CN104622817B - A kind of protein polymer composite Nano carrier and preparation method thereof - Google Patents
A kind of protein polymer composite Nano carrier and preparation method thereof Download PDFInfo
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- CN104622817B CN104622817B CN201510038139.XA CN201510038139A CN104622817B CN 104622817 B CN104622817 B CN 104622817B CN 201510038139 A CN201510038139 A CN 201510038139A CN 104622817 B CN104622817 B CN 104622817B
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Abstract
The present invention is a kind of protein polymer composite Nano carrier and preparation method thereof, it is related to the nanometer formulation field of antineoplastic, it is to be designed and developed based on the natural lipoprotein structure feature of human body, with polymer core, hydrophilic protein shell, and flexibly modification target ligand can be needed according to treatment, construct the protein polymer composite Nano carrier with active targeting, stability in medicine body is improved, increases tumor-targeting；In order to improve load factor of the protein on polymer core, cation additions are added to be prepared for cationic polymer nanoparticle by prescription；The nanosuspension is milky white liquid, more uniform through the distribution of transmission electron microscopy visible particles, rounded；In order that it is more prone to preserve, stability is improved, add freeze drying protectant to carry out freeze-drying and form powdery；Protein polymer composite Nano diameter of carrier prepared by the present invention is uniform, and good stability can effectively encapsulate fat-soluble antineoplastic.
Description
Technical field
It is specifically a kind of to contain fat-soluble antineoplastic the present invention relates to the nanometer formulation field of antineoplastic
Albumen-composite nano-polymers carrier and preparation method thereof.
Background technology
Malignant tumour is to be only second to heart disease, threatens the second largest killer of global human health and lives.At present, chemical drugs
Thing treats the three big means that oncotherapy is together constituted with operative treatment, radiotherapy.
Chemotherapy is the treatment method that tumour cell is killed with chemicals.But because most of chemotherapeutics belongs to fat
Soluble drug, poorly water-soluble largely have impact on the curative effect of medicine.Further, since chemicals has stronger cell
Toxic action, lacks targeting characteristic again, can cause larger toxic and side effect, and pain is brought to patient, and treatment can be even caused sometimes
Interrupt.
The fat-soluble chemotherapeutics of listing at present, mostly using organic solvent and surfactant as solubilized means, no
Only targeting is poor, and the surfactant component for being used can also bring excitant or anaphylaxis, increases the pain of patient.Such as
Taxol and paclitaxel injection of dwelling, in order to reach the purpose of dissolving medicine, are respectively necessary for using surfactant polyoxy second more
Alkene castor oil and Tween-80（Polyoxyethylene sorbitan monoleate）And cosolvent ethanol；For another example etoposide injection, need to add Tween-80,
Polyethylene glycol, ethanol etc. reach the purpose of solubilized medicine.The use of the solubilizing components such as these surfactants is possible to cause one
The adverse reaction of series, such as allergic reaction, neurotoxicity, bone marrow suppression, fluid retention.Also some cancer therapy drugs, thin
The active anticancer of brilliance is shown in born of the same parents' experiment, but because dissolubility and irritating problem not yet have formulation application in clinic.
It is a kind of pharmaceutical component of the Chinese medicine dried venom of toads such as Bufalin, is one of most strong dried venom of toads diene acid lactone of antitumor action in the dried venom of toads.
Document report Bufalin can suppress tumor cell proliferation, promote tumor cell differentiation, apoptosis-induced, suppression at much lower concentrations
Tumor Angiongesis, and sensitiveness of the tumour cell to chemotherapeutics can be recovered, suppress Tumor Angiongesis etc..Bufalin is to more
Kind solid tumor is inhibited, and the inhibition to HCC is especially notable, and in cell experiment, Bufalin shows ratio
The stronger tumor cell of liver inhibition of many kinases targeting inhibitor Sorafenibs（Cao Y, Li HX, Xu LT, et al.
Bufalin enhances the anti-proliferative effect of sorafenib on hμman
hepatocellular carcinoma cells through downregulation of ERK [J]. Mol Biol
Rep, 2012, 39(2):1683）.But, the poorly water-soluble of Bufalin, half-life short produces excitant and the heart to human body
Dysentery（Ying Li , 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）, a variety of undesirable elements limit
The application of Bufalin, not yet has monomer formulation application in clinic.From from the aspect of the effectiveness and reliability of medication, this is developed
The cancer target nanometer formulation of class medicine is the means for most possibly solving its dissolubility and stimulating sex chromosome mosaicism, reduces the poison of medicine
Side effect, increases drug safety, plays its antitumous effect.
Poly- a-cyanoacrylate class material has good biocompatibility, is biodegradable the features such as nontoxic
High molecular polymer, has been widely used in the preparation of nanoparticulate carriers.Prepared with poly- a-cyanoacrylate class material
Nanoparticle can improve the stability of preparation, increase the solubility of medicine, be now widely used for compound and traditional Chinese medicine monomer with
And protein and polypeptide delivery systematic research.But individually using cyanoacrylate as carrier, exist and most of polymerizations
The similar problem of species carrier, after polymer nanoparticle drug carriers enter blood circulation, easily and immunoglobulin G
（immunoglobulin G）, complement protein C3(complement C3) and fibronectin（fibronectin）Deng in blood plasma
Opsonin combine, and absorbed by mononuclear phagocyte system, internal stability is not good；Do not possess tumor-targeting.
Albumin is natural hydrophilic protein matter, with safety non-toxic, non-immunogenicity, biodegradable, biofacies
Capacitive is good, the advantages of cheap and easy to get, and property quite stable, stabilization can be kept in the range of pH4-9.Made using albumin
It is carrier shell, by increasing capacitance it is possible to increase the hydrophily of carrier, improves carrier stability in blood, and make carrier that there is certain swelling
Knurl taxis, albumin is also available for modification containing many surface active groups, and target ligand, such as cholic acid can be modified on albumen,
Urso, enoxolone, antibody etc., existing lot of documents show that tumor cell surface has corresponding acceptor, can pass through
Intake of the mediation enhancing tumour cell of these parts to carrier, makes carrier have active targeting.
This patent, as shell, using polymer support as kernel, devises a kind of new targeting using hydrophilic protein
Albumen-polymer nanoparticle drug carriers, is used to contain antineoplastic.This carrier is based on the natural lipoprotein structure feature of human body
Design and develop, lipoprotein is that the naturally occurring lipid of human body transports carrier, with lipophilic kernel and hydrophilic protein shell mechanism.
According to the literature, lipoprotein can effectively transport fat-soluble cancer therapy drug, and with tumor-targeting（Can be targeted to mistake
The tumour cell of degree expression LDL receptor）.But lipoprotein needs to be extracted from fresh plasma, and extraction process is very multiple
It is miscellaneous, and apolipoprotein is unstable, easily aggregation, is difficult commercialization；In addition lipoprotein is only capable of targeted to LDL receptor
The tumour of overexpression, its application is restricted.Albumen-the polymer support of this patent design construction, with hydrophilic protein outside
Shell and the polymer support core of fat-soluble medicine can be accommodated, and can as needed modify different target ligands, with excellent
Drug carrying ability, remarkable internal compatibility and stability, and flexible targeting are the targeting vectors of great functionalization.
It is conventional increase polymer or liposome using hydrophilic material polyethylene glycol as the modification shell of carrier surface
Deng the method for stability in nano-carrier body, and using albumin as the shell mechanism of carrier, compared with polyethylene glycol, with spy
Other advantage,（1）It is easy to carry out surface modification, each albumin molecule has multiple carboxyls and amino, can modifies as needed
Functional group；（2）More preferable biocompatibility and opsonic action is gone, albumin is the natural hydrophilic protein existed in human body,
Non-immunogenicity, albumin can effectively reduce absorption of the albumen on carrier in blood plasma, when extending carrier circulation in blood
Between.（3）Itself there is certain tumour taxis, the vasopermeability increase of tumour and inflammatory tissue, albumin can pass through
GP60 (60-kDa glycoprotein) acceptor of tumor vascular endothelium, or by with tumor tissues in be rich in cysteine
Acidic secretion protein（Secreted protein acid and rich in cysteine, SPARC) promote carrier swollen
Intake in tumor tissue.
Also there are many researchs and patent that albumin is used alone as carrier at present, but albumin is used alone as load
Body there may be problems with.（1）Need to typically be solidified by heating or chemical cross-linking agent.May influence medicine stability or
The hydrophily of albumen；（2）The nanoparticle or microspherulite diameter prepared as material with albumin are larger, typically more than 500 μm.The U.S.
Biological Science Co., Ltd develops Nab technologies and prepares medicine albumin nano granular, and the technology is had using the high pressure even technology of breast to pastille
The mixed system of machine phase and the phase of water containing albumen carries out high-pressure homogenising, prepares nanoparticle, and the technology is selected the physicochemical property of medicine
Property is stronger, and medicine need to have stronger compatibility with albumin.In addition, according to《Pharmacy》（Cui Fude is edited,《Pharmacy》
The second edition, 2011:536.）Classification, these carriers belong to passive targeted preparation, and passive targeted preparation is only capable of using in body
The retention or phagocytosis of Different Organs, tissue or reticuloendothelial system to different size of particulate, conduct drugs to target portion
Position, Targeting Effect is not good enough.
The content of the invention
In order to solve the above technical problems, the invention provides a kind of albumen-composite nano-polymers carrier, to contain liposoluble
The polymer of property antineoplastic as kernel, using the albumin with targeting as hydrophilic outer shell；The present invention is also carried
The preparation method of above albumen-composite nano-polymers carrier is supplied, according to treatment needs, target can have been modified on albumin in advance
To group, the albumen with active targeting-composite nano-polymers carrier is constructed.
The present invention is achieved through the following technical solutions：A kind of albumen-composite nano-polymers carrier, including polymer core
With targeting protein shell；The polymer core is alpha-cyanoacrylate alkyl ester kernel, the alpha-cyanoacrylate alkyl ester kernel bag
Carry fat-soluble antineoplastic；The targeting protein shell is wrapped in the surface of the alpha-cyanoacrylate alkyl ester kernel；Institute
Targeting protein shell is stated for hydrophilic protein shell.
The albumen-composite nano-polymers carrier is calculated including following components by mass percentage：1 ~ 10% it is antitumor
Medicine, 10 ~ 60% alpha-cyanoacrylate alkyl ester, 0.5 ~ 10% cation additions, 1 ~ 10% protein matter, 20 ~ 60%
Stabilizer and 5 ~ 30% freeze drying protectant.
The antineoplastic is Bufalin, Etoposide, taxol, dwell taxol, Gefitinib, HCPT more
With the one kind in adriamycin, curcumin, fluorouracil, endoxan, Irinotecan, mitoxantrone, cis-platinum.
The alpha-cyanoacrylate alkyl ester be Methyl 2-cyanoacrylate MCA, cyanacrylate ECA, alpha-cyanoacrylate just
One kind in the own ester HCA of butyl ester BCA, isobutylcyanoacrylate IBCA, alpha-cyanoacrylate and alpha-cyanoacrylate dissident's ester IHCA.
The cation additions are the one kind or its composition in octadecylamine, Polyethylenimine, 1B and arginine.
The protein matter is that native protein or synthetic protein or albuminoid and its derivative or modified thing are repaiied
One kind or its mixture in the protein of decorations.
The stabilizer is Pluronic F68, pluronic F123, pluronic F127, macrodex, dextran
40th, one kind or its mixture in Dextran-20, polyvinyl alcohol, Tween-80 and Arlacel-80.
The freeze drying protectant be it is following in one kind：（1）Carbohydrate/polyalcohol：Sucrose, trehalose, mannitol, lactose,
Glucose, maltose；（2）Polymer：HES, PVP, PEG, glucan, albumin；（3）Amino acid：Serine, glutamic acid
Sodium, alanine, glycine, methyl amimoacetic acid；（4）Salt and amine：One kind or its mixture in phosphate, acetate, citrate.
The protein matter is albumin.
The trim is cholic acid, urso, enoxolone, hyaluronic acid, galactolipin, folic acid and lipoprotein receptor
Identification recombinant polypeptide in one or more；The trim can pass through with the amino of the protein matter, carboxyl or sulfydryl
Covalent bond is connected.
The preparation method of the albumen-composite nano-polymers carrier, comprises the following steps：
1）Preparation contains the alpha-cyanoacrylate alkyl ester kernel of the antineoplastic；
Calculate by mass percentage, weigh 1 ~ 10% antineoplastic, 0.5 ~ 8% cation additions and 5 ~ 40% stabilizers
Dissolved with oil phase, oil phase mixed solution, rotating speed is controlled for 100 ~ 1000rpm, slowly by 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers
It is added dropwise over containing 5 ~ 40% stabilizer concentrations to form aqueous phase system in the watery hydrochloric acid of 0 ~ 10M；
Or 1 ~ 10% antineoplastic and the dissolving of 5 ~ 40% stabilizer oil phases are taken, oil phase mixed solution controls the rotating speed to be
100 ~ 1000rpm, slowly by 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers dropwises add containing 5 ~ 40% stabilizers and 0.5 ~ 5% sun from
Sub- additive concentration for 0 ~ 10M watery hydrochloric acid in form aqueous phase system；
Or take 1 ~ 10% antineoplastic, the dissolving of 0.5 ~ 8% cation additions oil phase, oil phase mixed solution, control
Rotating speed is 100 ~ 1000rpm, is containing 5-50% stabilizer concentrations by the addition of 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers dropwises slowly
Aqueous phase system is formed in the watery hydrochloric acid of 0 ~ 10M；
Or the dissolving of 1 ~ 10% antineoplastic oil phase is taken, oil phase mixed solution controls rotating speed for 100 ~ 1000rpm, delays
It is slow to be with 0.5 ~ 5% cation additions concentration containing 5-50% stabilizers by the addition of 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers dropwises
Aqueous phase system is formed in the watery hydrochloric acid of 0 ~ 10M；
Or 1 ~ 10% antineoplastic, 0.5 ~ 8% cation additions are taken with the dissolving of 5 ~ 40% stabilizer oil phases, oil phase
Mixed solution, controls rotating speed for 100 ~ 1000rpm, slowly add containing 5 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers dropwises ~
Aqueous phase system is formed in 40% stabilizer and the watery hydrochloric acid that 0.5 ~ 5% cation additions concentration is 0 ~ 10M；
Or take 1 ~ 10% antineoplastic and the dissolving of 0.5 ~ 8% cation additions oil phase, oil phase mixed solution, control
Rotating speed is 100 ~ 1000rpm, slowly add containing 5 ~ 50% stabilizers and 0.5 10 ~ 60% alpha-cyanoacrylate alkyl ester monomers dropwises ~
5% cation additions concentration for 0 ~ 10M watery hydrochloric acid in form aqueous phase system；
Oil phase mixed solution is slowly added dropwise in aqueous phase system, after 2 ~ 8h of stirring, is formed and is contained the alpha-cyanoacrylate
The mixed liquor of alkyl ester kernel；
Mass percent is calculated, and the oil phase and the water phase cationic additive total amount are 0.5 ~ 10%；Oil phase and water
Stabilizer total amount is 20 ~ 60% in phase.
The oil phase is one kind or its mixture in ethyl acetate, dichloromethane, chloroform, acetone, ether.
2）The native protein extraction purification is modified on protein, obtains targeting protein shell；
The method of modifying of wherein protein is concretely comprised the following steps：Trim is added into tetrahydrofuran or dimethylformamide DMF
Dissolving, then it is separately added into 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate and N-hydroxy-succinamide；0
DEG C stirring 2 ~ 6 h, continue to be stirred overnight at room temperature, recrystallize, obtain the active ester of trim, be dissolved in dimethylformamide DMF
In, it is added dropwise in the solution dissolved with albumin, stirring while adding, room temperature continues to stir 2 ~ 6 h；48 h of reaction solution dialysis
Afterwards, 12000 rpm centrifugations 10min, removes unreacted trim active ester and its hydrolysate, the lyophilized product for obtaining white
Thing, the albumin as modified, albumin is 1 with the mol ratio of trim:5~1:60.
3）In targeting protein shell described in the alpha-cyanoacrylate alkyl ester kernel outer cladding, formed and contain albumen-polymerization
The suspension of thing composite Nano carrier；
Concretely comprise the following steps：Mixed liquor containing alpha-cyanoacrylate alkyl ester kernel is adjusted into pH to neutrality with NaOH, plus
Enter 1 ~ 10% protein matter of calculation by mass percentage, continue to stir 0.5 ~ 6 h, 30 ~ 120min of rotary evaporation, remove remnants'
Organic solvent, 3000 ~ 10000 rpm are centrifuged 2 ~ 15 min, and crossing 0.2 ~ 0.8 μm of miillpore filter carries out aseptic filtration, obtains final product egg
In vain-composite nano-polymers vehicle suspension.
Protein matter is combined by charge effect, hydrophobic force, Van der Waals force equimolecular intermolecular forces with non-covalent bond
The surface of alpha-cyanoacrylate alkyl ester kernel.
4）The freeze drying protectant is added in suspension carries out freeze-drying, forms powdery.
Concretely comprise the following steps：5 ~ 30% frozen-dried protective is added in the albumen-composite nano-polymers vehicle suspension
Agent, freezes 24 hours, forms powdery.
The temperature of the rotary evaporation is 0 ~ 60 DEG C, preferably 20 ~ 30 DEG C.
The average grain diameter of the albumen-composite nano-polymers carrier is 20 ~ 1000nm, preferably 80 ~ 200nm.
The albumen-composite nano-polymers carrier is used to prepare antineoplastic.
Beneficial effect：The present invention compared with prior art, is designed and developed based on the natural lipoprotein structure feature of human body
, with polymer core, hydrophilic protein shell, and target ligand can be flexibly modified, fat-soluble medicine can be effectively contained,
Stability in medicine body is improved, increases tumor-targeting；In order to improve load factor of the protein on polymer core, by
Cation additions are added to be prepared for cationic polymer nanoparticle in prescription, albumin isoelectric point is 4.5 or so, is situated between in neutrality
It is negatively charged in matter, can be combined in polymer nanoparticle table by charge effect, hydrophobic force, Van der Waals force equimolecular intermolecular forces
Face；Targeting group can be modified on albumen in advance according to treatment needs, thus construct the albumen with active targeting-poly-
Compound composite Nano carrier；The nanosuspension is milky white liquid, more uniform through the distribution of transmission electron microscopy visible particles,
It is rounded；In order that it is more prone to preserve, stability is improved, add freeze drying protectant to carry out freeze-drying and form powdery.
Brief description of the drawings
Fig. 1 is to carry Bufalin-urso modified albumin-composite nano-polymers grain according to prepared by example 1
Grain size distribution.
Fig. 2 is to carry Bufalin-urso modified albumin-composite nano-polymers grain according to prepared by example 1
Transmission electron microscope picture.
Fig. 3 is Bufalin bulk drug and the load Bufalin-urso modified albumin-polymerization prepared according to example 1
The In-vitro release curves of medicine in thing composite nano-granule.
Specific embodiment
Below by embodiment combination accompanying drawing, the present invention is described further, but protection scope of the present invention is not limited
In this.
Embodiment 1：Carry the preparation of Bufalin-urso modified protein-composite nano-polymers grain.
3.5 mg Bufalins, 5 mg octadecylamines and 30mg pluronic F127 dichloromethane is weighed respectively to dissolve.Slowly
By 60 mg BCAs（BCA）It is added dropwise over 5 mL and contains macrodex and each 25 mg's of Pluronic F68
Watery hydrochloric acid（0.1 M）In, dichloromethane mixed solution is slowly added dropwise in system, side stirring is added dropwise.After stirring 4 h, hydrogen is used
Sodium oxide molybdena adjusts pH to neutrality, adds the bovine serum albumin(BSA) of 5 mg ursos modification, continues to stir 1 h.Rotary evaporation
Remaining dichloromethane is removed, 5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load Bufalin-urso modification egg
In vain-composite nano-polymers vehicle suspension.
30mg sweet dews are added in Bufalin-urso modified protein-composite nano-polymers vehicle suspension is carried
Alcohol, 30mg trehaloses are lyophilized 24 hours, obtain powder, can again disperse to form egg by adding sterilized water or physiological saline
In vain-composite nano-polymers vehicle suspension, particle size is almost preceding unchanged with lyophilized.
Embodiment 2：Carry the preparation of Bufalin-hyaluronic acid decorated albumen-composite nano-polymers grain.
10 mg Bufalins, 10 mg octadecylamine dichloromethane are weighed respectively to dissolve.Slowly by 100 mg alpha-cyanoacrylates
Ethyl ester（ECA）It is added dropwise over the watery hydrochloric acid that 10 ml contain macrodex and each 50 mg of Pluronic F68（0.1M）In, will
Dichloromethane mixed solution is slowly added dropwise in system, and side stirring is added dropwise.After stirring 4 h, pH is adjusted to neutrality with NaOH,
The bovine serum albumin(BSA) for adding 20 mg hyaluronic acid decorated, continues to stir 1 h.Rotary evaporation removes remaining dichloromethane,
5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load Bufalin-hyaluronic acid decorated albumen-composite nano-polymers carrier
Suspension.
50 mg sucrose are added in Bufalin-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension is carried
Lyophilized 24 hours, powder is obtained, can again disperse to be formed that albumen-polymer is compound to be received by adding sterilized water or physiological saline
Rice vehicle suspension, particle size is almost preceding unchanged with lyophilized.
Embodiment 3：Carry the preparation of Etoposide-enoxolone modified protein-composite nano-polymers grain.
30 mg Etoposides, 3 mg octadecylamines are weighed respectively to be dissolved with 50 mg pluronic F127 dichloromethane.It is slow
Slowly by 210 mg Methyl 2-cyanoacrylates（MCA）It is added dropwise over 5 mL and contains macrodex and each 10 mg of Pluronic F68
Watery hydrochloric acid（0.1 M）In, dichloromethane mixed solution is slowly added dropwise in system, side stirring is added dropwise.After stirring 4 h, use
NaOH adjusts pH to neutrality, adds the bovine serum albumin(BSA) of 5 mg enoxolones modification, continues to stir 1 h.Rotary evaporation
Remaining dichloromethane is removed, 5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load Etoposide-enoxolone modification egg
In vain-composite nano-polymers vehicle suspension.
20 mg PEG are added in Etoposide-enoxolone modified protein-composite nano-polymers vehicle suspension is carried
400,10 mg glycine are lyophilized 24 hours, obtain powder, can again disperse to be formed by adding sterilized water or physiological saline
Albumen-composite nano-polymers vehicle suspension, particle size is almost preceding unchanged with lyophilized.
Embodiment 4：Carry the preparation of taxol-modified with folic acid albumen-composite nano-polymers grain.
5 mg taxols, 12mg octadecylamines is weighed respectively to be dissolved with 60 mg pluronic F127 dichloromethane.Slowly
By 40 mg isobutylcyanoacrylates（IBCA）It is added dropwise over 10 ml and contains 10 mg PVAC polyvinylalcohols 1788 and 1.5 mg
Arginic watery hydrochloric acid（0.1M）In, dichloromethane mixed solution is slowly added dropwise in system, side stirring is added dropwise.Stir 4 h
Afterwards, pH to neutrality is adjusted with NaOH, adds the bovine serum albumin(BSA) of 15 mg modified with folic acid, continue to stir 1 h.Rotation is steamed
Hair removes remaining dichloromethane, and 5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load taxol-modified with folic acid albumen-
Composite nano-polymers vehicle suspension.
10 mg mannitol, 10 are added in taxol-modified with folic acid albumen-composite nano-polymers vehicle suspension is carried
Mg trisodium citrates are lyophilized 24 hours, obtain powder, can again disperse to form egg by adding sterilized water or physiological saline
In vain-composite nano-polymers vehicle suspension, particle size is almost preceding unchanged with lyophilized.
Embodiment 5：Carry the preparation of taxol-urso modified proteins of dwelling-composite nano-polymers grain more.
Weigh and dwell 10 mg taxol dichloromethane more and dissolve.Slowly by 60 mg BCAs（BCA）By
It is added dropwise to 10 ml and contains the mg pluronic F127 of 25 mg PVAC polyvinylalcohols 1788,15, macrodex and pluronic
The watery hydrochloric acid of each 20 mg of F68 and 10 mg Polyethylenimines 1200（0.1 M）In, dichloromethane mixed solution is slowly added dropwise into body
In system, side stirring is added dropwise.After stirring 4 h, pH to neutrality is adjusted with NaOH, add the ox of 10 mg ursos modification
Seralbumin, continues to stir 1 h.Rotary evaporation removes remaining dichloromethane, and 5000 rpm are centrifuged 3 min, crosses micropore filter
Film, obtains final product load taxol-urso modified protein-composite nano-polymers vehicle suspensions of dwelling more.
25 mg are added in taxol-urso modified protein-composite nano-polymers vehicle suspensions of dwelling are carried more
Mannitol, 20 mg PVP K30,30mg sodium glutamates are lyophilized 24 hours, obtain powder, can be by adding sterilized water or physiology
Salt solution disperses to form albumen-composite nano-polymers vehicle suspension again, and particle size is almost preceding unchanged with lyophilized.
Embodiment 6：Carry the preparation of Gefitinib-urso modified protein-composite nano-polymers grain.
3 mg Gefitinibs are weighed respectively to be dissolved with 90 mg pluronic F127 chloroforms.Slowly by 25 mg cyanogen
Base Hexyl 2-propenoate（HCA）It is added dropwise over 10 ml and contains macrodex and each 25 mg of Pluronic F68,10 mg arginine
Watery hydrochloric acid（0.1 M）In, chloroform mixed solution is slowly added dropwise in system, side stirring is added dropwise.After stirring 4 h, use
NaOH adjusts pH to neutrality, adds the bovine serum albumin(BSA) of 10 mg ursos modification, continues to stir 1 h.Rotation is steamed
Hair removes remaining chloroform, and 5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load Gefitinib-urso and repair
Decorations albumen-composite nano-polymers vehicle suspension.
25 mg are added in Gefitinib-urso modified protein-composite nano-polymers vehicle suspension is carried
Lactose, 20 mg glycine trisodiums are lyophilized 24 hours, obtain powder, can again be disperseed by adding sterilized water or physiological saline
Albumen-composite nano-polymers vehicle suspension is formed, particle size is almost preceding unchanged with lyophilized.
Embodiment 7：Carry the preparation that hydroxyl carries camptothecine-hyaluronic acid decorated albumen-composite nano-polymers grain.
5 mg HCPTs, 5 mg octadecylamine dichloromethane are weighed respectively to dissolve.Slowly by 50 mg alpha-cyanoacrylates
Dissident's ester（IHCA）It is added dropwise over 5 ml and contains 75 mg PVAC polyvinylalcohols 1788 and 20 mg pluronic F127, and 5mg
The watery hydrochloric acid of lysine（0.1M）In, dichloromethane mixed solution is slowly added dropwise in system, side stirring is added dropwise.Stir 4 h
Afterwards, pH to neutrality is adjusted with NaOH, the bovine serum albumin(BSA) for adding 5 mg hyaluronic acid decorated continues to stir 1 h.Rotation
Evaporation removes remaining dichloromethane, and 5000 rpm are centrifuged 3 min, cross miillpore filter, obtains final product load hydroxyl and carries camptothecine-hyalomitome
Sour modified protein-composite nano-polymers vehicle suspension.
20 mg are added carrying during hydroxyl carries camptothecine-hyaluronic acid decorated albumen-composite nano-polymers vehicle suspension
Macrodex, 30 mg trehaloses are lyophilized 24 hours, obtain powder, can again be disperseed by adding sterilized water or physiological saline
Albumen-composite nano-polymers vehicle suspension is formed, particle size is almost preceding unchanged with lyophilized.
Embodiment 8：Carry the preparation of adriamycin-enoxolone modified protein-composite nano-polymers grain.
7.5 mg adriamycins, 10 mg octadecylamines are weighed respectively to be dissolved with 20 mg pluronic F127 dichloromethane.It is slow
Slowly by 75 mg isobutylcyanoacrylates（IBCA）It is added dropwise over dilute salt that 10 ml contain 50 mg PVAC polyvinylalcohols 1788
Acid（0.1M）In, dichloromethane mixed solution is slowly added dropwise in system, side stirring is added dropwise.After stirring 4 h, NaOH is used
Regulation pH adds the bovine serum albumin(BSA) of 10 mg enoxolones modification to neutrality, continues to stir 1 h.Rotary evaporation removes remaining
Dichloromethane, 5000 rpm be centrifuged 3 min, cross miillpore filter, obtain final product load adriamycin-enoxolone modified protein-polymer
Composite Nano vehicle suspension.
The sweet ammonia of 20 mg is added in adriamycin-enoxolone modified protein-composite nano-polymers vehicle suspension is carried
Acid, 20mg PEG400,10 mg trisodium citrates are lyophilized 24 hours, obtain powder, can be by adding sterilized water or physiology salt
Water disperses to form albumen-composite nano-polymers vehicle suspension again, and particle size is almost preceding unchanged with lyophilized.
Embodiment 9：Carry the preparation of taxol-enoxolone modified protein-composite nano-polymers grain.
20 mg taxols, 15 mg octadecylamines are weighed respectively to be dissolved with 10 mg Pluronic F68 dichloromethane.Slowly
By 70 mg BCAs（BCA）It is added dropwise over 10 ml and contains 20 mg PVAC polyvinylalcohols 1788,20mg dextrorotation
Sugared acid anhydride 70 and the arginic watery hydrochloric acid of 4mg（0.1M）In, dichloromethane mixed solution is slowly added dropwise in system, side is added dropwise and stirs
Mix.After stirring 4 h, pH to neutrality is adjusted with NaOH, add the bovine serum albumin(BSA) of 20 mg enoxolones modification, continued
Stir 1 h.Rotary evaporation removes remaining dichloromethane, and 5000 rpm are centrifuged 3 min, cross miillpore filter, obtain final product load taxol-
Enoxolone modified protein-composite nano-polymers vehicle suspension.
10 mg trehaloses are added in taxol-enoxolone modified protein-composite nano-polymers vehicle suspension is carried
Lyophilized 24 hours, powder is obtained, can again disperse to be formed that albumen-polymer is compound to be received by adding sterilized water or physiological saline
Rice vehicle suspension, particle size is almost preceding unchanged with lyophilized.
Experimental example 1：Particle diameter and distribution.
Using Malvern laser granulometry（Zetasizer Nano ZS90 type laser particle analyzers, Britain's Malvern is public
Department）Particle diameter is determined, the measurement result of embodiment 1 is as shown in Figure 2.Measurement result shows that the Bufalin nanometer prepared by the present invention is made
The average grain diameter of agent is 149.3 nm, and polydispersity coefficient is 0.073.
Experimental example 2：Morphology is distributed.
Take preparation appropriate, with distilled water diluting after, take it is one after another drop of placed in a moment on the copper mesh for being covered with carbon and supporting film,
2% phosphotungstic acid is added dropwise carries out negative staining, uses transmission electron microscope（Tecnai 12, Philips company, Holland）Observation
Particle shape, the observed result of embodiment 1 is shown in Fig. 2.From Figure 2 it can be seen that particle distribution is more uniform, it is rounded.
Experimental example 3：Protein load rate.
Albumen loads with rate：Albumen loads with rate（%）=（1-MIt is free/MThrow）*100
In formula, MIt is freeRefer to free BSA（modified）Amount（μg）, MThrowRefer to the BSA of input（modified）Amount（μg）.
Urso modified albumin is 94.33% in the load factor of carrier surface in embodiment 1；It is hyaluronic acid decorated in embodiment 2
Albumin carrier surface load factor be 89.72%；The albumin of enoxolone modification is in the negative of carrier surface in embodiment 3
Load rate is 91.19%；The albumin of the Folic Acid of embodiment 4 modification is 85.42% in the load factor of carrier surface；Bear in embodiment 5
The albumin of deoxycholic aicd modification is 92.25% in carrier surface load factor；The albumin of urso modification in embodiment 6
It is 95.18% in carrier surface load factor；Hyaluronic acid decorated albumin is in carrier surface load factor in embodiment 7
87.49%；The albumin of enoxolone modification is 92.31% in the load factor of carrier surface in embodiment 8；Radix Glycyrrhizae in experimental example 9
The albumin of hypo acid modification is 90.76% in the load factor of carrier surface.
Experimental example 4：Release in vitro.
Phosphate buffer with pH7.4 investigates the release in vitro behavior of preparation as dissolution medium.Bufalin saturated solution
Preparation of traditional Chinese medicine prepared by middle medicine and embodiment 1（Bufalin）Release profiles it is as shown in Figure 3.As seen from Figure 3, in preparation
The release of medicine has certain slow release effect compared with saturated solution, but be can reach substantially in 24h and discharge completely.
Experimental example 5：Cell inhibitory rate and Evaluation on Its Targeting Performance.
Investigate load Bufalin-urso modified protein-composite nano-polymers grain prepared by embodiment 1 and uncoated
IC of the common Bufalin-polymer beads of modified protein to tumor cell of liver HepG250Respectively 32.7nM and 98.6nM,
Can be seen that suppression effect of the load Bufalin-urso modified protein-composite nano-polymers grain to tumor cell of liver HepG2
Fruit is significantly higher than the common Bufalin-polymer beads of uncoated modified protein.Show to coat the albumen that urso is modified
Afterwards, intake of the tumour cell to carrier can be increased, with tumour cell targeting.Load Etoposide prepared by investigation embodiment 3-
Common Etoposide-the polymer beads pair of enoxolone modified protein-composite nano-polymers grain and uncoated modified protein
The IC of tumor cell of liver HepG250Respectively 97.3 μM and 148.8 μM, it can be seen that load Etoposide-enoxolone modified protein-
Composite nano-polymers grain is significantly higher than the common support of uncoated modified protein to the inhibition of tumor cell of liver HepG2
Pool glycosides-polymer beads.After showing coating decoration albumen, intake of the tumour cell to carrier can be increased, with tumour cell target
Tropism.Investigate and dwell load prepared by embodiment 5 taxol-urso modified protein-composite nano-polymers grain more and do not wrap
Cover the common ICs of the taxol-polymer beads to tumor cell of liver HepG2 of dwelling of modified protein more50Respectively 62.7nM and
128.9nM, it can be seen that carry taxol-urso modified proteins of dwelling-composite nano-polymers grain to tumor cell of liver more
The inhibition of HepG2 is significantly higher than common taxol-polymer beads of dwelling of uncoated modified protein more.Show coating decoration
After albumen, intake of the tumour cell to carrier can be increased, with tumour cell targeting.
Preferred embodiment but the present invention is not limited to above-mentioned implementation method to the embodiment for of the invention, but should
This recognizes that those skilled in the art may make various modifications and transformation to the present invention, and these modifications and transformation are also belonged to
In the scope of the present invention defined in appended claims.
Claims (9)
1. a kind of albumen-composite nano-polymers carrier, it is characterised in that including polymer core and targeting protein shell；
The polymer core is alpha-cyanoacrylate alkyl ester kernel, and the alpha-cyanoacrylate alkyl ester kernel contains fat-soluble anti-swollen
Tumor medicine；
The targeting protein shell is wrapped in the surface of the alpha-cyanoacrylate alkyl ester kernel；
The targeting protein shell is hydrophilic protein shell；
Albumen-composite nano-polymers carrier is calculated including following components by mass percentage：1~10% antineoplastic,
10~60% alpha-cyanoacrylate alkyl ester, 0.5~10% cation additions, 1~10% protein matter, 20~60%
Stabilizer and 5~30% freeze drying protectant.
2. a kind of albumen-composite nano-polymers carrier according to claim 1, it is characterised in that
The antineoplastic be Bufalin, Etoposide, taxol, taxol of dwelling more, Gefitinib, HCPT and Ah
One kind in mycin, curcumin, fluorouracil, endoxan, Irinotecan, mitoxantrone, cis-platinum；
The alpha-cyanoacrylate alkyl ester is Methyl 2-cyanoacrylate MCA, cyanacrylate ECA, BCA
One kind in the own ester HCA of BCA, isobutylcyanoacrylate IBCA, alpha-cyanoacrylate and alpha-cyanoacrylate dissident's ester IHCA；
The cation additions are the one kind or its composition in octadecylamine, Polyethylenimine, 1B and arginine；
The protein matter is the egg that native protein, synthetic protein, albuminoid and its derivative or modified thing are modified
One kind or its mixture in white matter；
The stabilizer be Pluronic F68, pluronic F123, pluronic F127, macrodex, Dextran 40,
One kind or its mixture in Dextran-20, polyvinyl alcohol, Tween-80 and Arlacel-80；
The freeze drying protectant is one kind as described below：
(1) carbohydrate/polyalcohol：Sucrose, trehalose, mannitol, lactose, glucose, maltose；
(2) polymer：HES, PVP, PEG, glucan, albumin；
(3) amino acid：Serine, sodium glutamate, alanine, glycine, methyl amimoacetic acid；
(4) salt and amine：One kind or its mixture in phosphate, acetate, citrate.
3. a kind of albumen-composite nano-polymers carrier according to claim 2, it is characterised in that the protein matter
It is albumin；The trim is cholic acid, urso, enoxolone, hyaluronic acid, galactolipin, folic acid and lipoprotein receptor
Body identification recombinant polypeptide in one or more.
4. a kind of preparation method of albumen-composite nano-polymers carrier according to claim 1, comprises the following steps：
1) the alpha-cyanoacrylate alkyl ester kernel for containing the antineoplastic is prepared；
2) protein matter is prepared, targeting protein shell is obtained；
3) in targeting protein shell described in the alpha-cyanoacrylate alkyl ester kernel outer cladding, form multiple containing albumen-polymer
Close the suspension of nano-carrier；
4) freeze drying protectant is added in suspension carries out freeze-drying, forms powdery.
5. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 4, it is characterised in that described
Step 1) it is specially：
Calculate by mass percentage, weigh 1~10% antineoplastic, 0~8% cation additions and 0~40% stabilizer
Dissolved with oil phase, oil phase mixed solution, rotating speed is controlled for 100~1000rpm, slowly by 10~60% alpha-cyanoacrylate alkyl ester lists
Body is added dropwise over containing forming water phase in 0~5% cation additions and watery hydrochloric acid that 5~50% stabilizer concentrations are 0~10M
System；
Oil phase mixed solution is slowly added dropwise in aqueous phase system, after 2~8h of stirring, is formed and is contained the alpha-cyanoacrylate alkyl ester
The mixed liquor of kernel；
The step 3) it is specially：Mixed liquor containing alpha-cyanoacrylate alkyl ester kernel is adjusted into pH to neutrality with NaOH,
Add and calculate 1~10% protein matter by mass percentage, continue to stir 0.5~6h, 30~120min of rotary evaporation, remove residual
Remaining organic solvent, 3000~10000rpm is centrifuged 2~15min, and crossing 0.2~0.8 μm of miillpore filter carries out aseptic filtration, obtains final product
Albumen-composite nano-polymers vehicle suspension；
The step 4) it is specially：5~30% lyophilized guarantor is added in the albumen-composite nano-polymers vehicle suspension
Shield agent, freezes 24 hours, forms powdery.
6. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 5, it is characterised in that described
Oil phase is one kind or its mixture in ethyl acetate, dichloromethane, chloroform, acetone, ether.
7. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 5, it is characterised in that described
The average grain diameter of albumen-composite nano-polymers carrier is 20~1000nm.
8. the preparation method of a kind of albumen-composite nano-polymers carrier according to claim 7, it is characterised in that described
The average grain diameter of albumen-composite nano-polymers carrier is 80~200nm.
9. a kind of albumen-composite nano-polymers carrier according to claim 1, it is characterised in that the albumen-polymerization
Thing composite Nano carrier is used to prepare antineoplastic.
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| CN105688228A (en) * | 2016-03-03 | 2016-06-22 | 中国医学科学院生物医学工程研究所 | Preparation method and application of cationic polymer-protein nano-carrier system |
| 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 |
| CN108743956B (en) * | 2018-04-24 | 2021-09-21 | 四川百利药业有限责任公司 | Albumin-bound anthracycline antitumor antibiotic preparation and preparation method thereof |
| CN109453138B (en) * | 2018-11-28 | 2022-03-22 | 江苏大学 | Drug-loaded albumin micro-particle or nanoparticle and preparation method thereof |
| CN112245409B (en) * | 2020-10-23 | 2022-03-15 | 安徽大学 | Vegetable protein-ursodesoxycholic acid sustained-release nanoparticle composite microcapsule and preparation method thereof |
| CN112972429A (en) * | 2021-03-24 | 2021-06-18 | 齐鲁工业大学 | Bovine serum albumin-nano silver modified chitosan nano drug delivery system and preparation method thereof |
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