CN107982543A - A kind of protein-isosulfocyanate key compound and its application - Google Patents
A kind of protein-isosulfocyanate key compound and its application Download PDFInfo
- Publication number
- CN107982543A CN107982543A CN201610951594.3A CN201610951594A CN107982543A CN 107982543 A CN107982543 A CN 107982543A CN 201610951594 A CN201610951594 A CN 201610951594A CN 107982543 A CN107982543 A CN 107982543A
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- CN
- China
- Prior art keywords
- isosulfocyanate
- compound
- protein
- key
- key compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 80
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 38
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Classifications
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- A—HUMAN NECESSITIES
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- A61K31/26—Cyanate or isocyanate esters; Thiocyanate or isothiocyanate esters
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- A—HUMAN NECESSITIES
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- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
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- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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Abstract
The invention discloses a kind of protein isosulfocyanate key compound, react to obtain by the amino group on the isothiocyanate group and protein in isosulfocyanate compound.The present invention is using protein and isosulfocyanate compound as raw material, both are bonded using radical reaction between the two, water soluble macromolecular substance is prepared, so as to carry out a series of researchs, there is provided the another possibility of isosulfocyanate medicine tumor suppression mechanism.The protein isosulfocyanate key compound has excellent water solubility, and excellent anti-cancer properties, also while solves the problems, such as internal conveying when being used as cancer therapy drug.The protein isosulfocyanate key compound also has hydrophobic compound load, covered effect well, can be used as its carrier, only can prepare that particle diameter is small, be evenly distributed and coats close nano particle by being simply blended.
Description
Technical field
The present invention relates to the technical field of antitumor drug, more particularly to a kind of protein-isosulfocyanate key compound
And its application.
Background technology
The intake of brassicaceous vegetable can prevent Several Kinds of Malignancy in meals, and anti-tumor biologically active substance therein is
The hydrolysate isothiocyanates (isothiocyanates, ITCs) of glucosinolate.But most of isosulfocyanates
Compound or derivatives thereof, is mostly slightly water-soluble compound, is used directly as inhibiting tumor medicine, can soon reaction degraded in vivo
Fall or dispose；And due to its hydrophobicity and activity, administering mode is also restricted very much, and bioavilability is low.
From 1966, (Sidransky.H, N.Ito, E.Verney, Influence of Alpha- such as Sidransky
Naphthyl-Isothiocyanate on Liver Tumorigenesis in Rats Ingesting Ethionine
And N-2-Fluorenylacetamide, J Natl Cancer I, 37 (1966) 677-686.) report isothiocyanates tune
Since controlling growth of tumour cell, the research of its antitumor mechanism starts to receive significant attention.Although isosulfocyanate medicine resists
Tumour mechanism not yet illustrates completely, but the mechanism of following aspect has been accepted, such as：Suppress I phase enzymatic activitys, prevent carcinogenic
Precursor activates；II phases dehydrogenation/antioxidase is activated, accelerates the metabolism of carcinogenic precursor in vivo；Activate Nrf2 or suppress NF-
The cells such as kb adjust the important transcription factor of oxidative stress, resistance of the increase cell to oxidative stress；Adjust cell cycle, induction
Apoptosis of tumor cells etc..
However, research is carried out in cellular level above, and isothiocyanates group is all based on as antitumor
Active group.It is very active in view of isothiocyanate group, once enter blood, adsorbed soon and with various albumen etc. in blood
Reaction, forms the chemical groups such as thiocarbamide and is bonded on albumen, and when it reaches tumor locus, the overwhelming majority has not been with original
Isothiocyanate group exist.Based on this, it is believed that, it may be possible to there is also certain suppression tumour in body for protein conjugates
The mechanism of growth, plays certain tumor killing effect.
Protein has the characteristics that safe and non-toxic, non-immunogenicity, biodegradable, current most widely used protein
There are bovine serum albumin(BSA) and human serum albumins etc..Albumin is a kind of endogenous protein, and there are multi-medicament combination in structure
Site, its surface group such as amino and carboxyl can be coupled other functional moleculars, have in terms of drug delivery and have many advantages,
Such as albumin solvent-borne type nano-paclitaxel (AbraxaneTM) by Life Sciences of the U.S. (American Bioscience)
Production, in 2005 by FDA (Food and Drug Adminstration) approval listing (M.J.Hawkins, P.Soon-Shiong, N.Desai,
Protein nanoparticles as drug carriers in clinical medicine,Adv Drug Deliver
Rev,60(2008)876-885.).The medicine application human serum albumin forms 130 nanosizeds as carrier, high pressure homogenization method
Paclitaxel particles, carry the albumin of taxane molecule, by being combined with the Albumin receptor Gp60 on cell membrane, active cell
Caveolin on film, and what current research found control caveolin is rich in cysteine acid acidic secretion albumen (SPARC),
Usually very abundant in tumor tissues, therefore, albumin combination type medicine has certain target function；In addition, effectively keep away
The allergic reaction that Emulsifier EL-60 and Tween-80 surfactant are brought is exempted from.But it is compared with Taxol (polyoxyethylene casters
Oil and absolute ethyl alcohol 1:1 mixed liquor is as cosolvent) for, clinical test results do not have soul-stirring improvement
(N.K.Ibrahim,B.Samuels,R.Page,D.Doval,K.M.Patel,S.C.Rao,M.K.Nair,P.Bhar,
N.Desai,G.N.Hortobagyi,Multicenter phase II trial of ABI-007,an albumin-bound
paclitaxel,in women with metastatic breast cancer,J ClinOncol,23(2005)6019-
6026.).Do not contained well in albumin to find out its cause, being largely because taxol, nano particle pine
Dissipate, once into blood, particle disperses immediately, and is eliminated rapidly, causes its pharmacokinetics almost identical with Taxol.
The content of the invention
The present invention, will both keys using radical reaction between the two using protein and isosulfocyanate compound as raw material
Altogether, water soluble macromolecular substance is prepared, so as to carry out a series of researchs, there is provided isosulfocyanate medicine tumor suppression
The another possibility of mechanism.The protein-isosulfocyanate key compound has excellent water solubility, and excellent cancer resistance
Can, also while solve the problems, such as internal conveying when being used as cancer therapy drug.In addition, the protein-isothiocyanates generic key
Compound also has hydrophobic compound load, covered effect well, its carrier can be used as, only by being simply blended i.e.
It can prepare that particle diameter is small, be evenly distributed and coats close nano particle.
The present invention provides a kind of protein-isosulfocyanate key compound, by the different sulphur in isosulfocyanate compound
Cyanic acid group reacts to obtain with the amino group on protein.Specific preparation method is as follows：
(1) isosulfocyanate compound being mixed with organic solvent A, protein is mixed with water, then both are mixed, and
The monitoring extent of reaction in real time；
(2) reaction product that step (1) obtains is after centrifuging, be filtered to remove precipitation, then dialyses and remove organic solvent A, obtains
To the protein-isosulfocyanate key compound.
The isosulfocyanate compound can derive from brassicaceous vegetable, including benzyl isothiocyanate,
Phenethyl isosulfocyanate, benzoyl isothiocyanate, benzyloxy-phenyl isothiocyanates, hexyl isothiocyanates, different sulphur
Cyanic acid allyl ester, Sulforaphane, 3- first thiopropyl isothiocyanates etc.；Also can by artificial synthesized,
Including benzylisothiocyanate-N-acetylcysteine conjugates, phenethyl isosulfocyanate-N-acetylcystein conjugation
Thing, 1- cyclopropyl naphthalene -4- bases isothiocyanates, arylsulfonyl isothiocyanic acid etc..
The protein is selected from animal serum albumin, casein, collagen or silk-fibroin；
Preferably, the molar ratio of the protein and isosulfocyanate compound is 1:10~50；
Isosulfocyanate compound and the mass volume ratio of organic solvent A are 2~100mg/ml；
Organic solvent A and the volume ratio of water are 1~4:10.
Further preferably, the isosulfocyanate compound is phenethyl isosulfocyanate, the different sulphur cyanogen of benzoyl
Acid esters, benzyloxy-phenyl isothiocyanates or hexyl isothiocyanates；The protein is human serum albumins or cow's serum
Albumin.
Further preferably, the isosulfocyanate compound is phenethyl isosulfocyanate or benzoyl isothiocyanic acid
The molar ratio of ester, protein and isosulfocyanate compound is 1:30~50.
Preferably, the organic solvent A is selected from dimethyl sulfoxide (DMSO), ethanol or methanol.Consider further that organic solvent
Residue problem, more preferably ethanol.
In step (1), isosulfocyanate compound in monitoring product in real time is carried out by high performance liquid chromatography (HPLC)
Residual quantity, when testing result twice change≤5% when can stop reacting.
The invention also discloses the protein prepared according to the above method-isosulfocyanate key compound to contain as carrier
The method of hydrophobic compound, step are as follows：
Protein-isosulfocyanate key compound is mixed with water, obtains carrier aqueous solution I, by hydrophobic compound with
Organic solvent B is mixed to get solution II, then both are mixed, the post-treated nanometer for obtaining containing hydrophobic compound
Grain.
Albumin is hydroaropic substance, when directly being contained to hydrophobic compound, it is understood that there may be stability problem；When
After being bonded hydrophobic isothiocyanic acid class compound, the hydrophobic region of albumin increases and is easier to be exposed, it is easier to
Some hydrophobic compounds are contained, the nano particle being prepared more is stablized, and burst drug release is reduced.
Preferably, the hydrophobic compound includes small molecule, anti-tumor drug, photosensitizer, contrast agent or fluorescence dye
Material.
It can be realized by containing small molecule, anti-tumor drug and be used in combination with other medicines.
It can be realized by containing photosensitizer, contrast agent and be used in combination with radiation treatment.
It can be realized by containing fluorescent dye and be used in combination with gene therapy.
Preferably, the small molecule, anti-tumor drug includes taxol, adriamycin or camptothecine.
Further preferably, the hydrophobic compound is taxol.
Preferably, the one kind of the organic solvent B in dichloromethane, ethanol, methanol, acetone, tetrahydrofuran
It is or a variety of.The ethanol of further preferred small toxicity.
Preferably, the concentration of the carrier aqueous solution I is 5~10g/L, the concentration of solution II is 80~120g/L；Carry
The volume ratio of body aqueous solution I and solution II is 100:1~4, more preferably 100:1；Further preferably, carrier aqueous solution I is dense
Spend for 9g/L, the concentration of solution II is 100g/L.
Further preferably, in the protein for the being used to prepare carrier aqueous solution I-isosulfocyanate key compound, egg
The molar ratio of white matter and isosulfocyanate compound is 1:50, isosulfocyanate compound is phenethyl isothiocyanic acid
Ester or benzoyl isothiocyanate.
The nano particle average grain diameter being prepared by the above method is less than 0.2 micron, dissolves in water for injection, glucose
And physiological saline remakes the injectable aqueous solutions to form pH between 5.0~7.0.
Compared with prior art, the invention has the advantages that：
(1) present invention angle out of organism understands the anticancer mechanism of isosulfocyanate medicine, rather than merely from thin
Born of the same parents' aspect, proposes that isothiocyanate group also plays tumor killing effect with protein conjugates, considers isothiocyanic acid from another aspect
The antitumor mechanism of esters medicine.
(2) to be insoluble in water class material, the present invention is bonded on protein most isosulfocyanate medicines, is formed
Water soluble protein mating type medicine, solves the problems, such as internal conveying.
(3) nano particle prepared by general extruding filtration method or high pressure homogenization method, can be there are high amount of drug in nanometer
Grain surface and the problem of cause burst drug release, contained using the method in the present invention, preparation process is simple, and bag medicine process
Without the input for being related to high pressure high energy.Using protein-isosulfocyanate key compound as carrier, due to adding hydrophobic region,
Hydrophobic compound can be preferably contained, by taking taxol as an example, significantly improves the stability that protein is combined with taxol, is carried
Medicine is efficient, the uniform particle sizes of nano particle.
(4) protein-isosulfocyanate key compound prepared by the present invention contains cancer therapy drug, the experiment in Mice Body
Obtain well as a result, being significantly better than and medicine is directly contained using albumin.
Brief description of the drawings
Fig. 1 is the albumin-isosulfocyanate key compound for preparing respectively of embodiment 1~3 in human breast cancer cell
Cytotoxicity experiment on BCap37 is as a result, and provide cell of the phenethyl isosulfocyanate on human breast cancer cell BCap37
Toxicity test result is as a comparison；
Fig. 2 is the albumin-isosulfocyanate key compound for preparing respectively of embodiment 1~3 in human colon cancer cell HT29
On cytotoxicity experiment as a result, and providing cytotoxicity experiment of the phenethyl isosulfocyanate on human colon cancer cell HT29
As a result as a comparison；
Fig. 3 is the albumin-isosulfocyanate key compound for preparing respectively of embodiment 1~3 on human lung cancer cell A549
Cytotoxicity experiment as a result, and providing cytotoxicity experiment result of the phenethyl isosulfocyanate on human lung cancer cell A549
As a comparison；
Fig. 4 is tumor volume versus time curve of more group reagents in vitro obtained in antitumor activity experiment；
Fig. 5 is that albumin-isothiocyanates key compound prepared by embodiment 17 contains dynamic optical after taxol in water
Scatter diagram；
Fig. 6 is that albumin prepared by comparative example contains dynamic light scattering diagram after taxol in water；
Fig. 7 is the nanometer that albumin-isothiocyanates key compound prepared by embodiment 17 contains taxol as carrier
Transmission electron microscope (TEM) figure of grain；
Fig. 8 is the transmission electron microscope for the nano particle that albumin prepared by comparative example contains taxol as carrier
(TEM) figure；
Fig. 9 is tumor volume versus time curve of more group reagents in vitro obtained in antitumor activity experiment；
Figure 10 is the mouse tumor photo that more group reagents are peeled off after antitumor activity is tested in vitro.
Embodiment
The present invention provides some specific implementation cases, but the present invention is from the limitation of these cases.
Embodiment 1：The preparation of protein-isothiocyanates key compound
A. with the molar ratio 1 of bovine serum albumin(BSA) and phenethyl isosulfocyanate：10 feed intake：By phenethyl isosulfocyanate
(Phenethylisothiocyanate, PEITC, Mw=163.24) 10mg is dissolved in 4ml dimethyl sulfoxide (DMSO)s；Bovine serum albumin
(BSA, Mw=67000) 410mg is dissolved in 20mL water in vain.
B. above two solution is mixed respectively, adjusts pH to 8.5 with 1M NaOH, 37 DEG C of reactions, take a small amount of solution to use
High performance liquid chromatography (HPLC) monitors extent of reaction in real time.
C.3000rpm, 10min is centrifuged.Be filtered to remove precipitation, dialysis removes organic solvent, obtain water miscible albumin-
Isothiocyanates key compound.
In the present embodiment, the molar ratio of albumin and phenethyl isosulfocyanate is 1:10, i.e., it is theoretical on an albumin
10 isothiocyanates groups are connect, are denoted as BSA-PEITC10.
Embodiment 2~3：
Preparation process is identical with embodiment 1, differs only in bovine serum albumin(BSA) and phenethyl isosulfocyanate
Molar ratio is respectively 1:30 and 1:50, BSA-PEITC30 and BSA-PEITC50 are denoted as respectively.
Embodiment 4：
Preparation process is identical with embodiment 1, differs only in, and dimethyl sulfoxide (DMSO) is replaced with absolute ethyl alcohol, is denoted as
BSA-PEITC10-1。
Embodiment 5：
Preparation process is identical with embodiment 3, differs only in, and dimethyl sulfoxide (DMSO) is replaced with absolute ethyl alcohol, is denoted as
BSA-PEITC30-1。
Embodiment 6：
A. with the molar ratio 1 of bovine serum albumin(BSA) and benzoyl isothiocyanate：10 feed intake, by the different sulphur cyanogen of benzoyl
Acid esters (Benzoyl isothiocyanate, BITC, Mw=163.20) 10mg is dissolved in 4ml absolute ethyl alcohols；Ox blood is pure
Protein 41 0mg is dissolved in 20mL water.
B. above two solution is mixed respectively, 1M NaOH adjust pH to 8.5, and 37 DEG C of reactions, take a small amount of solution to use
HPLC monitors extent of reaction in real time.
C.3000rpm, 10min is centrifuged.Be filtered to remove precipitation, revolving removes organic solvent, obtain water miscible albumin-
Isothiocyanates key compound, is denoted as BSA-BITC10.
Embodiment 7~8：
Preparation process is identical with embodiment 6, differs only in bovine serum albumin(BSA) and benzoyl isothiocyanate
Molar ratio be respectively 1:30 and 1:50, BSA-BITC30 and BSA-BITC35 are denoted as respectively.
Embodiment 9：
A. with the molar ratio 1 of bovine serum albumin(BSA) and benzyloxy-phenyl isothiocyanates：10 feed intake：Benzyloxy-phenyl is different
Thiocyanates (Benzyloxyphenylisothiocyanate, BOPITC, Mw=241.31) 15mg is dissolved in 4ml absolute ethyl alcohols
In；Bovine serum albumin(BSA) (BSA, Mw=67000) 410mg is dissolved in 20mL water.
B. above two solution is mixed respectively, adjusts pH to 8.5 with 1M NaOH, 37 DEG C of reactions, take a small amount of solution to use
High performance liquid chromatography (HPLC) monitors extent of reaction in real time.
C.3000rpm, 10min is centrifuged.Be filtered to remove precipitation, dialysis removes organic solvent, obtain water miscible albumin-
Isothiocyanates key compound, is denoted as BSA-BOPITC10.
Embodiment 10~11：
Preparation process is identical with embodiment 9, differs only in bovine serum albumin(BSA) and benzyloxy-phenyl isothiocyanic acid
The molar ratio of ester is respectively 1:30 and 1:50, BSA-BOPITC20 and BSA-BOPITC35 are denoted as respectively.
Embodiment 12：
A. with the molar ratio 1 of bovine serum albumin(BSA) and hexyl isothiocyanates：10 feed intake：By hexyl isothiocyanates
(Hexyl isothiocyanate, HITC, Mw=143.25) 9mg is dissolved in 4ml absolute ethyl alcohols；Bovine serum albumin(BSA) (BSA,
Mw=67000) 410mg is dissolved in 20mL water.
B. above two solution is mixed respectively, adjusts pH to 8.5 with 1M NaOH, 37 DEG C of reactions, take a small amount of solution to use
High performance liquid chromatography (HPLC) monitors extent of reaction in real time.
C.3000rpm, 10min is centrifuged.Be filtered to remove precipitation, dialysis removes organic solvent, obtain water miscible albumin-
Isothiocyanates key compound, is denoted as BSA-HITC10.
Embodiment 13~14：
Preparation process is identical with embodiment 12, differs only in bovine serum albumin(BSA) and benzyloxy-phenyl isothiocyanic acid
The molar ratio of ester is respectively 1:30 and 1:50, BSA-HITC20 and BSA-HITC40 are denoted as respectively.
After reacted 24h, the bonding rate for albumin-isothiocyanates key compound that the various embodiments described above are prepared collects
In table 1 below：
Table 1
Performance test 1：External inhibiting tumor cell proliferation experiment
Albumin-isosulfocyanate key compound of the different bonding ratios prepared respectively with embodiment 1~3 is respectively in human milk
When progress 48 is small in gland cancer BCap37 cells (Fig. 1), Human colorectal carcinoma HT29 cells (Fig. 2) and typeⅡ pneumocyte (Fig. 3)
The experiment of inhibiting tumor cell cultivation effect, and phenethyl isosulfocyanate (being denoted as free PEITC) is provided respectively in human breast carcinoma
Cytotoxicity experiment result in BCap37 cells, Human colorectal carcinoma HT29 cells and typeⅡ pneumocyte is contrasted.
It can be seen from the figure that the phenethyl isosulfocyanate of small molecule has very strong cytotoxicity, and it is incorporated in
After on albumin, half lethal dose IC50Value increases 10~100 times, illustrates propagation of the key compound for cell certain dense
Had little to no effect under degree scope, meanwhile, small molecule isosulfocyanate medicine is also illustrate that in vivo once being incorporated in albumen
In matter, its strong toxicity on a cellular level is just lost, the mechanism experiment of cell aspect is directly carried out using small-molecule drug
Its tumor suppression mechanism in vivo cannot be represented.
Performance test 2：Anti-tumor activity test
Inhibitory action of five groups of difference preparations to BCap37 breast cancer cell tumor bearing nude mice tumours is investigated, is specially：
BALB/c-nu/nu nude mices oxter transplanting 1 × 106A BCap37 tumour cells, treat tumour length to about 100mm3After start to be administered, often
It carries out tail vein injection, successive administration seven days.
Group I：Albumin-isothiocyanates key compound (BSA-BITC10, the equivalent ITC dosage prepared using embodiment 6：
50mg/kg nude mices).
Group II：Using albumin-isothiocyanates key compound (BSA-BITC20, equivalent ITC dosage：50mg/kg nude mices).
Preparation process is identical with embodiment 6, differs only in bovine serum albumin(BSA) and feeding intake for benzoyl isothiocyanate is rubbed
You are than being respectively 1:20.
Group III：Albumin-isothiocyanates key compound (BSA-BITC30, the equivalent ITC dosage prepared using embodiment 7：
50mg/kg nude mices).
Group IV：Albumin-isothiocyanates key compound (BSA-PEITC30, the equivalent ITC agent prepared using embodiment 2
Amount：50mg/kg nude mices).
Group V：Using small molecule benzoyl isothiocyanate (Free-BITC, equivalent ITC dosage：50mg/kg nude mices).
Phosphate buffer negative control group (PBS).
It is administered seven times altogether, records the line of apsides and nude mice weight of tumour every time, calculates gross tumor volume V (V=[Length
×(Width)2]/2), draw tumor volume versus time change curve.Experimental result is as shown in Figure 4.
Figure 4, it is seen that BSA-BITC30 groups and BSA-PEITC30 groups and the tumour of small molecule BITC control groups have
Significant difference (p<0.0005) there is suppression after, illustrating benzoyl isothiocyanate or phenethyl isosulfocyanate albumin-binding
The effect of tumour growth processed, and bonding rate is higher, and tumor killing effect is better.
Embodiment 15：Protein-isothiocyanates key compound contains taxol
Taxol is dissolved in ethanol (10mg, 100 μ L), stir while be slowly added dropwise to embodiment 1 prepare it is white
In albumen-isothiocyanates key compound aqueous solution (90mg, 10mL), that is, form nano particle.Vacuum rotary steam removes a small amount of second
Alcohol, is filtered to remove precipitation, that is, obtains the nano particle that albumin-isothiocyanates key compound contains taxol as carrier.
Embodiment 16~17：
Preparation process is identical with embodiment 15, differs only in albumin-isothiocyanates key compound point of use
It is not prepared by embodiment 2~3.
Embodiment 18~19：
Preparation process is identical with embodiment 15, differs only in albumin-isothiocyanates key compound point of use
It is not prepared by embodiment 6, embodiment 8.
Embodiment 20~21：
Preparation process is identical with embodiment 15, differs only in albumin-isothiocyanates key compound point of use
It is not prepared by embodiment 9, embodiment 11.
Embodiment 22~24：
Preparation process is identical with embodiment 15, differs only in albumin-isothiocyanates key compound point of use
It is not prepared by embodiment 12~14.
Comparative example：Albumin contains taxol
Taxol is dissolved in ethanol (10mg, 100 μ L), is stirred while being slowly added dropwise water-soluble to bovine serum albumin(BSA)
In liquid (90mg, 10mL), that is, form nano particle.Vacuum rotary steam removes a small amount of ethanol, is filtered to remove precipitation, that is, obtains white egg
The white nano particle for containing taxol.
Performance test 3：Drugloading rate and particle diameter research in nano particle
Measured in embodiment 15~24 and made respectively using dynamic light scattering (Dynamic Light Scattering, DLS)
Standby albumin-isothiocyanates key compound contains granularity, distribution and the potential of taxol nanoparticle, sees table 2 below, and give
Go out each albumin-isothiocyanates key compound and contain envelop rate and drugloading rate in taxol nanoparticle.
Table 2
As can be seen from the above table, the bonding degree in albumin-isothiocyanates key compound is higher, then after containing taxol
The nanoparticle particle diameter of formation is smaller, and drugloading rate is higher；The isothiocyanates for having phenyl ring contains taxol effect as hydrophobic patch
Rate higher, can form the nano particle that size is homogeneous, well dispersed.
As shown in figure 5, albumin isothiocyanates key compound prepared by embodiment 17 contains taxol in water as carrier
In be assembled into dynamic particle size and be distributed as 0.167, average-size is the particle of 140.7nm.As shown in fig. 6, comparative example preparation is white
Albumen taxol nanoparticle is then assembled into dynamic particle size and is distributed as 0.370 in water, and average-size is the particle of 255.6nm.
In addition, using the form and size of transmission electron microscope observation two kinds of particles, as shown in Figure 7,8, taxol is very
Contain well in albumin-isothiocyanates key compound, form uniform nano particle；And albumin and taxol are then
The particle of irregular form is formed, and it is loosely organized.
Performance test 4：Nano particle freezes stability study
Taxol (the BSA-BITC35/ that the albumin-benzoyl isothiocyanate prepared in measure embodiment 19 contains
PTX) the lyophilized stability of nano particle, concrete technology are as follows：
220nm filters filter, particle diameter 122.9nm, add 3% glucose, vacuum freeze drying, obtains loose white
Powder.Redissolved with deionized water, be positioned over room temperature, the average grain diameter of nano particle is stablized in 8h, see the table below 3.
Table 3
| Time/h | 0 | 3 | 6 | 8 |
| Particle diameter/nm | 128.9 | 130.1 | 120.8 | 121.1 |
| Distribution | 0.217 | 0.218 | 0.207 | 0.199 |
Observe upper table, it can be seen that particle diameter is stablized after the nano particle freezes redissolution, and distribution is good.
Performance test 5：Anti-tumor activity test
Inhibitory action of five groups of difference preparations to BCap37 breast cancer cell tumor bearing nude mice tumours is investigated, is specially：
BALB/c-nu/nu nude mices oxter transplanting 1 × 106A BCap37 tumour cells, treat tumour length to about 100mm3After start to be administered, often
Tail vein injection was carried out every two days.
Group I：Albumin-isothiocyanates key compound (BSA-PEITC50, the equivalent ITC dosage prepared using embodiment 3：
100mg/kg nude mices).
Group II：Albumin-isothiocyanates the key compound prepared using embodiment 17 contains receiving for taxol as carrier
Rice grain (BSA-PEITC50/PTX, equivalent PTX dosage：10mg/kg nude mices).
Group III：The albumin prepared using comparative example contains nano particle (BSA/PTX, the equivalent PTX dosage of taxol：
10mg/kg nude mices).
Group IV：Using small molecule phenethyl isosulfocyanate and PEG2000 key compounds (PEITC-PEG, equivalent ITC dosage：
100mg/kg nude mices), specific preparation process is：
With PEG2000-NH2With the molar ratio 1 of phenethyl isosulfocyanate：1 feeds intake：By PEG2000-NH2(Mw=2000)
200mg is dissolved in 4mL water；Phenethyl isosulfocyanate (Phenethylisothiocyanate, PEITC, Mw=163.24)
16mg is dissolved in 4ml dimethyl sulfoxide (DMSO)s.Above two solution is mixed, adds triethylamine (Triethylamine, TEA, Mw=
101.19) 10mg, 40 DEG C of reaction 12h, dialysis remove organic solvent.
Group V：Phosphate buffer negative control group (PBS).
It is administered five times altogether, records the line of apsides and nude mice weight of tumour every time, calculates gross tumor volume V (V=[Length
×(Width)2]/2), draw tumor volume versus time change curve.Continue the growing state of observation tumour after drug withdrawal, in
Put to death mouse within the 16th day after administration, peel off tumour.Experimental result is as shown in Figure 9 and Figure 10.
It can be seen in figure 9 that BSA-PEITC50 groups and the tumour of PBS control group have significant difference (p=
0.0048) still play the role of necessarily suppressing tumour growth after, illustrating phenethyl isosulfocyanate albumin-binding, and small molecule
PEITC abdominal cavities are directly administered (Wu W J, Zhang Y, Zeng Z L, et al.beta-
phenylethylisothiocyanate reverses platinum resistance by a GSH-dependent
mechanism in cancer cells with epithelial-mesenchymal transition phenotype
[J] .BiochemPharmacol.85 (2013) 486-496.) or bonding PEG formation water soluble molecules intravenous injections, do not have
Tumor killing effect；On the other hand, the growth for carrying the mouse tumor of the BSA/PTX groups of taxol is subject to a degree of suppression (p=
0.0025), but tumour inhibiting rate is still less than 50% (the 15th day), and BSA-PEITC50/PTX groups, and the tumour of mouse is during the administration
Volume persistently reduces, and starts (the p that rebounds at 15 days<0.0005), tumour inhibiting rate 82%.The mouse of all groups was being tested
Weight does not decline in journey.
Claims (10)
1. a kind of protein-isosulfocyanate key compound, it is characterised in that by the different sulphur cyanogen in isosulfocyanate compound
Acid groups react to obtain with the amino group on protein.
2. protein according to claim 1-isosulfocyanate key compound, it is characterised in that specific preparation method is such as
Under：
(1) isosulfocyanate compound being mixed with organic solvent A, protein is mixed with water, then both are mixed, and in real time
Monitor the extent of reaction；
(2) reaction product that step (1) obtains is after centrifuging, be filtered to remove precipitation, then dialyses and remove organic solvent A, obtains institute
The protein stated-isosulfocyanate key compound.
3. protein according to claim 2-isosulfocyanate key compound, it is characterised in that the isothiocyanic acid
Ester type compound is selected from benzyl isothiocyanate, phenethyl isosulfocyanate, benzoyl isothiocyanate, benzyloxy-phenyl
Isothiocyanates, hexyl isothiocyanates, allyl isothiocyanate, Sulforaphane, 3- first thiopropyls
Isothiocyanates, benzylisothiocyanate-N-acetylcysteine conjugates, half Guang of phenethyl isosulfocyanate-N- acetyl
At least one of propylhomoserin conjugate, 1- cyclopropyl naphthalene -4- bases isothiocyanates, arylsulfonyl isothiocyanic acid；
The protein is selected from animal serum albumin, casein, collagen or silk-fibroin；
The organic solvent A is selected from dimethyl sulfoxide (DMSO), ethanol or methanol.
4. protein according to claim 3-isosulfocyanate key compound, it is characterised in that the protein with it is different
The molar ratio of thiocyanate ester compound is 1:10~50；
Isosulfocyanate compound and the mass volume ratio of organic solvent A are 2~100mg/ml；
Organic solvent A and the volume ratio of water are 1~4:10.
5. protein according to claim 4-isosulfocyanate key compound, it is characterised in that the isothiocyanic acid
Ester type compound is phenethyl isosulfocyanate, benzoyl isothiocyanate, benzyloxy-phenyl isothiocyanates or hexyl are different
Thiocyanates；
The protein is human serum albumins or bovine serum albumin(BSA).
6. a kind of protein-isosulfocyanate key compound according to Claims 1 to 5 any claim is as carrier
The method for containing hydrophobic compound, it is characterised in that step is as follows：
Protein-isosulfocyanate key compound is mixed with water, obtains carrier aqueous solution I, by hydrophobic compound with it is organic
Solvent B is mixed to get solution II, then both are mixed, the post-treated nano particle for obtaining containing hydrophobic compound.
7. protein according to claim 6-isosulfocyanate key compound contains hydrophobic compound as carrier
Method, it is characterised in that the hydrophobic compound includes small molecule, anti-tumor drug, photosensitizer, contrast agent or fluorescence dye
Material.
8. protein according to claim 7-isosulfocyanate key compound contains hydrophobic compound as carrier
Method, it is characterised in that the small molecule, anti-tumor drug includes taxol, adriamycin or camptothecine.
9. protein according to claim 6-isosulfocyanate key compound contains hydrophobic compound as carrier
Method, it is characterised in that one kind in dichloromethane, ethanol, methanol, acetone, tetrahydrofuran of the organic solvent B or
It is a variety of.
10. protein according to claim 6-isosulfocyanate key compound contains hydrophobic compound as carrier
Method, it is characterised in that
The concentration of the carrier aqueous solution I is 5~10g/L, and the concentration of solution II is 80~120g/L；
Carrier aqueous solution I and the volume ratio of solution II are 100:1~4.
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| CN109569347A (en) * | 2018-12-14 | 2019-04-05 | 苏州大学 | A kind of method of hydrophobic material water phase transfer |
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| CN109569347A (en) * | 2018-12-14 | 2019-04-05 | 苏州大学 | A kind of method of hydrophobic material water phase transfer |
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