according to claim 1, is characterized in that described denaturant or applicable Denaturing are glycine, Tris, glucose, ethanol, acetone, 2 mercapto ethanol, carbamide or their combination.
11. methods according to claim 1, is characterized in that described method comprises further: (c) is by the nanoparticle dialysis unnecessary micromolecular compound of removing or concentrate further.
12. methods according to claim 11, is characterized in that described method comprises further: the nanoparticle after dialysis is prepared into pharmaceutical preparation through dehydration by (d).
13. methods according to claim 12, is characterized in that described dehydration is lyophilizing, distilling under reduced pressure or spraying dry.
14. 1 kinds, for the preparation of the method for protein nano grain of sending lyophobic dust in body, comprise the following steps:
A (), at 50 ~ 85 DEG C, under the condition of pH5 ~ 8.5, obtains protein solution with albumen described in the first dissolution with solvents; B pharmacological active substance, under denaturant or suitable Denaturing, joins in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmaceutical pack is rolled in 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 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 or their combination,
Described albumen is albumin, transferrins, insulin, endostatin research, hemoglobin, myosin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, lamin, collagen protein, man-made protein or their combination; When described albumen is albumin, described hydrophobicity pharmacological active substance does not comprise paclitaxel or Docetaxel;
The first described solvent is water, normal saline, phosphate buffer, acetum, glycine solution, TRIS buffer, hydrogen peroxide, glutathion aqueous solution, glucose solution, aqueous trehalose, mannitol solution, sucrose solution, acetyltryptophan solution, sodium caprylate solution or their combination;
Described denaturant or suitable Denaturing glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, isopropyl alcohol, formalin, acetone, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine, trichloroacetic acid or their combination.
15. methods according to claim 14, is characterized in that described method comprises further:
C () is by the nanoparticle dialysis unnecessary micromolecular compound of removing or concentrate further.
16. methods according to claim 15, is characterized in that described method comprises further:
D nanoparticle after dialysis is prepared into pharmaceutical preparation through dehydration by ().
17. methods according to claim 16, is characterized in that described dehydration is lyophilizing, distilling under reduced pressure or spraying dry.
18. 1 kinds of protein nano grains including pharmacological active substance, for vivo drug delivery; It is characterized in that the preparation process of described nanoparticle by the following method:
A (), at 50 ~ 85 DEG C, under the condition of pH5 ~ 8.5, obtains protein solution with albumen described in the first dissolution with solvents;
B pharmacological active substance, under denaturant or applicable Denaturing, joins in the protein solution described in step (a) by (), albumen is launched and refolding or self assembly, pharmaceutical pack is rolled in 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 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, or their combination,
Described albumen is albumin, transferrins, insulin, endostatin research, hemoglobin, myosin, lysozyme, immunoglobulin, α-2-macroglobulin, fibronectin, lamin, collagen protein, man-made protein or their combination; When described albumen is albumin, described pharmacological active substance does not comprise paclitaxel or Docetaxel;
The first described solvent is water, normal saline, phosphate buffer, acetum, glycine solution, TRIS buffer, hydrogen peroxide, glutathion aqueous solution, glucose solution, aqueous trehalose, mannitol solution, sucrose solution, acetyltryptophan solution, sodium caprylate solution or their combination;
Described denaturant or applicable Denaturing are glycine, Tris, hydrogen peroxide, glutathion, methanol, ethanol, propanol, isopropyl alcohol, formalin, acetone, 2 mercapto ethanol, dithiothreitol, DTT, guanidine hydrochloride, carbamide, perchloric acid, tributylphosphine, Capoten, performic acid, penicillamine, glutathion, methimazole, acetylcysteine or their combination.
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| CN104306339A (en) * | 2014-10-22 | 2015-01-28 | 南京大学 | Thymidylate synthase preparation loaded albumin nano-microsphere and preparation method thereof |
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| CN107157950B (en) * | 2016-03-07 | 2021-05-25 | 中国科学院上海药物研究所 | Albumin nanoparticles and preparation method and application thereof |
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| CN108114273B (en) * | 2018-02-02 | 2020-12-22 | 南京大学 | Perfluorocarbon albumin nanoparticles and preparation method and application thereof |
| CN111202849B (en) * | 2018-11-02 | 2022-06-03 | 四川大学 | Albumin-bound nitrosourea antitumor pharmaceutical preparation and preparation method thereof |
| CN109806404B (en) * | 2019-04-03 | 2023-08-29 | 南京从一医药科技有限公司 | Albumin nanoparticle capable of achieving two-stage oxygen delivery and preparation method and application thereof |
| CN113425700B (en) * | 2021-06-28 | 2022-08-23 | 中山大学 | Construction and application of nano-drug delivery system based on protein self-assembly |
| CN114948784A (en) * | 2022-04-20 | 2022-08-30 | 重庆医科大学 | Bionic nano-particles and preparation method and application thereof |
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