CN101648022A - Nanometer microsphere or hollow sphere of casein or casein-polyacrylic acid compound and preparation method and application thereof - Google Patents
Nanometer microsphere or hollow sphere of casein or casein-polyacrylic acid compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a nanometer microsphere or a nanometer hollow sphere of casein or casein-polyacrylic acid compound, and a nanometer microsphere or a nanometer hollow sphere of cross-linking casein, wherein the nanometer microsphere or the nanometer hollow sphere of the casein or casein-polyacrylic acid compound consists of a compound of casein and polyacrylic acid, and the average grain diameter of the nanometer microsphere or the nanometer hollow sphere is 170-370nm; the nanometer microsphere or the nanometer hollow sphere of cross-linking casein consists of cross-linking casein, andthe average grain diameter of the nanometer microsphere or the nanometer hollow sphere is 240-500nm. The nanometer microsphere or the nanometer hollow sphere of cross-linking casein has good biocompatibility and biodegradability without causing organism immunoreaction; degradation products of the nanometer microsphere or the nanometer hollow sphere of cross-linking casein can be absorbed and metabolized by a human body and has the treatment action, nutritive efficiency and the like on the human body, and therefore, the nanometer microsphere or the nanometer hollow sphere of cross-linking casein can be used as a carrier of drugs. The invention discloses a preparation method of the nanometer microsphere or the nanometer hollow sphere of the casein or casein-polyacrylic acid compound, and apreparation method of the nanometer microsphere or the nanometer hollow sphere of cross-linking casein.
Description
Technical field
The present invention relates to casein nano material and method for making thereof and as the application in the pharmaceutical carrier
Background technology
Delivery system combines medicine or other bioactive substance and carrier material exactly and makes medicine pass through modes such as diffusion in the regular hour, be discharged in the environment or be transported to the particular target tissue with a certain speed, to body health generation effect, mainly constitute by medicine and carrier two parts.Nano-medicament carrier means dissolving or is dispersed with the various nanoparticles of medicine, as nanometer liposome, polymer nanocomposite capsule, nanosphere, nano-micelle etc., made by natural or synthesized polymer material usually.The nano-medicament carrier preparation method is more, and wherein commonly used have cross-linking method, precipitation/coacervation, solvent evaporated method, spray drying method, porous centrifuging, salting out method and a ultrasonic method etc.Using these technology, to prepare microsphere be to realize that according to the different physical and chemical performances of material as temperature sensitivity, dissolubility etc., the particle size of preparation does not wait to the hundreds of micron from tens nanometers.The outstanding advantage of nano-medicament carrier is: the targeted medicine is promptly controlled medicine and is entered specific target organ or target cell; Slow releasing pharmaceutical prolong drug action time; Improve stability of formulation and oral administration biaavailability; Carrier material is biodegradable and have a biocompatibility.
Macromolecular material as drug release carrier, need have biocompatibility and biological degradability, can degradation in vivo be micromolecular compounds such as water, carbon dioxide just, thereby by organism metabolism, absorption or drainage, human body is had no side effect, and degradation process take place the time confidential suitable.The biological degradable medicine slow-releasing of developing at present mainly is synthetic high polymer and natural polymer with macromolecular material.It is natural and modified natural is biological that synthetic high polymer mainly contains polylactic acid, poly-anhydride, amino acids polymer, poly phosphate, poly phosphazene, Merlon etc.Natural polymer has polysaccharide (as chitosan, pectic substance, hyaluronic acid, cyclodextrin, cellulose, sodium alginate, amylose etc.) and protein-based (as collagen, gelatin, albumin etc.).
The research of material in modern medicinal agents biodegradable and that have a biocompatibility is used, develop novel pharmaceutical formulation with special efficacy, alleviate patient's misery, improving and bringing into play more and more heavier effect in the quality of life, becoming the research focus of present material science and pharmaceutics.Biodegradable high-molecular is very extensive in the research of medical domain, but for body, it is foreign body after all, when macromolecule contacts with organism, if the material biocompatibility is not good enough, organism just shows the instinct of repelling foreign body, inflammation, allergy etc. can occur.When blood contacted with the polymeric biomaterial surface, various plasma proteinss can be adsorbed on the foreign body surface to some extent very soon with the material surface different in kind, cause hematoblastic adhesion subsequently, and haemolysis or blood coagulation may take place, thereby formed thrombosis.Therefore, the selection of plasma proteins absorption has reflected the blood compatibility of biomaterial.Most of biological degradation polyalcohols, especially synthetic macromolecule, it is very slow to degrade, catabolite in vivo the holdup time long, and synthetic high polymer is generally pressed repetitive, regular connection, easier crystallization not too is beneficial to the effect of enzyme.In addition on the surface, as if natural polymer is more suitable in as bio-medical material because its structure and tissue are approaching more.Yet there are many deadly defects in natural polymer, is subjected to the bad mechanical property that human body repels, is difficult to estimate its vivo degradation speed, natural polymer too by force as physiologically active.Because these shortcomings have limited the application of natural polymer.
Albumen in the milk is mainly casein, lactalbumin and immunoglobulin, and wherein casein (casein) accounts for about 80% of milk gross protein, contain aminoacid and a large amount of immunoglobulin of whole needed by human body, be white in color amorphous powder or granule have hygroscopicity.Be dissolved in diluted alkaline and concentrated acid, water insoluble and organic solvent.Casein is not single protein, but is become with more proximate protein group in nature in structure by a class, comprises α s1-casein, α s2-casein, beta-casein and κ-casein, molecular weight are in the scope of 19000-25000Da, and the average isoelectric point, IP of these mixture is about 4.7.They all are catenate amphipathic protein, because the construction features of casein molecule, it can be regarded as the block type copolymer that is made of hydrophilic amino acid and hydrophobic amino acid.The primary structure of these four kinds of compositions all is determined, and main feature is that high-load phosphoserine residue and considerable proline are arranged.Phosphoserine residue structure is relevant to the binding ability of calcium ion with casein, also caseic character and function is had great importance.Beta-casein in the milk has stronger anti-mutagen mechanism, can reduce canceration.As drug carrier material, mostly the casein microsphere is to adopt glutaraldehyde as cross linker to be prepared from (macromolecule circular, 2006. (8) .1-9).
Summary of the invention
Have excellent biological compatibility and biodegradability based on casein, be difficult for causing organism immune response, the metabolism that not only can be absorbed by the body of its catabolite, and human body had characteristics such as therapeutical effect and nutritive effect, the present invention as the pharmaceutical carrier drug loading, particularly loads cancer therapy drugs such as cisplatin with the nanosphere of casein-polyacrylic acid composite or hollow nano-sphere and pure caseic nanosphere thereof or hollow nano-sphere.
The objective of the invention is:
1. a kind of nanosphere or hollow nano-sphere of the biodegradable and good casein-polyacrylic acid composite of biocompatibility are provided.
Above-mentioned nanoparticle is crosslinked with glutaraldehyde or calcium salt 2., as if being dialysed, this Nano microsphere can obtain the nanosphere or the hollow nano-sphere of crosslinked casein-polyacrylic acid composite then in the water of pH=3; As if being dialysed, this Nano microsphere can obtain pure caseic nanosphere or hollow nano-sphere in the water of pH=7.
3. a kind of method for making of above-mentioned various Nano microspheres is provided.
4. above-mentioned various Nano microspheres are loaded cancer therapy drugs such as cisplatin as pharmaceutical carrier.
Technical scheme of the present invention is as follows:
A kind of Nano microsphere of casein-polyacrylic acid composite or nano-hollow ball, it is made up of casein and polyacrylic acid compound, the mean diameter of Nano microsphere or nano-hollow ball is respectively 170-480nm and 360-500nm, and wherein polyacrylic percentage composition is 75%-85% in the complex microsphere.Change preparation condition, it is controlled to obtain particle diameter, the Nano microsphere of casein-polyacrylic acid composite that pattern is adjustable or nano-hollow ball.
Above-mentioned Nano microsphere can be crosslinked with glutaraldehyde or calcium salt, obtains the Nano microsphere or the nano-hollow ball of crosslinked casein-polyacrylic acid composite all stable under acid and alkaline environment.
A kind of crosslinked caseic Nano microsphere or nano-hollow ball, it is made up of crosslinked casein, and the mean diameter of Nano microsphere or nano-hollow ball is respectively 240-480nm and 490-500nm.
The Nano microsphere of above-mentioned casein-polyacrylic acid composite or nano-hollow ball or crosslinked caseic Nano microsphere or nano-hollow ball, described casein can also be α s1-casein or beta-casein.
A kind of method for making of Nano microsphere of above-mentioned casein-polyacrylic acid composite, it comprises the steps:
Step 1. adds the 50ml distilled water in the there-necked flask of 100ml, 0.1 gram casein and 0.5-0.75 gram acrylic acid stir, and are heated to 70-90 ℃ then,
After step 2. treated that casein all dissolves, logical nitrogen protection after the whole eliminatings of the air in the system, added 0.0125-0.0187 and restrains potassium peroxydisulfate, and along with the carrying out of reaction, the system color gradually becomes light blue until newborn blueness from colourless,
Step 3. removes thermal source, stops to stir, cools off, filters, do the dialysis of dialysis medium with the water of pH=3, obtain the Nano microsphere that mean diameter is casein-polyacrylic acid composite of 170-320nm, wherein polyacrylic content percentage composition is 75%-85% in the complex microsphere.It can be placed in 4 ℃ of refrigerators and preserve.
The method for making of the Nano microsphere of above-mentioned casein-polyacrylic acid composite in the described step 1, can be added 0.25-0.5 gram propanoic acid, makes the quality that adds potassium peroxydisulfate in the step 2 and acrylic acid mass ratio smaller or equal to 0.025 simultaneously.Adding propanoic acid mainly is in order to improve caseic water solublity.
A kind of method for making of Nano microsphere of above-mentioned crosslinked casein-polyacrylic acid composite, it is that the Nano microsphere of the above-mentioned casein-polyacrylic acid composite that makes is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or calcium salt are crosslinked, filtered then in 24 hours 40 ℃ of reactions, dialysis obtains the Nano microsphere of crosslinked casein-polyacrylic acid composite in the water of pH=3.
The method for making of the Nano microsphere of above-mentioned crosslinked casein-polyacrylic acid composite, described calcium salt can be calcium carbonate, calcium sulfate or calcium phosphate.
A kind of method for making of above-mentioned crosslinked casein Nano microsphere, it is that the Nano microsphere of the above-mentioned casein-polyacrylic acid composite that makes is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, 40 ℃ of reactions 24 hours, filter then, nano microsphere system after crosslinked is regulated pH to 7, and dialysis obtains crosslinked casein Nano microsphere in the water of pH=7 again.
A kind of method for making of nano-hollow ball of above-mentioned casein-polyacrylic acid composite, it comprises the steps:
Step 1 '. add the 50ml distilled water in the there-necked flask of 100ml, 0.1 gram casein, 0.5-0.75 gram acrylic acid and 0.2-0.4 gram propanoic acid stir, and are heated to 70-90 ℃ then,
Step 2 '. after treating that casein all dissolves, logical nitrogen protection is after the whole eliminatings of the air in the system; add potassium peroxydisulfate, quality and acrylic acid mass ratio that potassium peroxydisulfate adds are 0.03-0.05, along with the carrying out of reaction; the system color gradually becomes light blue blue until breast from colourless
Step 3 '. remove thermal source, stop to stir, cool off, filter, dialysis medium dialysis with analytically pure hydrochloric acid and distilled water modulation pH=3, obtaining mean diameter is the nano-hollow microsphere of casein-polyacrylic acid composite of 360-370nm, and wherein polyacrylic content percentage composition is 75%-85% in the compound hollow microballoon.It can be placed in 4 ℃ of refrigerators and preserve.
A kind of method for making of nano-hollow ball of above-mentioned crosslinked casein-polyacrylic acid composite, it is that the nano-hollow ball of the above-mentioned casein-polyacrylic acid composite that makes is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, filtered then in 24 hours 40 ℃ of reactions, dialysis obtains the nano-hollow ball of crosslinked casein-polyacrylic acid composite in the water of pH=3.
A kind of method for making of above-mentioned crosslinked casein nano-hollow ball, it is that the nano-hollow ball of the above-mentioned casein-polyacrylic acid composite that makes is added 50-75 μ L mass percent concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, 40 ℃ of reactions 24 hours, filter then, nano microsphere system after crosslinked is regulated pH to 7, and dialysis obtains crosslinked casein nano-hollow ball in the water of pH=7 again.
The casein that is used to prepare nanoparticle among the present invention also can replace with α s1-casein or beta-casein.
Nanoparticle of the present invention can particularly can be used as the carrier of antitumor drug as the carrier of medicine.
As the carrier of medicine, it can add the cancer therapy drugs such as cisplatin of wanting load in various microgranules of the present invention, through 37 ℃ of vibration a few hours, can obtain being enclosed with the Nano microsphere of medicine.
Crosslinked caseic Nano microsphere of the present invention or nano-hollow ball, have excellent biological compatibility and biodegradability, be difficult for causing organism immune response, the metabolism that not only can be absorbed by the body of its catabolite, and human body had characteristics such as therapeutical effect and nutritive effect, therefore can be used as the carrier of medicine.
Description of drawings
Fig. 1 is the particle size distribution cartogram of the Nano microsphere of each embodiment gained, wherein Fig. 1-1～Fig. 1-5 is respectively embodiment 1, the uncrosslinked composite nano-microsphere of embodiment 7 gained, the uncrosslinked compound nano-hollow sphere of embodiment 12 gained, embodiment 15, the uncrosslinked composite nano-microsphere of embodiment 16 gained.Fig. 2 is the transmission electron microscope photo of the uncrosslinked composite nano-microsphere of embodiment 1.
Fig. 3 is the electromicroscopic photograph of the uncrosslinked compound nano-hollow sphere of embodiment 12, and wherein Fig. 3-1～Fig. 3-3 is respectively a transmission electron microscope picture, section transmission electron microscope picture, sem photograph.
Fig. 4 is the infrared spectrogram of the Nano microsphere of embodiment 12 gained, Fig. 4-1 wherein, Fig. 4-the 2nd, embodiment 12 pure crosslinked casein nano-hollow ball that dialysis obtains the nano-hollow ball of crosslinked casein-polyacrylic acid composite and dialysis obtains in the water of pH=7 in the water of pH=3 respectively.
Fig. 5 is the section transmission electron microscope picture of the uncrosslinked compound nano-hollow sphere of embodiment 13.
Fig. 6 is the transmission electron microscope photo of the uncrosslinked composite nano-microsphere of embodiment 15.
Fig. 7 is the transmission electron microscope photo of the uncrosslinked composite nano-microsphere of embodiment 16.
Fig. 8 is the drug release curve of embodiment 17.
Fig. 9 is the drug release curve of embodiment 18.
Figure 10 is the drug release curve of embodiment 19.
The specific embodiment
Below by embodiment technical scheme of the present invention is further described.
Embodiment 1:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis medium dialysis with analytically pure hydrochloric acid and distilled water modulation pH=3 adds water 250ml at every turn, changes water 20 times; dialysis is with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator, and mean diameter is 170nm; particle size distribution is seen Fig. 1-1, and transmission electron microscope photo is seen Fig. 2.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, the dialysis medium dialysis of analytically pure hydrochloric acid of reuse and distilled water modulation pH=3 (down together) adds water 250ml at every turn, changes water 20 times, dialyse a couple of days in the water of pH=3, can obtain Nano microsphere 0.39 gram of crosslinked casein-polyacrylic acid composite, mean diameter is 200nm, and polyacrylic content is 81% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can be compound, pure crosslinked caseic Nano microsphere 0.065 gram in the water of pH=7 again, and mean diameter is 240nm.
Embodiment 2:
In the there-necked flask of 100ml, add 40ml distilled water, 0.08g casein, acrylic acid 0.6g, be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source, stop to stir, cool off, filter, dialyse with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.52 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 210nm, and polyacrylic content is 85% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.068 gram in the water of pH=7 again, and mean diameter is 250nm.
Embodiment 3:
In the there-necked flask of 100ml, add 40ml distilled water, 0.12g casein, acrylic acid 0.6g, be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source, stop to stir, cool off, filter, dialyse with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 60ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.06g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.6 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 190nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.098 gram in the water of pH=7 again, and mean diameter is 240nm.
Embodiment 4:
In the there-necked flask of 100ml, add 40ml distilled water, 0.08g casein, acrylic acid 0.4g, be heated to 80 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source, stop to stir, cool off, filter, dialyse with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.39 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 200nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.063 gram in the water of pH=7 again, and mean diameter is 270nm.
Embodiment 5:
In the there-necked flask of 100ml, add 40ml distilled water, 0.08g casein, acrylic acid 0.4g, be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source, stop to stir, cool off, filter, dialyse with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.41 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 210nm, and polyacrylic content is 83% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.059 gram in the water of pH=7 again, and mean diameter is 270nm.
Embodiment 6:
In the there-necked flask of 100ml, add 40ml distilled water, 0.08g casein, acrylic acid 0.4g, be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source, stop to stir, cool off, filter, dialyse with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 60ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.06g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.40 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 180nm, and polyacrylic content is 83% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.066 gram in the water of pH=7 again, and mean diameter is 270nm.
Embodiment 7:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless, at this moment, removes thermal source; stop to stir; cooling; filter; dialysis is with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere; the gained nanoparticle is put into 4 ℃ of preservations of refrigerator, and mean diameter is 200nm, and particle size distribution is seen Fig. 1-2.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.41 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 220nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.068 gram in the water of pH=7 again, and mean diameter is 280nm.
Embodiment 8:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.6g; propanoic acid 0.3g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.54 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 230nm, and polyacrylic content is 85% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.061 gram in the water of pH=7 again, and mean diameter is 290nm.
Embodiment 9:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.4g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.43 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 230nm, and polyacrylic content is 84% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.065 gram in the water of pH=7 again, and mean diameter is 290nm.
Implement 10:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.12g casein; acrylic acid 0.6g; propanoic acid 0.3g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 60ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.06g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.63 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 240nm, and polyacrylic content is 83% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.099 gram in the water of pH=7 again, and mean diameter is 310nm.
Embodiment 11:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 80 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.41 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 240nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.067 gram in the water of pH=7 again, and mean diameter is 300nm.
Embodiment 12:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.015g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Transmission electron microscope through this nanoparticle section turns out to be hollow-core construction, and mean diameter is 360nm, and particle size distribution is seen Fig. 1-3, and electromicroscopic photograph is seen Fig. 3.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, hollow Nano microsphere 0.38 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this hollow Nano microsphere is dialysed, mean diameter is 360nm, polyacrylic content is 81% in the complex microsphere, and its infrared spectrum is seen Fig. 4-1; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic hollow Nano microsphere 0.064 gram in the water of pH=7 again, and mean diameter is 490nm, and its infrared spectrum is seen Fig. 4-2.
Embodiment 13:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.012g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Transmission electron microscope through this nanoparticle section turns out to be hollow-core construction, and its electromicroscopic photograph is seen Fig. 5.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, hollow Nano microsphere 0.39 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this hollow Nano microsphere is dialysed, mean diameter is 370nm, and polyacrylic content is 80% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic hollow Nano microsphere 0.061 gram in the water of pH=7 again, and mean diameter is 500nm.
Embodiment 14:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; carrying out along with reaction; the system color gradually becomes light blue blue until breast from colourless; at this moment; remove thermal source; stop to stir; cooling; filter; dialysis can obtain composite nano-microsphere with the inorganic salt micromolecule and the unreacted monomer of the system of removing, and the gained nanoparticle is put into 4 ℃ of preservations of refrigerator.Take out aforesaid liquid, it is crosslinked to add the 60ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.06g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.43 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 230nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain pure crosslinked caseic Nano microsphere 0.069 gram in the water of pH=7 again, and mean diameter is 290nm.
Embodiment 15:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g α s1-casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; along with the carrying out of reaction, the system color gradually becomes light blue blue until breast from colourless, at this moment; remove thermal source; stop to stir; cooling; filter; dialysis is with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator, and mean diameter is 320nm; particle size distribution is seen Fig. 1-4, and electromicroscopic photograph is seen Fig. 6.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.35 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 350nm, and polyacrylic content is 79% in the complex microsphere; If this nano microsphere system is regulated pH=7, dialysis can obtain caseic Nano microsphere 0.064 gram of pure crosslinked α s1-in the water of pH=7 again, and mean diameter is 470nm.
Embodiment 16:
In the there-necked flask of 100ml, add the 40ml distilled water; 0.08g beta-casein; acrylic acid 0.4g; propanoic acid 0.2g; be heated to 90 ℃ then; after treating that casein all dissolves; logical nitrogen protection; after the whole eliminatings of the air in the system; add potassium peroxydisulfate 0.01g; along with the carrying out of reaction, the system color gradually becomes light blue blue until breast from colourless, at this moment; remove thermal source; stop to stir; cooling; filter; dialysis is with the inorganic salt micromolecule and the unreacted monomer of the system of removing; can obtain composite nano-microsphere, the gained nanoparticle is put into 4 ℃ of preservations of refrigerator, and mean diameter is 320nm; particle size distribution is seen Fig. 1-5, and electromicroscopic photograph is seen Fig. 7.Take out aforesaid liquid, it is crosslinked to add the 40ul mass percent and be 25% glutaraldehyde or calcium sulphate dihydrate 0.04g, filtered then in 24 hours 40 ℃ of reactions, Nano microsphere 0.38 gram that can obtain crosslinked casein-polyacrylic acid composite in the water of pH=3 as if this Nano microsphere is dialysed, mean diameter is 360nm, and polyacrylic content is 82% in the complex microsphere; If this nano microsphere system is regulated pH=7, Nano microsphere 0.065 gram of in the water of pH=7, dialysing again and can obtain pure crosslinked beta-casein, mean diameter is 480nm.
Embodiment 17:
The pure crosslinked caseic Nano microsphere of dialysing in the water of pH=7 that embodiment 1 is obtained adds the 0.01g cisplatin, and 37 ℃ were stirred 72 hours down.Get part medicine-carried system high speed centrifugation under 400000 gravity, collect the supernatant.With the Pt ionic concentration of inductively coupled plasma mass spectrograph (ICP-MS) in the mensuration stillness of night, the carrying drug ratio and the clad ratio that can calculate cisplatin are about 8% and 70% respectively.And finally in normal saline, measure its drug release curve, its drug release curve is seen Fig. 8.
Embodiment 18:
The pure caseic Nano microsphere of dialysing in the water of pH=7 that embodiment 7 is obtained adds the 0.01g cisplatin, and 37 ℃ were stirred 72 hours down.Get part medicine-carried system high speed centrifugation under 400000 gravity, collect the upper strata stillness of night.With the Pt ionic concentration of inductively coupled plasma mass spectrograph (ICP-MS) in the mensuration stillness of night, the carrying drug ratio and the clad ratio that can calculate cisplatin are about 9% and 80% respectively.And finally in normal saline, measure its drug release curve, the drug release curve is seen Fig. 9.
Embodiment 19:
The pure caseic Nano microsphere of dialysing in the water of pH=7 that embodiment 12 is obtained adds the 0.01g cisplatin, and 37 ℃ were stirred 72 hours down.Get part medicine-carried system high speed centrifugation under 400000 gravity, collect the upper strata stillness of night.With the Pt ionic concentration of inductively coupled plasma mass spectrograph (ICP-MS) in the mensuration stillness of night, the carrying drug ratio and the clad ratio that can calculate cisplatin are about 8% and 60% respectively.And finally in normal saline, measure its drug release curve, the drug release curve is seen Figure 10.
Claims (15)
1. the Nano microsphere of a casein-polyacrylic acid composite or nano-hollow ball, it is characterized in that: it is made up of casein and polyacrylic acid compound, the mean diameter of Nano microsphere or nano-hollow ball is respectively 170-480nm and 360-500nm, and wherein polyacrylic percentage composition is 75%-85% in the complex microsphere.
2. Nano microsphere according to claim 1 is characterized in that: it is crosslinked with glutaraldehyde or calcium salt, the Nano microsphere or the nano-hollow ball of all stable crosslinked casein-polyacrylic acid composite under acid and alkaline environment that obtains.
3. crosslinked caseic Nano microsphere or nano-hollow ball, it is characterized in that: it is made up of crosslinked casein, and the mean diameter of Nano microsphere or nano-hollow ball is respectively 240-480nm and 490-500nm.
4. according to Nano microsphere or nano-hollow ball or the crosslinked caseic Nano microsphere or the nano-hollow ball of claim 1,2 or 3 described casein-polyacrylic acid composites, it is characterized in that: described casein is α s1-casein or beta-casein.
5. a method for preparing the Nano microsphere of the described casein-polyacrylic acid composite of claim 1 is characterized in that it comprises the steps:
Step 1. adds the 50ml distilled water in the there-necked flask of 100ml, 0.1 gram casein and 0.5-0.75 gram acrylic acid stir, and are heated to 70-90 ℃ then,
After step 2. treated that casein all dissolves, logical nitrogen protection after the whole eliminatings of the air in the system, added 0.0125-0.0187 and restrains potassium peroxydisulfate, and along with the carrying out of reaction, the system color gradually becomes light blue until newborn blueness from colourless,
Step 3. removes thermal source, stops to stir, cooling off then, filter, and dialysis obtains the Nano microsphere that mean diameter is casein-polyacrylic acid composite of 170-320nm again, and wherein polyacrylic percentage composition is 75%-85% in the complex microsphere.
The method for making of the Nano microsphere of 6 casein-polyacrylic acid composites according to claim 5, it is characterized in that: in the described step 1, add 0.25-0.375 gram propanoic acid, make the quality that adds potassium peroxydisulfate in the step 2 and acrylic acid mass ratio simultaneously smaller or equal to 0.025.
7 one kinds of methods that prepare the Nano microsphere of the described crosslinked casein-polyacrylic acid composite of claim 2, it is characterized in that: it is that the Nano microsphere of claim 5 or the 6 described casein-polyacrylic acid composites that make is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or calcium salt are crosslinked, filtered then in 24 hours 40 ℃ of reactions, dialysis obtains the Nano microsphere of crosslinked casein-polyacrylic acid composite in the water of pH=3.
8. the method for making of the Nano microsphere of crosslinked casein-polyacrylic acid composite according to claim 7, it is characterized in that: described calcium salt is calcium carbonate, calcium sulfate or calcium phosphate.
9. method for making for preparing the described crosslinked casein Nano microsphere of claim 3, it is characterized in that: it is that the Nano microsphere of the described casein-polyacrylic acid composite that makes of claim 1 is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, 40 ℃ of reactions 24 hours, filter then, this nano microsphere system is regulated pH=7, and dialysis obtains crosslinked casein Nano microsphere in the water of pH=7 again.
10. a method for preparing the nano-hollow ball of the described casein-polyacrylic acid composite of claim 1 is characterized in that it comprises the steps:
Step 1 '. add the 50ml distilled water in the there-necked flask of 100ml, 0.1 gram casein, 0.5-0.75 gram acrylic acid and 0.2-0.4 gram propanoic acid stir, and are heated to 80-90 ℃ then,
Step 2 '. after treating that casein all dissolves, logical nitrogen protection is after the whole eliminatings of the air in the system; add potassium peroxydisulfate, quality and acrylic acid mass ratio that potassium peroxydisulfate adds are 0.03-0.05, along with the carrying out of reaction; the system color gradually becomes light blue blue until breast from colourless
Step 3 '. remove thermal source, stop to stir, cool off, filter, dialyse, obtain the nano-hollow ball that mean diameter is casein-polyacrylic acid composite of 360-370nm, wherein polyacrylic content percentage composition is 75%-85% in the complex microsphere.
11. method for preparing the nano-hollow ball of the described crosslinked casein-polyacrylic acid composite of claim 2, it is characterized in that: it is that the nano-hollow ball of the above-mentioned casein-polyacrylic acid composite that makes is added 50-75 μ L mass percentage concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, filtered then in 24 hours 40 ℃ of reactions, dialysis obtains the nano-hollow ball of crosslinked casein-polyacrylic acid composite in the water of pH=3.
12. method for preparing the described crosslinked casein nano-hollow ball of claim 3, it is characterized in that: it is that the nano-hollow ball of the described casein-polyacrylic acid composite that makes of claim 10 is added 50-75 μ L mass percent concentration is that 25% glutaraldehyde or 0.04-0.06g calcium sulphate dihydrate are crosslinked, 40 ℃ of reactions 24 hours, filter then, this nano microsphere system is regulated pH=7, and dialysis obtains crosslinked casein nano-hollow ball in the water of pH=7 again.
13. according to the Nano microsphere of the Nano microsphere of the arbitrary described preparation casein-polyacrylic acid composite of claim 5-12 or hollow ball or crosslinked casein-polyacrylic acid composite or hollow ball or the crosslinked crosslinked casein Nano microsphere or the method for hollow ball, it is characterized in that: the casein that is used to prepare nanoparticle replaces with α s1-casein or beta-casein.
14. claim 3 or 4 described casein Nano microspheres or nano-hollow ball are as the application in the pharmaceutical carrier.
15. according to claim 14 as the application in the pharmaceutical carrier, it is characterized in that: as the application in the carrier of antitumor drug.
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|---|---|---|---|---|
| CN103169979A (en) * | 2012-12-14 | 2013-06-26 | 江南大学 | Preparation method of soybean protein-acrylic acid compound |
| CN104622817B (en) * | 2015-01-27 | 2017-06-27 | 江苏大学 | A kind of protein polymer composite Nano carrier and preparation method thereof |
| CN107552024A (en) * | 2017-08-06 | 2018-01-09 | 桂林理工大学 | Preparation method for the crosslinking casein porous microsphere of lead absorption |
| CN109701066A (en) * | 2018-12-25 | 2019-05-03 | 合肥必更赢科技有限公司 | A kind of ventilative casein complex microsphere and its preparation method and application |
| CN113318264A (en) * | 2021-05-28 | 2021-08-31 | 瑞聚再生(厦门)医学科技有限公司 | Degradable polyurethane biomaterial and preparation method and application thereof |
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2009
- 2009-08-21 CN CN2009101846079A patent/CN101648022B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103169979A (en) * | 2012-12-14 | 2013-06-26 | 江南大学 | Preparation method of soybean protein-acrylic acid compound |
| CN104622817B (en) * | 2015-01-27 | 2017-06-27 | 江苏大学 | A kind of protein polymer composite Nano carrier and preparation method thereof |
| CN107552024A (en) * | 2017-08-06 | 2018-01-09 | 桂林理工大学 | Preparation method for the crosslinking casein porous microsphere of lead absorption |
| CN109701066A (en) * | 2018-12-25 | 2019-05-03 | 合肥必更赢科技有限公司 | A kind of ventilative casein complex microsphere and its preparation method and application |
| CN109701066B (en) * | 2018-12-25 | 2021-09-21 | 德晟康(苏州)生物科技有限公司 | Breathable casein composite microsphere and preparation method and application thereof |
| CN113318264A (en) * | 2021-05-28 | 2021-08-31 | 瑞聚再生(厦门)医学科技有限公司 | Degradable polyurethane biomaterial and preparation method and application thereof |
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