CN109553684A - A kind of nano-carrier albumen and its preparation method and application - Google Patents
A kind of nano-carrier albumen and its preparation method and application Download PDFInfo
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- CN109553684A CN109553684A CN201710875115.9A CN201710875115A CN109553684A CN 109553684 A CN109553684 A CN 109553684A CN 201710875115 A CN201710875115 A CN 201710875115A CN 109553684 A CN109553684 A CN 109553684A
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- carrier albumen
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- albumen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5169—Proteins, e.g. albumin, gelatin
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2710/10011—Adenoviridae
- C12N2710/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2720/12322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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Abstract
The present invention provides a kind of nano-carrier albumen and its preparation method and application, the nano-carrier albumen includes protein nano particle, flexible peptide and albumin binding peptide, is connected wherein the protein nano particle passes through flexible peptide with albumin binding peptide.Nano-carrier albumen of the invention has reversible closing hollow dome structure, surface amalgamation and expression has albumin binding domain, on the one hand the targeting transport of small molecule, anti-tumor drug may be implemented, on the other hand the load medicine albumen that partial size is big, blood halflife is long can be formed in conjunction with seralbumin, improve the targeted delivery efficiency of tumour medicine.
Description
Technical field
The invention belongs to biopharmaceutical technology, it is related to a kind of nano-carrier albumen and its preparation method and application, tool
Body is related to a kind of nano-carrier albumen and its preparation method and application of albumin in combination.
Background technique
For malignant tumour as one of the disease for seriously threatening human life and health, treatment is always global problem.Mesh
The small molecule chemotherapeutics drugs such as adriamycin, taxol, the oxaliplatin of the preceding research and development that succeeded, although can effectively inhibit tumour cell
Growth, but these drug side-effects are big, also can serious injuring normal cell while killing tumor cell；Major partization
Treating drug, accretion rate is fast in vivo, half-life short, therefore in order to maintain drug effect, it is necessary to patient's frequent drug administration, not only cause
The waste of drug, and medical expense has been aggravated, seriously limit the application of anticancer drug in the course of disease treatment.
Utilize high-permeability and retention effect (the enhanced permeability and retention of solid tumor
Effect, EPR), nano-carrier has significant advantage in terms of improving anti-tumor drug to the targeted delivery of tumor locus, various
The nano-medicament carrier of type is designed and develops, such as liposome, polymeric micelle, carbon nanotube and protein nano particle etc..
Wherein, protein nano particle is due to receiving significant attention with good biocompatibility and biodegradability.
Protein nano particle be it is a kind of by dozens of into a spontaneous inside for assembling formation in an orderly manner of protein protomer up to a hundred
Empty ball-type or class ball-type nano particle, particle size range 10-200nm.Common protein nano particle includes that ferritin, heat are stopped
Gram albumen and viruslike particle (virus-like particles, VLP), these protein bodies are in vivo or actively or passively
Ground is gathered in tumor tissues, realizes the targeting to tumour.The primary amino acid sequences of protein nano particle are carried out suitably
Increase or delete, will not influence the enclosed construction of protein nano particle；And under the influence of the factors such as temperature, pH, denaturant, egg
The closing ball shape structure of white nano particle, which can become loose, even will be completely dissociated, and after removing these factors, enclosed construction again may be used
To restore.Therefore, the reversible protein nano particle of this inner hollow, structure is a kind of drug delivery vehicle of great potential.
106110333 A of CN discloses a kind of using ferritin as anti-tumor drug of carrier and preparation method thereof, this method
By the mixed solution of ferritin and anti-tumor drug at 200-800MPa HIGH PRESSURE TREATMENT 6-20h, product is isolated and purified
The anti-tumor drug using ferritin as carrier is obtained afterwards, realizes the targeted delivery of anti-tumor drug, but can not still solve to resist
The problem of tumour medicine accretion rate is fast, half-life short.
Carrying residence time of the medicine medium in blood circulation system is an important factor for influencing drug targeting delivery efficiency.Prolong
The half-life period of long protein nano particle in vivo generallys use two methods: (1) chemical modification: by high molecular weight hydrophilic polymer
Polyethylene glycol covalent coupling blocks the enzyme of protease in the surface of protein nano particle to increase the size of protein nano particle
Solution effect, to extend its circulation time in blood, the shortcomings that this method is process complexity, and product homogenieity is difficult to control
System, modification will increase the risk of drug leakage；(2) Gene Fusion: gene fusion technique is used, in protein nano particle table
The shortcomings that low charged polypeptide sequence of face fusion hydrophily, the effect of the similar chemical modification of realization, this method is can not to increase egg
The hollow structure of white nano particle causes drug/protein loaded than decline.
Ratio of the human serum albumins in blood protein is high, and metabolic half life is about 19 days, the extension based on albumin
The strategy of drug metabolism half-life period is the hot spot studied at present.Albumin binding peptide is that a kind of and albumin has high affinity
The small peptide of power, such as albumin binding domain (Albumin binding domain, ABD) He Kangbai in streptococcal protein G
Protein antibodies Variable domain (Domain of antibody).Such structural domain molecular weight is small, and plasticity is strong, by albumin
After binding peptide and other protein fusion expressions, fusion protein still has the ability in conjunction with albumin, can rely on albumin
Long-term effect and increase molecular volume advantage achieve the purpose that extend target protein metabolic half life.
Xu etc. by ciliary neurotrophic factor and albumin binding domain amalgamation and expression, realize ciliary nerve nutrition because
Long-term effect (L.Xu, C.Zhang, L.Liu, et al., Purification and characterization of a of son
long-acting ciliary neurotrophic factor via genetically fused with an
Albumin-binding domain, Protein Expression and Purification, 2017,139:14-20), eyelash
Shape neurotrophic factor itself has therapeutic effect to neurodegenerative disorders or metabolic disease, but does not have hollow reversible
Spherical structure, it is weak to the targeting of tumour, therefore oncotherapy can not be used for as medicine medium is carried.
Therefore it provides a kind of not only have longer half-life to tumor locus with targeting but also in blood circulation system
Load medicine albumen, be of great significance in biopharmaceutical technology.
Summary of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of nano-carrier albumen and its preparation method and application,
The nano-carrier albumen has reversible closing hollow structure, and surface amalgamation and expression has albumin binding domain, on the one hand may be used
With realize small molecule, anti-tumor drug targeting transport, on the other hand can be formed with seralbumin ining conjunction with partial size greatly, blood
The load medicine albumen of long half time, further increases the targeted delivery efficiency of tumour medicine.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the nano-carrier albumen includes protein nano the present invention provides a kind of nano-carrier albumen
Grain, flexible peptide and albumin binding peptide are connected wherein the protein nano particle passes through flexible peptide with albumin binding peptide.
Nano-carrier albumen of the invention is melted in natural protein nano particle surface amalgamation and expression albumin binding peptide
The closing hollow dome structure for not influencing protein body after conjunction albumin binding peptide, realizes the loading to anti-tumor drug, carries
Medicine albumen enter in vivo after by albumin binding peptide rapidly in conjunction with seralbumin, increase the partial size of protein body, prolong
The half-life period of protein body in blood has been grown, the long-acting circulation for carrying medicine albumen is realized.
Preferably, the protein nano particle is assembled by 5-1000 protein protomer forms dome structure, such as can be 5
A, 50,100,150,200,250,300,350,400,450,500,550,600,650
A, 700,750,800,850,900,950 or 1000.
Preferably, the protein nano particle be Ferritin particles, heat shock protein particle, hepatitis B surface antigen particle,
Adenovirus capsid proteins, poultry tumor leukemia virus capsid protein, Cowpea virus capsid protein, cucumber mosaic virus capsid
In albumen, Rotavirus capsid protein or DNA binding protein any one or at least two combination, preferably ferritin
Grain.
Preferably, the molecular formula of the flexible peptide is (GxSy)nAnd/or (PAS)n；Wherein, x is the arbitrary integer of 1-10, y
For the arbitrary integer of 1-10, n is the arbitrary integer of 1-200.
Preferably, x is the arbitrary integer of 1-10, such as can be 1,2,3,4,5,6,7,8,9 or 10.
Preferably, y is the arbitrary integer of 1-10, such as can be 1,2,3,4,5,6,7,8,9 or 10.
Preferably, n is the arbitrary integer of 1-200, for example, can be 1,10,20,30,40,50,60,70,80,90,100,
110,120,130,140,150,160,170,180,190 or 200.
Preferably, the albumin binding peptide is the albumin binding domain and/or antialbumin in streptococcal protein G
Antibody variable region domain, the preferably albumin binding domain in streptococcal protein G.
Preferably, the length of the albumin binding peptide be 5-1000 amino acid residue, such as can be 5,50,
100,150,200,250,300,350,400,450,500,550,600,650,700
A, 750,800,850,900,950 or 1000.
Preferably, the albumin binding peptide is blended in the surface of protein nano particle.
In the present invention, albumin binding peptide be blended in by flexible peptide the end N- of protein protomer, the end C- or other in
Between sequence location, be blended in the surface for closing the protein nano particle of hollow ball-type, extend protein nano particle in blood
Half-life period.
Preferably, the amino acid sequence of the nano-carrier albumen is as shown in SEQ ID NO.1.
Second aspect, the present invention provides a kind of methods for preparing nano-carrier albumen as described in relation to the first aspect, including such as
Lower step:
(1) nano-carrier protein fusion gene is cloned into construction recombination plasmid on pET-30a carrier, then switches into large intestine
The prokaryotic expression of nano-carrier albumen is carried out in bacillus；
(2) supernatant is collected after the Escherichia coli ultrasonication that expression is had to nano-carrier albumen；
(3) supernatant is purified to obtain the nano-carrier albumen.
The third aspect, the present invention provides a kind of nano-carrier albumen as described in relation to the first aspect in loading anti-tumor drug
Application.
Preferably, the anti-tumor drug be in adriamycin, taxol, auspicious statin difficult to understand or maytansine any one or extremely
Few two kinds of combination.
Fourth aspect, the present invention provides a kind of antitumor load medicine protein nano particle, including as described in relation to the first aspect
Nano-carrier albumen.
5th aspect prepares the antitumor side for carrying medicine protein nano particle as described in fourth aspect the present invention provides a kind of
Method includes the following steps:
(1) nano-carrier albumen is mixed with anti-tumor drug；
(2) denaturation treatment is carried out to nano-carrier albumen, anti-tumor drug is made to enter nano-carrier albumen；
(3) renaturation process is carried out to nano-carrier albumen, removes the anti-tumor drug of unloaded, obtains the antitumor load
Medicine protein nano particle.
Preferably, step (1) anti-tumor drug is small molecule, anti-tumor drug.
Preferably, the molecular weight of step (1) described anti-tumor drug be 100-2000Da, such as can be 100Da,
200Da、300Da、400Da、500Da、600Da、700Da、800Da、900Da、1000Da、1100Da、1200Da、1300Da、
1400Da, 1500Da, 1600Da, 1700Da, 1800Da, 1900Da or 2000Da.
Preferably, step (1) anti-tumor drug is any in adriamycin, taxol, auspicious statin difficult to understand or maytansine
It is a kind of or at least two combination.
Preferably, the mass ratio of step (1) the nano-carrier albumen and the anti-tumor drug is (1-10): 1, such as
It can be 1:1,2:1,3:1,4:1,5:1,6:1,7:1,8:1,9:1 or 10:1, preferably 5:1.
Preferably, denaturation treatment described in step (2) is heating, denaturant is added or adjusts any one in hydrostatic pressure
Kind or at least two combination.
Preferably, the denaturant is urea and/or guanidine hydrochloride.
Preferably, the anti-tumor drug of step (3) the removal unloaded is real using dialysis and/or gel permeation chromatography
It is existing.
In the present invention, the time of denaturation treatment is 0.1-72 hours, and the time of renaturation process is 0.1-20 hours.Nanometer carries
Body protein can be heated 1-6 hours at 30-80 DEG C and is denaturalized, cooling 0.1-2 hours progress renaturation；It can be in 1-10M urea
And/or keep being denaturalized for 1-50 hours in 0.5-8M guanidine hydrochloride, it keeps carrying out for 0.1-20 hours after removing urea and/or guanidine hydrochloride
Renaturation；It can also keep being denaturalized for 1-50 hours under the hydrostatic pressure of 50-800MPa, remove and keep 0.1- under hydrostatic pressure
20 hours progress renaturation.Denaturation treatment and renaturation process are carried out to nano-carrier albumen, realize the loading to anti-tumor drug
And targeted delivery.
Compared with prior art, the invention has the following beneficial effects:
(1) nano-carrier albumen of the invention has tumor-targeting strong, raw using natural protein nano particle as carrier
Object compatibility is high, immunogenicity is low, the efficient and convenient advantages such as cheap of prokaryotic expression；
(2) protein nano particle surface amalgamation and expression of the invention has albumin binding domain, with the white egg in serum
It is white to combine, the metabolism time of nano-carrier albumen in blood is significantly increased, realizes and carries the length of medicine albumen in blood
Effect property, improves the targeted delivery efficiency of drug；
(3) nano-carrier purity of protein of the invention is greater than 95%, and it is small to load adriamycin etc. in such a way that embedding carries medicine
Molecule antineoplastic medicament solves the problems, such as that drug is water-soluble low, avoids using organic solvent and surfactant bring pair
Effect；
(4) the nano-carrier protein on cells small toxicity prepared by the present invention for being mounted with adriamycin, there is significantly tumour
Effect inhibits, and side effect is much smaller than free DOX.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of nano-carrier albumen；
Fig. 2 (a) is the polyacrylamide gel electrophoresis map of nano-carrier albumen, and Fig. 2 (b) is the circle of nano-carrier albumen
Two color test maps, Fig. 2 (c) are that the Electronic Speculum of nano-carrier albumen detects figure, and Fig. 2 (d) is the gel detection of nano-carrier albumen
Map；
Fig. 3 is that the desalination after nano-carrier protein loaded drug chromatographs map；
Fig. 4 is the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin, free adriamycin (DOX) and blank
Growth inhibition curve of the nano-carrier albumen (HFn-ABD) to lung cell A549；
Fig. 5 be lung cell A549 to the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin and to free Ah
The intake situation of mycin (DOX)；
Fig. 6 is the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin and free adriamycin (DOX) to swollen in vivo
The growth inhibition curve of tumor；
Fig. 7 is the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin and free adriamycin (DOX) to nude mouse
The influence curve of weight；
Fig. 8 is the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin and free adriamycin (DOX) to nude mice life
Deposit the influence of rate；
Fig. 9 is the nano-carrier albumen (HFn-ABD/DOX) for being mounted with adriamycin and free adriamycin (DOX) in SD rat
Intracorporal pharmacokinetics measurement result figure；
Figure 10 is the external in conjunction with qualification figure of nano-carrier albumen (HFn-ABD) and seralbumin (HSA).
Specific embodiment
The technological means and its effect taken for the present invention is further explained, with reference to embodiments with attached drawing to this hair
It is bright to be further described.It is understood that the specific embodiments described herein are used only for explaining the present invention, rather than
Limitation of the invention.
In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art,
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer, be can be by regular channel commercially available from
The conventional products of acquisition.
The preparation of 1 nano-carrier albumen of embodiment
(1) nano-carrier protein fusion gene is cloned into construction recombination plasmid on pET-30a carrier, then switches into large intestine
It is cultivated in bacillus BL21 using LB culture medium, to OD600nmWhen up to 3.0, the isopropylthio gala of final concentration of 1mM is added
Glucosides (IPTG) induces the prokaryotic expression of target gene, and after 3 hours, centrifugation obtains thallus；
(2) Escherichia coli of nano-carrier albumen carry out ultrasonication expression, specially ultrasound 4s stops 6s, power
20%, ultrasound about 10min are then centrifuged 30min at 10000rpm and collect supernatant；
(3) supernatant is centrifuged 15min at 10000rpm after 0.5M ammonium sulfate precipitation and removes ammonium sulfate precipitation, obtains
Supernatant carry out butyl-Sepharose column hydrophobic chromatography, 35% step elution, 90% step elution, collection obtain purity compared with
High destination protein.
Fig. 1 is the structural schematic diagram of nano-carrier albumen.The nano-carrier albumen of preparation is detected, such as Fig. 2 (a) institute
Show, the purity of the nano-carrier albumen (HFn-ABD) of preparation is greater than 95%；As shown in Fig. 2 (b), albumin integrated structure is accessed
Behind domain (albumin-binding domain, ABD), the secondary structure of protein nano particle (HFn) does not almost change, contains
There are a large amount of αhelix；As shown in Fig. 2 (c), HFn-ABD is in hollow-sphere structures, and partial size slightly increases compared with HFn；Such as Fig. 2 (d)
Shown, the peak position out of HFn-ABD and HFn are close, the possible reason is the molecular weight of the two is close, therefore Superdex 200
HFn-ABD and HFn cannot be distinguished well.
Embodiment 2 is mounted with the preparation of the nano-carrier albumen of adriamycin
(1) adriamycin (DOX) of final concentration of 0.2mg/mL is added dropwise to the HFn-ABD solution that concentration is 1mg/mL
In, it is uniformly mixed；
(2) in light protected environment, the mixed liquor of HFn-ABD and DOX is heated to 50 DEG C, is saved 6 hours, then by mixed liquor
It cools down 2 hours at normal temperature, albumen is made to restore structure；
(3) it is centrifuged 30min at 10000rpm, obtained load medicine albumen supernatant is removed into unloaded using desalting column chromatography
Free DOX drug, finally use UV spectrophotometer measuring DOX concentration, dying method with coomassie brilliant blue detect protein concentration,
Calculate drug load.
Drug load calculation formula are as follows:
Wherein, N indicates useful load of the DOX in HFn-ABD, and C indicates molar concentration, and MW indicates relative molecular mass.
As shown in figure 3, the HFn-ABD (HFn-ABD/DOX) for being mounted with DOX has at the characteristic absorption wavelength 480nm of DOX
It is obvious to absorb, illustrate that DOX is successfully loaded to HFn-ABD；Absorptance by HFn-ABD/DOX in the position 280nm and 480nm
It can infer roughly, DOX charging ratio is higher；According to drug load calculation formula, 89 DOX are calculated and are loaded in HFn-ABD
In.
Toxicity of 3 HFn-ABD/DOX of embodiment to A549 cell
(1) it is in the lung cell A549 of logarithmic growth phase using trypsin digestion processing, is centrifuged 2min under 1200rpm
Trypsase is removed, culture medium is added and adjusts cell concentration to 1 × 105Cells/mL takes 100 μ L to be laid on 96 orifice plates；
(2) 96 orifice plates are placed in 5%CO2In incubator, after being incubated for for 24 hours at 37 DEG C, culture medium is removed, PBS cleaning divides
100 complete mediums of the μ L containing DOX, HFn-ABD/DOX and HFn/DOX are not added, are arranged 8 concentration gradients (DOX equivalent), it is dense
From 0 to 62.5 μM of range of degree, while the control group that HFn-ABD and HFn is added is set；
(3) after cultivating 60h, culture medium is removed, 100 μ L complete mediums and 10 μ L CCK-8 solution are added in PBS cleaning,
After reacting 2h, light absorption value of each hole at 450nm (a length of 630nm of background wave) is measured using microplate reader.
The calculation formula of cell viability are as follows:
Cell viability (%)=[A (dosing group)-A (blank group)]/[A (0 medicine group)-A (blank group)] × 100%
Wherein, A (blank group) indicates that the light absorption value of complete medium, A (0 medicine group) indicate only complete culture containing cell
The light absorption value of base, A (dosing group) indicate the light absorption value of the complete medium containing cell and drug.
As shown in figure 4, the IC50 of DOX and HFn-ABD/DOX is respectively 0.88 μM and 10.06 μM.
External intake of the 4 A549 cell of embodiment to HFn-ABD/DOX
(1) the A549 cell of logarithmic growth phase is in using trypsin digestion processing, centrifugation 2min removal under 1200rpm
Pancreatin is added culture medium and adjusts cell concentration to 1 × 106Cells/mL takes 100 μ L to be laid on 96 orifice plates；
(2) 96 orifice plates are placed in 5%CO2In incubator, after being incubated for for 24 hours at 37 DEG C, culture medium is removed, PBS cleaning divides
100 complete mediums of the μ L containing DOX and HFn-ABD/DOX are not added, DOX concentration is 5 μM；
(3) after cultivating 1,2,4 and 8h respectively, culture medium is removed, 100 μ L PBS buffer solution and final concentration is added in PBS cleaning
The fluorescent absorption value in each hole is measured using fluorescence microplate reader, excitation wavelength is after acting on 0.5h for the hydrochloric acid solution of 0.1M
450nm, a length of 580nm of diverging wave.
As shown in figure 5, A549 cellular uptake dissociates, the speed of DOX is faster than the speed of intake HFn-ABD/DOX.
The Anticancer effect in vivo of 5 HFn-ABD/DOX of embodiment
(1) choosing and weighing about within 6 weeks the BALB/c nude mice of 20g is experimental subjects, injects 100 between the left armpit and left chest of nude mice
μ L concentration is 1 × 107The A549 cell suspension of cells/mL, carries out kind of a tumor；
(2) after two weeks, nude mice is randomly divided into 3 groups, every group 6, each group injects PBS, DOX and HFn-ABD/DOX respectively,
The injection concentration of DOX is 5mg/kg, and administration frequency is 3 days/time；
(3) tumor size and quality change situation of real-time monitoring nude mice, detection frequency are 2 days/time.
The calculation formula of gross tumor volume are as follows:
Gross tumor volume (mm3)=(maximum gauge × minimum diameter2)/2
As shown in figs 6-8, the nude mouse tumor sustainable growth of PBS is injected, but nude mice weight does not change, tested 16 days,
Death does not occur for nude mice；The nude mouse tumor volume for injecting DOX reduces, and nude mice weight is down to the 72% of substance weight, after administration 12 days
Nude mice is all dead, it can thus be appreciated that side effect is too strong, threatens although DOX has the effect of significantly inhibiting tumour growth
The life of nude mice；The nude mouse tumor volume for injecting HFn-ABD/DOX slightly increases, and nude mice weight is down to the 86% of substance weight,
Experiment 16 days, death does not occur for nude mice, illustrates that HFn-ABD/DOX has apparent effect to inhibit tumour, and side effect is much smaller than
Free DOX.
The pharmacokinetics of 6 HFn-ABD/DOX of embodiment detects
(1) male SD rat for choosing weight about 200g is experimental subjects, is randomly divided into 3 groups, every group 3, each group is distinguished
By tail vein injection PBS, DOX and HFn-ABD/DOX, the administration concentration of DOX is 3mg/kg；
(2) wherein, the rat for injecting HFn-ABD/DOX, administration 10min, 30min, 1h, 2h, 4h, 8h, 12h, for 24 hours,
Eye socket blood sampling (about 0.5mL) is carried out after 36h, 48h and 60h；Inject DOX rat, administration 10min, 25min, 1h, 2h, 4h,
8h, 12h, for 24 hours with blood was collected after 36h；The rat of PBS is injected, blood sampling is primary daily, as background deduction sample；
(3) after blood clotting, serum is separated, is added in 96 orifice plates, the fluorescent absorption in each hole is measured using fluorescence microplate reader
Value, while it is 0.25,0.5,1.25,2.5,5,10,15,20,25 μ g/mL that DOX concentration gradient, which is arranged, measures fluorescent absorption value,
Excitation wavelength is 480nm, launch wavelength 580nm.
As shown in Fig. 9 and table 1, the half-life period of HFn-ABD/DOX is up to 12 hours, is longer than free DOX (1 hour),
Also longer than HFn/DOX reported in the literature (about 1.6 hours), it follows that in protein nano particle surface amalgamation and expression albumin
Binding structural domain significantly increases the metabolism time of nano-carrier albumen in blood, realizes and carries medicine albumen in blood
Long-term effect.
IC50, half-life period and the area under the drug-time curve of table 1 HFn-ABD/DOX and free DOX
| Drug | IC50(μm) | t1/2(h) | AUC(mg/L·h) |
| DOX | 0.88 | 1.029 | 1.55 |
| HFn-ABD/DOX | 10.06 | 17.476 | 30.211 |
7 nano-carrier albumen of embodiment and human serum albumins it is external in conjunction with situation
(1) compound concentration is human serum albumins (human serum albumin, HSA) mother liquor of 6mg/mL, and dilutes
For various concentration；
(2) the HSA solution of 0.5mL various concentration is taken to mix with the HFn-ABD that 0.5mL concentration is 2mg/mL, so that HFn-
The mass ratio of ABD and HSA is respectively 1:0,1:0.5,1:1,1:1.5,1:2 and 1:3, separately takes the HSA solution of 0.5mL various concentration
After being mixed with the buffer of 0.5mL as a control group；
(3) it is incubated overnight, in buffer (0.5M Na2SO4, 50mM PB, PH=7) in carry out Superose6 gel chromatography
Detection.
As shown in Figure 10, HFn-ABD can be in conjunction with HSA, and when the mass ratio of HFn-ABD and HSA is 1:1, HSA goes out
Peak position is at 17mL, and the peak value of HSA is substantially reduced after addition HFn-ABD, illustrates that most of HSA have been integrated on HFn-ABD.
In conclusion nano-carrier protein surface amalgamation and expression of the invention has albumin binding domain, secondary structure
It does not change, in closing hollow dome structure, partial size slightly increases；Nano-carrier purity of protein obtained is greater than 95%, can
Load the small molecule, anti-tumor drugs such as adriamycin；It is mounted with the nano-carrier protein on cells small toxicity of adriamycin, is had to tumour
Apparent effect inhibits, and side effect is much smaller than free DOX；The albumin binding domain and blood of protein nano particle surface
Albumin in clear combines, and significantly increases the metabolism time of nano-carrier albumen in blood, realizes load medicine albumen and exists
Long-term effect in blood.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (10)
1. a kind of nano-carrier albumen, which is characterized in that the nano-carrier albumen includes protein nano particle, flexible peptide and white
Protein binding peptide is connected wherein the protein nano particle passes through flexible peptide with albumin binding peptide.
2. nano-carrier albumen according to claim 1, which is characterized in that the protein nano particle is by 5-1000 egg
Bai Yaji assembles to form dome structure；
Preferably, the protein nano particle is Ferritin particles, heat shock protein particle, hepatitis B surface antigen particle, adenopathy
Virus capsid protein, poultry tumor leukemia virus capsid protein, Cowpea virus capsid protein, cucumber mosaic virus capsid egg
In white, Rotavirus capsid protein or DNA binding protein any one or at least two combination, preferably ferritin
Grain.
3. nano-carrier albumen according to claim 1 or 2, which is characterized in that it is described flexibility peptide molecular formula be
(GxSy)nAnd/or (PAS)n；
Wherein, x is the arbitrary integer of 1-10, and y is the arbitrary integer of 1-10, and n is the arbitrary integer of 1-200.
4. nano-carrier albumen according to claim 1-3, which is characterized in that the albumin binding peptide is chain
Albumin binding domain and/or anti-albumin antibodies Variable domain in coccus G-protein, preferably streptococcal protein G
In albumin binding domain；
Preferably, the length of the albumin binding peptide is 5-1000 amino acid residue；
Preferably, the albumin binding peptide is blended in the surface of protein nano particle.
5. any one of -4 nano-carrier albumen according to claim 1, which is characterized in that the amino of the nano-carrier albumen
Acid sequence is as shown in SEQ ID NO.1.
6. a kind of method for preparing the nano-carrier albumen as described in claim any one of 1-5, which is characterized in that including walking as follows
It is rapid:
(1) nano-carrier protein fusion gene is cloned into construction recombination plasmid on pET-30a carrier, then switches into Escherichia coli
The middle prokaryotic expression for carrying out nano-carrier albumen；
(2) supernatant is collected after the Escherichia coli ultrasonication that expression is had to nano-carrier albumen；
(3) supernatant is purified to obtain the nano-carrier albumen.
7. a kind of nano-carrier albumen as described in claim any one of 1-5 is loading the application in anti-tumor drug；
Preferably, the anti-tumor drug is any one in adriamycin, taxol, auspicious statin difficult to understand or maytansine or at least two
The combination of kind.
8. a kind of antitumor load medicine protein nano particle, which is characterized in that including nanometer as described in any one in claim 1-5
Carrier protein.
9. a kind of prepare the antitumor method for carrying medicine protein nano particle as claimed in claim 8, which is characterized in that including as follows
Step:
(1) nano-carrier albumen is mixed with anti-tumor drug；
(2) denaturation treatment is carried out to nano-carrier albumen, anti-tumor drug is made to enter nano-carrier albumen；
(3) renaturation process is carried out to nano-carrier albumen, removes the anti-tumor drug of unloaded, obtains the antitumor load medicine egg
White nano particle.
10. according to the method described in claim 9, it is characterized in that, step (1) anti-tumor drug is small molecule, anti-tumor
Drug；
Preferably, the molecular weight of step (1) described anti-tumor drug is 100-2000Da；
Preferably, step (1) anti-tumor drug is any one in adriamycin, taxol, auspicious statin difficult to understand or maytansine
Or at least two combination；
Preferably, the mass ratio of step (1) the nano-carrier albumen and the anti-tumor drug is (1-10): 1, preferably 5:
1；
Preferably, denaturation treatment described in step (2) be heating, be added denaturant or adjust hydrostatic pressure in any one or
At least two combination；
Preferably, the denaturant is urea and/or guanidine hydrochloride；
Preferably, the anti-tumor drug of step (3) the removal unloaded is realized using dialysis and/or gel permeation chromatography.
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| WO2022229220A1 (en) * | 2021-04-26 | 2022-11-03 | Evox Therapeutics Limited | Modified extracellular vesicles (evs) with improved half-life |
| CN114668771A (en) * | 2022-03-18 | 2022-06-28 | 南京林业大学 | Preparation method and application of ferritin nanoparticles loaded with adriamycin and ursolic acid together |
| CN114668771B (en) * | 2022-03-18 | 2022-12-06 | 南京林业大学 | A preparation method and application of ferritin nanoparticles co-loaded with doxorubicin and ursolic acid |
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