CN104622817A - Protein-polymer composite nano-carrier and preparation method thereof - Google Patents
Protein-polymer composite nano-carrier and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a protein-polymer composite nano-carrier and a preparation method thereof and relates to the field of nano preparations of antitumor medicines. The protein-polymer composite nano-carrier is designed and developed on the basis of the characteristics of a lipoprotein structure of a human body and comprises a polymer core and a hydrophilic protein shell; a targeting ligand can be flexibly modified according to the treatment requirement, so as to construct the protein-polymer composite nano-carrier with active targeting property; the in vivo stability of the medicine is improved; the tumor targeting property is increased. In order to increase the load rate of the protein on the polymer kernel, a cationic additive is added to the formula to prepare a cationic polymer nanoparticle; nano suspension is milk white liquid; spherical particles with relatively uniform distribution can be seen by scanning through a transmission electron microscope. A freeze-drying protective additive is added for freeze-drying the nano suspension to form powder, so that the protein-polymer composite nano-carrier is relatively easy to store and the stability is improved. The protein-polymer composite nano-carrier prepared by the method is uniform in particle size and good in stability; and a fat-soluble antitumor medicine can be effectively encapsulated.
Description
Technical field
The present invention relates to the nanometer formulation field of antitumor drug, specifically a kind of energy bag carries albumen-composite nano-polymers carrier of fat-soluble antitumor drug and preparation method thereof.
Background technology
Malignant tumor is only second to heart disease, threatens the second largest killer of global human health and lives.At present, chemotherapy and operative treatment, radiotherapy together constitute three large means of oncotherapy.
Chemotherapy is with the Therapeutic Method of chemicals killing off tumor cells.But because most of chemotherapeutics belongs to fat-soluble medicine, poorly water-soluble, have impact on the curative effect of medicine to a great extent.In addition, because chemicals has stronger cytotoxicity, lack again targeting characteristic, larger toxic and side effects can be caused, bring misery to patient, treatment sometimes even can be caused to interrupt.
The fat-soluble chemotherapeutics of current listing, mostly adopt organic solvent and surfactant as solubilising means, not only targeting is poor, and the surfactant component used also can bring zest or anaphylaxis, increases the misery of patient.As paclitaxel and paclitaxel injection of dwelling, in order to reach the object of dissolved substance, need respectively to use surfactant polyoxyethylene Oleum Ricini and tween 80 (polyoxyethylene sorbitan monoleate) and cosolvent ethanol more; For another example etoposide inj, need add the object that tween 80, Polyethylene Glycol, ethanol etc. reach solubilize drugs.The use of the solubilizing component such as these surfactants likely causes a series of untoward reaction, as anaphylaxis, neurotoxicity, bone marrow depression, fluid retention etc.Also have some cancer therapy drugs, in cell experiment, demonstrate remarkable active anticancer, but not yet have formulation application in clinical due to dissolubility and irritating problem.As Toadpoison Medicine, being a kind of pharmaceutical component of Chinese medicine Venenum Bufonis, is one of Venenum Bufonis dienoic acid lactone that in Venenum Bufonis, antitumor action is the strongest.Bibliographical information Toadpoison Medicine at much lower concentrations can inhibition tumor cell propagation, promote tumor cell differentiation, apoptosis-induced, Tumor suppression angiogenesis, and the sensitivity of tumor cell to chemotherapeutics can be recovered, Tumor suppression angiogenesis etc.Toadpoison Medicine is inhibited to multiple solid tumor, especially remarkable to the inhibition of hepatoma carcinoma cell, in cell experiment, Toadpoison Medicine demonstrates than many kinase targets to stronger tumor cell of liver inhibition (the Cao Y of inhibitor BAY 43-9006, Li HX, Xu LT, et al. Bufalin enhances the anti-proliferative effect of sorafenib on h μm of an hepatocellular carcinoma cells through downregulation of ERK [J]. Mol Biol Rep, 2012,39 (2): 1683).But, the poorly water-soluble of Toadpoison Medicine, half-life is short, zest and cardiac toxicity (Ying Li are produced to human body, Hang Zhao, Lin-Rui Duan, et al. Preparation, characterization and evaluation of bufalin liposomes coated with citrus pectin [J]. Colloids and Surfaces A:Physicochem. Eng. Aspects, 2014,444:54 – 62), all undesirable elements limit the application of Toadpoison Medicine, not yet have monomer whose formulation application in clinical.From the viewpoint of the effectiveness and reliability of medication, the cancer target nanometer formulation developing this kind of medicine is the means most possibly solving its dissolubility and zest problem, reduces the toxic and side effects of medicine, increases drug safety, plays its antitumous effect.
Ju α ?cyanoacrylate material there is good biocompatibility, the feature such as nontoxic, is biodegradable high molecular polymer, has now been widely used in the preparation of nanoparticulate carriers.With Ju α ?the nanoparticle for preparing of cyanoacrylate material can improve the stability of preparation, increase the dissolubility of medicine, be widely used in compound and Chinese medicine monomer and protein and polypeptide delivery systematic research at present.But adopt separately cyanoacrylate as carrier, there is the problem similar with most polymers class carrier, after polymer nanoparticle drug carriers enters blood circulation, easy and immunoglobulin G (immunoglobulin G), complement protein C
3(complement C
3) and the blood plasma such as fibronectin (fibronectin) in opsonin combine, and absorbed by mononuclear phagocyte system, body internal stability is not good; Do not possess tumor-targeting.
The advantages such as albumin is natural hydrophilic protein matter, has safety non-toxic, non-immunogenicity, biodegradable, good biocompatibility, cheap and easy to get, and character quite stable, can keep stable in the scope of pH4-9.Adopt albumin as carrier shell, the hydrophilic of carrier can be increased, improve carrier stability in blood, and make carrier have certain tumor tropism, albumin also can for modifying containing many surface active groups, target ligand can be modified on albumen, as cholic acid, ursodesoxycholic acid, enoxolone, antibody etc., existing lot of documents shows, tumor cell surface has corresponding receptor, and the mediation by these parts strengthens tumor cell to the picked-up of carrier, makes carrier have active targeting.
This patent adopts hydrophilic protein as shell, using polymer support as kernel, devises a kind of novel targeting proteins-polymer nanoparticle drug carriers, in order to bag carrying anti-tumor medicine.This carrier designs and develops based on the lipoprotein structure feature that human body is natural, and lipoprotein is that the naturally occurring lipid of human body transports carrier, has lipophilic kernel and hydrophilic protein shell mechanism.According to bibliographical information, lipoprotein can effectively transport fat-soluble cancer therapy drug, and has tumor-targeting (can targeting to the tumor cell of overexpression low density lipoprotein receptor).But lipoprotein needs to extract from fresh plasma, and extraction process is very complicated, and apolipoprotein is unstable, easily assembles, not easily commercialization; In addition lipoprotein only can targeting to the tumor of low density lipoprotein receptor overexpression, its application is restricted.Albumen-the polymer support of this patent design construction, there is hydrophilic protein shell and the polymer support core of fat-soluble medicine can be held, and different target ligands can be modified as required, there is excellent drug carrying ability, remarkable body internal contact compatibility and stability, and targeting flexibly, be the targeting vector having functionalization.
Modification shell using hydrophilic material Polyethylene Glycol as carrier surface, it is the method for the nano-carrier body internal stability such as conventional increase polymer or liposome, and using albumin as the shell mechanism of carrier, compared with Polyethylene Glycol, there is special advantage, (1) be easy to carry out finishing, each albumin molecule has multiple carboxyl and amino, can rhetorical function group as required; (2) better biocompatibility and go opsonic action, albumin is the natural hydrophilic protein existed in human body, non-immunogenicity, and albumin effectively can reduce the absorption of albumen on carrier in blood plasma, extends carrier circulation time in blood.(3) self there is certain tumor tropism, the vascular permeability of tumor and inflammatory tissue increases, albumin can by the gP60 of tumor vascular endothelium (60-kDa glycoprotein) receptor, or by with tumor tissues in be rich in cysteine acidic secretion protein (secreted protein acid and rich in cysteine, the SPARC) picked-up that promotes carrier in tumor tissues.
Also have at present and be much used alone albumin as the research of carrier and patent, but be used alone albumin and may there is following problem as carrier.(1) generally need by heating or chemical cross-linking agent solidification.The stability of medicine or the hydrophilic of albumen may be affected; (2) be that the nanoparticle prepared of material or microspherulite diameter are comparatively large, generally more than 500 μm with albumin.America Biological Science Co., Ltd develops Nab technology and prepares medicine albumin nano granular, this technology adopts the even technology of high pressure breast to pastille organic facies and carries out high-pressure homogenising containing the mixed system of albumen aqueous phase, prepare nanoparticle, this technology is comparatively strong to the physicochemical property selectivity of medicine, and medicine need have stronger affinity with albumin.In addition, according to " pharmaceutics ", (Cui Fude edits, " pharmaceutics " second edition, classification 2011:536.), these carriers all belong to passive targeted preparation, passive targeted preparation only can utilize Different Organs in body, tissue or reticuloendothelial system retaining or phagocytosis the microgranule of different size, and conduct drugs to target site, Targeting Effect is not good enough.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of albumen-composite nano-polymers carrier, to wrap the polymer of year fat-soluble antitumor drug as kernel, adopting the albumin with targeting as hydrophilic outer shell; Present invention also offers the preparation method of above albumen-composite nano-polymers carrier, according to treatment needs, targeting group can be modified in advance on albumin, construct the albumen-composite nano-polymers carrier with active targeting.
The present invention is achieved through the following technical solutions: a kind of albumen-composite nano-polymers carrier, comprises polymer core and targeting proteins matter shell; Described polymer core is alpha-cyanoacrylate alkane ester kernel, and described alpha-cyanoacrylate alkane ester kernel bag carries fat-soluble antitumor drug; Described targeting proteins matter shell is wrapped in the surface of described alpha-cyanoacrylate alkane ester kernel; Described targeting proteins matter shell is hydrophilic protein shell.
Described albumen-composite nano-polymers carrier is calculated by mass percentage and is comprised following component: the antitumor drug of 1 ~ 10%, the alpha-cyanoacrylate alkane ester of 10 ~ 60%, 0.5 ~ 10% cation additions, the protein matter of 1 ~ 10%, the stabilizing agent of 20 ~ 60% and 5 ~ 30% freeze drying protectant.
Described antitumor drug is the one in Toadpoison Medicine, etoposide, paclitaxel, paclitaxel of dwelling, gefitinib, hydroxy camptothecin and amycin, curcumin, fluorouracil, cyclophosphamide, irinotecan, mitoxantrone, cisplatin more.
Described alpha-cyanoacrylate alkane ester is the one in methyl 2-cyanoacrylate MCA, cyanacrylate ECA, BCA BCA, isobutylcyanoacrylate IBCA, the own ester HCA of alpha-cyanoacrylate and alpha-cyanoacrylate dissident ester IHCA.
Described cation additions is one in 18-amine., PVOH amine, 1B and arginine or its compositions.
Described protein matter is one in the protein modified of native protein or synthetic protein or albuminoid and derivant thereof or modified thing or its mixture.
Described stabilizing agent is one in Pluronic F68, pluronic F123, pluronic F127, macrodex, Dextran 40, Dextran-20, polyvinyl alcohol, tween 80 and Arlacel-80 or its mixture.
Described freeze drying protectant be following in one: (1) saccharide/polyhydric alcohol: sucrose, trehalose, mannitol, lactose, glucose, maltose; (2) polymer: HES, PVP, PEG, glucosan, albumin; (3) aminoacid: Serine, sodium glutamate, alanine, glycine, sarcosine; (4) salt and amine: the one in phosphate, acetate, citrate or its mixture.
Described protein matter is albumin.
Described trim is one or more in cholic acid, ursodesoxycholic acid, enoxolone, hyaluronic acid, galactose, folic acid and lipoprotein receptor identification recombinant polypeptide; Described trim can be connected by covalent bond with the amino of described protein matter, carboxyl or sulfydryl.
The preparation method of described albumen-composite nano-polymers carrier, comprises the following steps:
1) preparation bag carries the alpha-cyanoacrylate alkane ester kernel of described antitumor drug;
Calculate by mass percentage, take 1 ~ 10% antitumor drug, 0.5 ~ 8% cation additions and 5 ~ 40% stabilizing agent oil phases to dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise containing 5 ~ 40% stabilizer concentrations is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Or take 1 ~ 10% antitumor drug and 5 ~ 40% stabilizing agent oil phases dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding containing 5 ~ 40% stabilizing agents and 0.5 ~ 5% cation additions concentration by 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Or take 1 ~ 10% antitumor drug, 0.5 ~ 8% cation additions oil phase dissolves, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise containing 5-50% stabilizer concentration is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Or take 1 ~ 10% antitumor drug oil phase and dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding containing 5-50% stabilizing agent and 0.5 ~ 5% cation additions concentration by 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Or take 1 ~ 10% antitumor drug, 0.5 ~ 8% cation additions and 5 ~ 40% stabilizing agent oil phases and dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding containing 5 ~ 40% stabilizing agents and 0.5 ~ 5% cation additions concentration by 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Or take 1 ~ 10% antitumor drug and 0.5 ~ 8% cation additions oil phase dissolves, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding containing 5 ~ 50% stabilizing agents and 0.5 ~ 5% cation additions concentration by 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Oil phase mixed solution is slowly added dropwise in aqueous phase system, after stirring 2 ~ 8h, forms the mixed liquor containing described alpha-cyanoacrylate alkane ester kernel;
Mass percent calculates, and described oil phase and described aqueous phase cationic additive total amount are 0.5 ~ 10%; In oil phase and aqueous phase, stabilizing agent total amount is 20 ~ 60%.
Described oil phase is one in ethyl acetate, dichloromethane, chloroform, acetone, ether or its mixture.
2) described native protein extraction purification or modify on protein, obtains targeting proteins matter shell;
Wherein the method for modifying concrete steps of protein are: trim is added oxolane or dimethyl formamide DMF dissolves, then add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide respectively; 0 DEG C is stirred 2 ~ 6 h, and continue stirred overnight at room temperature, recrystallization, obtains the active ester of trim, is dissolved in dimethyl formamide DMF, dropwise joins and is dissolved with in albuminous solution, and limit edged stirs, and room temperature continues stirring 2 ~ 6 h; Reactant liquor is dialysed after 48 h, and the centrifugal 10min of 12000 rpm, removes unreacted trim active ester and hydrolyzate thereof, and lyophilizing obtains the product of white, and be the albumin of modification, the mol ratio of albumin and trim is 1:5 ~ 1:60.
3) targeting proteins matter shell described in described alpha-cyanoacrylate alkane ester kernel outer cladding, forms the suspension containing albumen-composite nano-polymers carrier;
Concrete steps are: regulated by the mixed liquor sodium hydroxide containing alpha-cyanoacrylate alkane ester kernel pH to neutral, add and calculate 1 ~ 10% protein matter by mass percentage, continue stirring 0.5 ~ 6 h, rotary evaporation 30 ~ 120min, the organic solvent that removing is remaining, centrifugal 2 ~ 15 min of 3000 ~ 10000 rpm, cross 0.2 ~ 0.8 μm of microporous filter membrane and carry out aseptic filtration, obtain albumen-composite nano-polymers vehicle suspension.
Protein matter, by charge effect, hydrophobic force, Van der Waals force equimolecular intermolecular forces, is combined in the surface of alpha-cyanoacrylate alkane ester kernel with non-covalent bond.
4) in suspension, add described freeze drying protectant and carry out lyophilization, form powdery.
Concrete steps are: in described albumen-composite nano-polymers vehicle suspension, add the freeze drying protectant of 5 ~ 30%, lyophilizing 24 hours, form powdery.
The temperature of described rotary evaporation is 0 ~ 60 DEG C, preferably 20 ~ 30 DEG C.
The mean diameter of described albumen-composite nano-polymers carrier is 20 ~ 1000nm, preferably 80 ~ 200nm.
Described albumen-composite nano-polymers carrier is for the preparation of antitumor drug.
Beneficial effect: the present invention compared with prior art, design and develop based on the lipoprotein structure feature that human body is natural, there is polymer core, hydrophilic protein shell, and target ligand can be modified flexibly, effectively can wrap and carry fat-soluble medicine, improve medicine body internal stability, increase tumor-targeting; In order to improve the load factor of protein on polymer core, cationic polymer nanoparticle has been prepared by adding cation additions in prescription, albumin isoelectric point, IP is about 4.5, electronegative in neutral medium, be combined in polymer nanoparticle surface by charge effect, hydrophobic force, Van der Waals force equimolecular intermolecular forces; According to treatment needs, targeting group can be modified in advance on albumen, constructs the albumen-composite nano-polymers carrier with active targeting thus; This nanosuspension is milky white liquid, comparatively even through the distribution of transmission electron microscopy visible particles, in spheroidal; Being more prone to make it preserve, improving stability, add freeze drying protectant and carry out lyophilization formation powdery.
Accompanying drawing explanation
Fig. 1 is the grain size distribution carrying Toadpoison Medicine-ursodesoxycholic acid modified albumin-composite nano-polymers grain prepared according to example 1.
Fig. 2 is the transmission electron microscope picture carrying Toadpoison Medicine-ursodesoxycholic acid modified albumin-composite nano-polymers grain prepared according to example 1.
The In-vitro release curves carrying Toadpoison Medicine-ursodesoxycholic acid modified albumin-composite nano-polymers grain Chinese medicine that Fig. 3 is Toadpoison Medicine crude drug and prepares according to example 1.
Detailed description of the invention
Below by embodiment, the present invention is described further by reference to the accompanying drawings, but protection scope of the present invention is not limited to this.
Embodiment 1: the preparation of carrying Toadpoison Medicine-ursodesoxycholic acid modified protein-composite nano-polymers grain.
Take 3.5 mg Toadpoison Medicines, 5 mg 18-amine. and 30mg pluronic F127 dichloromethane respectively to dissolve.Slowly 60 mg BCAs (BCA) are dropwise added 5 mL to contain in the dilute hydrochloric acid (0.1 M) of macrodex and each 25 mg of Pluronic F68, dichloromethane mixed solution is slowly added dropwise in system, drip limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 5 mg ursodesoxycholic acid are modified, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry Toadpoison Medicine-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension.
30mg mannitol is added in year Toadpoison Medicine-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension, 30mg trehalose lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 2: the preparation of carrying Toadpoison Medicine-hyaluronic acid decorated albumen-composite nano-polymers grain.
Take 10 mg Toadpoison Medicines respectively, 10 mg 18-amine. dichloromethane dissolve.Slowly 100 mg cyanacrylates (ECA) are dropwise added 10 ml contain macrodex and each 50 mg of Pluronic F68 dilute hydrochloric acid (0.1M) in, dichloromethane mixed solution is slowly added dropwise in system, drips limit stirring.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 20 mg are hyaluronic acid decorated, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry Toadpoison Medicine-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension.
50 mg sucrose lyophilizing 24 hours is added in year Toadpoison Medicine-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 3: the preparation of carrying etoposide-enoxolone modified protein-composite nano-polymers grain.
Take 30 mg etoposides, 3 mg 18-amine. and 50 mg pluronic F127 dichloromethane respectively to dissolve.Slowly 210 mg methyl 2-cyanoacrylates (MCA) are dropwise added 5 mL to contain in the dilute hydrochloric acid (0.1 M) of macrodex and each 10 mg of Pluronic F68, dichloromethane mixed solution is slowly added dropwise in system, drip limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 5 mg enoxolone are modified, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry etoposide-enoxolone modified protein-composite nano-polymers vehicle suspension.
20 mg PEG 400 are added in year etoposide-enoxolone modified protein-composite nano-polymers vehicle suspension, 10 mg glycine lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 4: the preparation of carrying paclitaxel-modified with folic acid albumen-composite nano-polymers grain.
Take 5 mg paclitaxels, 12mg 18-amine. and 60 mg pluronic F127 dichloromethane respectively to dissolve.Slowly 40 mg isobutylcyanoacrylates (IBCA) are dropwise added 10 ml to contain in the 10 arginic dilute hydrochloric acid of mg PVAC polyvinylalcohol 1788 and 1.5 mg (0.1M), dichloromethane mixed solution is slowly added dropwise in system, drip limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin of 15 mg modified with folic acid, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry paclitaxel-modified with folic acid albumen-composite nano-polymers vehicle suspension.
10 mg mannitol are added in year paclitaxel-modified with folic acid albumen-composite nano-polymers vehicle suspension, 10 mg trisodium citrate lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 5: the preparation of carrying paclitaxel-ursodesoxycholic acid modified protein-composite nano-polymers grain of dwelling more.
Take 10 mg paclitaxel dichloromethane of dwelling to dissolve more.Slowly 60 mg BCAs (BCA) are dropwise added 10 ml contains 25 mg PVAC polyvinylalcohol 1788,15 mg pluronic F127, in the dilute hydrochloric acid (0.1 M) of macrodex and each 20 mg of Pluronic F68 and 10 mg PVOH amine 1200, dichloromethane mixed solution is slowly added dropwise in system, drips limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 10 mg ursodesoxycholic acid are modified, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry paclitaxel of dwelling-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension more.
Add 25 mg mannitol carrying to dwell in paclitaxel-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension more, 20 mg PVP K30,30mg sodium glutamate lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 6: the preparation of carrying gefitinib-ursodesoxycholic acid modified protein-composite nano-polymers grain.
Take 3 mg gefitinibs respectively and 90 mg pluronic F127 chloroform dissolve.Slowly the own ester of 25 mg alpha-cyanoacrylate (HCA) is dropwise added 10 ml to contain in macrodex and each 25 mg of Pluronic F68,10 mg arginic dilute hydrochloric acid (0.1 M), chloroform mixed solution is slowly added dropwise in system, drip limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 10 mg ursodesoxycholic acid are modified, continue stirring 1 h.The chloroform that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry gefitinib-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension.
25 mg lactose are added in year gefitinib-ursodesoxycholic acid modified protein-composite nano-polymers vehicle suspension, 20 mg glycine trisodium lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 7: carry the preparation that hydroxyl carries camptothecine-hyaluronic acid decorated albumen-composite nano-polymers grain.
Take 5 mg hydroxy camptothecins respectively, 5 mg 18-amine. dichloromethane dissolve.Slowly 50 mg alpha-cyanoacrylate dissidents ester (IHCA) are dropwise added 5 ml contains 75 mg PVAC polyvinylalcohol 1788 and 20 mg pluronic F127, and in the dilute hydrochloric acid of 5mg lysine (0.1M), dichloromethane mixed solution is slowly added dropwise in system, drips limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 5 mg are hyaluronic acid decorated, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry hydroxyl and carry camptothecine-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension.
Carry in camptothecine-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension at a year hydroxyl and add 20 mg macrodexs, 30 mg trehalose lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 8: the preparation of carrying amycin-enoxolone modified protein-composite nano-polymers grain.
Take 7.5 mg amycin, 10 mg 18-amine. and 20 mg pluronic F127 dichloromethane respectively to dissolve.Slowly 75 mg isobutylcyanoacrylates (IBCA) are dropwise added 10 ml to contain in the dilute hydrochloric acid (0.1M) of 50 mg PVAC polyvinylalcohol 1788, dichloromethane mixed solution is slowly added dropwise in system, drip limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 10 mg enoxolone are modified, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry amycin-enoxolone modified protein-composite nano-polymers vehicle suspension.
20 mg glycine are added in year amycin-enoxolone modified protein-composite nano-polymers vehicle suspension, 20mg PEG400,10 mg trisodium citrate lyophilizing 24 hours, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Embodiment 9: the preparation of carrying paclitaxel-enoxolone modified protein-composite nano-polymers grain.
Take 20 mg paclitaxels, 15 mg 18-amine. and 10 mg Pluronic F68 dichloromethane respectively to dissolve.Slowly 70 mg BCAs (BCA) are dropwise added 10 ml contains 20 mg PVAC polyvinylalcohol 1788, in 20mg macrodex and the arginic dilute hydrochloric acid of 4mg (0.1M), dichloromethane mixed solution is slowly added dropwise in system, drips limit and stir.After stirring 4 h, regulate pH to neutral with sodium hydroxide, add the bovine serum albumin that 20 mg enoxolone are modified, continue stirring 1 h.The dichloromethane that rotary evaporation removing is remaining, centrifugal 3 min of 5000 rpm, cross microporous filter membrane, must carry paclitaxel-enoxolone modified protein-composite nano-polymers vehicle suspension.
10 mg trehalose lyophilizing 24 hours is added in year paclitaxel-enoxolone modified protein-composite nano-polymers vehicle suspension, obtain powder, again disperse to form albumen-composite nano-polymers vehicle suspension by adding sterilized water or normal saline, size almost with lyophilizing before unchanged.
Experimental example 1: size and distribution.
Adopt Malvern laser granulometry (Zetasizer Nano ZS90 type laser particle analyzer, Malvern company of Britain) to measure particle diameter, embodiment 1 measurement result as shown in Figure 2.Measurement result shows, the mean diameter of the Toadpoison Medicine nanometer formulation prepared by the present invention is 149.3 nm, and polydispersity coefficient is 0.073.
Experimental example 2: morphology distributes.
Getting preparation appropriate, after distilled water diluting, getting on the one after another drop of copper mesh being covered with carbon supporting film, place in a moment, drip 2% phosphotungstic acid and carry out negative staining, with transmission electron microscope (Tecnai 12, Philips company, Holland) observe particle shape, the observed result of embodiment 1 is shown in Fig. 2.As seen from Figure 2, distribution of particles is comparatively even, in spheroidal.
Experimental example 3: protein load rate.
Albumen loads with rate: albumen loads with rate (%)=(1-M
free/ M
throw) * 100
In formula, M
freerefer to free BSA(modified) amount (μ g), M
throwrefer to drop into BSA(modified) amount (μ g).In embodiment 1, ursodesoxycholic acid modified albumin is 94.33% in the load factor of carrier surface; Albumin hyaluronic acid decorated in embodiment 2 is 89.72% in carrier surface load factor; The albumin that in embodiment 3, enoxolone is modified is 91.19% in the load factor of carrier surface; The albumin that embodiment 4 Folic Acid is modified is 85.42% in the load factor of carrier surface; The albumin that in embodiment 5, ursodesoxycholic acid is modified is 92.25% in carrier surface load factor; The albumin that in embodiment 6, ursodesoxycholic acid is modified is 95.18% in carrier surface load factor; Albumin hyaluronic acid decorated in embodiment 7 is 87.49% in carrier surface load factor; The albumin that in embodiment 8, enoxolone is modified is 92.31% in the load factor of carrier surface; The albumin that in experimental example 9, enoxolone is modified is 90.76% in the load factor of carrier surface.
Experimental example 4: release in vitro.
With the phosphate buffer of pH7.4 for release medium, investigate the release in vitro behavior of preparation.The release profiles of preparation of Chinese medicine (Toadpoison Medicine) prepared by Toadpoison Medicine saturated solution Chinese medicine and embodiment 1 as shown in Figure 3.As seen from Figure 3, the release of preparation of Chinese medicine, compared with saturated solution, has certain slow release effect, but substantially can reach at 24h and discharge completely.
Experimental example 5: cell inhibitory rate and Evaluation on Its Targeting Performance.
Investigate the common Toadpoison Medicine-polymer beads carrying Toadpoison Medicine-ursodesoxycholic acid modified protein-composite nano-polymers grain and non-coating decoration albumen of embodiment 1 preparation to the IC of tumor cell of liver HepG2
50be respectively 32.7nM and 98.6nM, can find out and carry common Toadpoison Medicine-polymer beads that the Toadpoison Medicine-ursodesoxycholic acid modified protein-inhibition of composite nano-polymers grain to tumor cell of liver HepG2 is significantly higher than non-coating decoration albumen.After showing the albumen that coated ursodesoxycholic acid is modified, the picked-up of tumor cell to carrier can be increased, there is tumor cell targeting.Investigate the common etoposide-polymer beads carrying etoposide-enoxolone modified protein-composite nano-polymers grain and non-coating decoration albumen of embodiment 3 preparation to the IC of tumor cell of liver HepG2
50be respectively 97.3 μMs and 148.8 μMs, can find out and carry common etoposide-polymer beads that the etoposide-enoxolone modified protein-inhibition of composite nano-polymers grain to tumor cell of liver HepG2 is significantly higher than non-coating decoration albumen.After showing coating decoration albumen, the picked-up of tumor cell to carrier can be increased, there is tumor cell targeting.Investigate prepared by embodiment 5 carry dwell paclitaxel-ursodesoxycholic acid modified protein-composite nano-polymers grain and non-coating decoration albumen more common more dwell paclitaxel-polymer beads to tumor cell of liver HepG2 IC
50be respectively 62.7nM and 128.9nM, can find out and carry common paclitaxel-polymer beads of dwelling that the paclitaxel-ursodesoxycholic acid modified protein-inhibition of composite nano-polymers grain to tumor cell of liver HepG2 of dwelling is significantly higher than non-coating decoration albumen more more.After showing coating decoration albumen, the picked-up of tumor cell to carrier can be increased, there is tumor cell targeting.
Described embodiment is preferred embodiment of the present invention, but the present invention is not limited to above-mentioned embodiment, but should admit, those skilled in the art may make various modification and transformation to the present invention, and these are modified and change and belong to equally in scope of the present invention that appended claims defines.
Claims (10)
1. albumen-composite nano-polymers carrier, is characterized in that, comprises polymer core and targeting proteins matter shell;
Described polymer core is alpha-cyanoacrylate alkane ester kernel, and described alpha-cyanoacrylate alkane ester kernel bag carries fat-soluble antitumor drug;
Described targeting proteins matter shell is wrapped in the surface of described alpha-cyanoacrylate alkane ester kernel;
Described targeting proteins matter shell is hydrophilic protein shell.
2. a kind of albumen-composite nano-polymers carrier according to claim 1; it is characterized in that, by mass percentage calculate comprise following component: the antitumor drug of 1 ~ 10%, the alpha-cyanoacrylate alkane ester of 10 ~ 60%, 0.5 ~ 10% cation additions, the protein matter of 1 ~ 10%, the stabilizing agent of 20 ~ 60% and 5 ~ 30% freeze drying protectant.
3. a kind of albumen-composite nano-polymers carrier according to claim 2, is characterized in that,
Described antitumor drug is the one in Toadpoison Medicine, etoposide, paclitaxel, paclitaxel of dwelling, gefitinib, hydroxy camptothecin and amycin, curcumin, fluorouracil, cyclophosphamide, irinotecan, mitoxantrone, cisplatin more;
Described alpha-cyanoacrylate alkane ester is the one in methyl 2-cyanoacrylate MCA, cyanacrylate ECA, BCA BCA, isobutylcyanoacrylate IBCA, the own ester HCA of alpha-cyanoacrylate and alpha-cyanoacrylate dissident ester IHCA;
Described cation additions is one in 18-amine., PVOH amine, 1B and arginine or its compositions;
Described protein matter is one in the protein modified of native protein, synthetic protein, albuminoid and derivant thereof or modified thing or its mixture;
Described stabilizing agent is one in Pluronic F68, pluronic F123, pluronic F127, macrodex, Dextran 40, Dextran-20, polyvinyl alcohol, tween 80 and Arlacel-80 or its mixture;
Described freeze drying protectant is the one of the following stated:
(1) saccharide/polyhydric alcohol: sucrose, trehalose, mannitol, lactose, glucose, maltose;
(2) polymer: HES, PVP, PEG, glucosan, albumin;
(3) aminoacid: Serine, sodium glutamate, alanine, glycine, sarcosine;
(4) salt and amine: the one in phosphate, acetate, citrate or its mixture.
4. a kind of albumen-composite nano-polymers carrier according to claim 3, it is characterized in that, described protein matter is albumin; Described trim is one or more in cholic acid, ursodesoxycholic acid, enoxolone, hyaluronic acid, galactose, folic acid and lipoprotein receptor identification recombinant polypeptide.
5. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 1, comprises the following steps:
1) preparation bag carries the alpha-cyanoacrylate alkane ester kernel of described antitumor drug;
2) described native protein extraction purification or modify on protein, obtains targeting proteins matter shell;
3) targeting proteins matter shell described in described alpha-cyanoacrylate alkane ester kernel outer cladding, forms the suspension containing albumen-composite nano-polymers carrier;
4) in suspension, add described freeze drying protectant and carry out lyophilization, form powdery.
6. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 5, it is characterized in that, described step 1) is specially:
Calculate by mass percentage, take 1 ~ 10% antitumor drug, 0 ~ 8% cation additions and 0 ~ 40% stabilizing agent oil phase to dissolve, oil phase mixed solution, control rotating speed is 100 ~ 1000rpm, and slowly adding 10 ~ 60% alpha-cyanoacrylate alkane ester monomers dropwise containing 0 ~ 5% cation additions and 5 ~ 50% stabilizer concentrations is form aqueous phase system in the dilute hydrochloric acid of 0 ~ 10M;
Oil phase mixed solution is slowly added dropwise in aqueous phase system, after stirring 2 ~ 8h, forms the mixed liquor containing described alpha-cyanoacrylate alkane ester kernel;
Described step 3) is specially: regulated by the mixed liquor sodium hydroxide containing alpha-cyanoacrylate alkane ester kernel pH to neutral; add and calculate 1 ~ 10% protein matter by mass percentage; continue stirring 0.5 ~ 6 h; rotary evaporation 30 ~ 120min; the organic solvent that removing is remaining; centrifugal 2 ~ 15 min of 3000 ~ 10000 rpm, cross 0.2 ~ 0.8 μm of microporous filter membrane and carry out aseptic filtration, obtain albumen-composite nano-polymers vehicle suspension;
Described step 4) is specially: in described albumen-composite nano-polymers vehicle suspension, add the freeze drying protectant of 5 ~ 30%, lyophilizing 24 hours, forms powdery.
7. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 6, is characterized in that, described oil phase is one in ethyl acetate, dichloromethane, chloroform, acetone, ether or its mixture.
8. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 6, it is characterized in that, the mean diameter of described albumen-composite nano-polymers carrier is 20 ~ 1000nm.
9. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 8, it is characterized in that, the mean diameter of described albumen-composite nano-polymers carrier is 80 ~ 200nm.
10. a kind of albumen-composite nano-polymers carrier according to claim 1, is characterized in that, described albumen-composite nano-polymers carrier is for the preparation of antitumor drug.
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| CN107982239B (en) * | 2017-12-08 | 2020-02-21 | 中国医学科学院生物医学工程研究所 | Protein-based non-spherical microcapsule with hydrophobic drug crystal as template and preparation method thereof |
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