CN105963703A - Preparation method of anti-tumor drug - Google Patents
Preparation method of anti-tumor drug Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
Abstract
The invention discloses a preparation method of an anti-tumor drug. The preparation method comprises the following steps: adding a polymer tetrazole derivative and a cross-linking agent containing methacrylate groups in water or a buffer solution, thus obtaining a carrier solution of which the concentration is 0.5 to 10 mg/mL; then adding a protein drug in the carrier solution, thus obtaining a mixed solution; then injecting the mixed solution in an organic solvent, thus obtaining a suspension solution; then carrying out an ultraviolet irradiation reaction, thus obtaining the anti-tumor drug. The preparation method disclosed by the invention has very strong selectivity, a carrier and an encapsulated drug, particularly, the protein drug and cells are not in reaction, so that the effects of the drug, protein and the cells can be well kept, complete and controllable release can be realized, a focus position can be reached, the drug is slowly released by nanogel, the practical and effective treatment effect can be achieved, and the problem of drug waste in the prior art cannot be caused.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to the preparation method of a kind of antitumor drug.
Background technology
In the past few decades, pharmaceutical grade protein based on antibody, cytokine, enzyme and transcription factor is used widely
Efficiently treatment (Vermonden T, Censi R, Hennink in diseases such as diabetes, cardiovascular disease and malignant tumor
WE. Chem. Rev. 2012, 112, 2853-2888; Walsh G. Nat. Biotech. 2000, 18, 831-
833).Compared with the chemotherapeutics with higher toxic and side effects, pharmaceutical grade protein is generally of higher specificity, preferably controls
Therapeutic effect and relatively low toxic and side effects, shown superior disease treatment effect clinically.But the egg of these Clinical practice
White matter medicine is all to work in extracellular.Although a lot of at the intracellular pharmaceutical grade protein worked, enter but without one
Enter Clinical practice, this is primarily due to, and the plasma half-life of these protein drugs is short, vivo degradation fast, cell endocytic efficiency is low,
Caused by the reasons such as transmitter loss process is slow (Lu Y, Sun W, Gu Z. J. Controlled Release 2014,194,
1-19).Nanogel is often referred to the three dimensional network formed by hydrophilic or amphipathy macromolecule chain by physics or chemical crosslinking
The hydrogel fines of shape structure, has high-moisture, good biocompatibility and loose structure, and nanogel can pass through physics
Crosslinking and chemical crosslinking preparation.Physical crosslinking includes hydrophobic effect, electrostatic interaction, hydrogen bond action etc., and physical gel preparation is generally
Mild condition, will not substantially damage the activity of bag medicine carrying thing；But existence and stability is poor, the medicine of release parcel lacks faster
Point.Chemical crosslinking includes radical polymerization, Michael addition reaction, amidation process, enzymic catalytic reaction, copper catalysis click chemistry
Reaction etc..But these chemical crosslink reactions the most do not possess specificity, this may make medicine participate in cross-linking reaction, causes degeneration.
And, although existing a lot of report uses nanogel conveying medicine, but it is less able to realize medicine targeting in vivo
Efficiently conveying.So need to develop specificity strong, the most quickly, without the cross-linking reaction method of catalysis, for preparation, there is target
The Biodegradable nano gel carrier of tropism, thus obtain antitumor drug of good performance.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of antitumor drug, nanogel bag carry pharmaceutical grade protein system
Standby obtain, the drug specificity obtained by force, the quickest.
To achieve the above object of the invention, the technical solution used in the present invention is: the preparation method of a kind of antitumor drug, bag
Include following steps:
(1) will polymer terazole derivatives and the addition water of the cross-linking agent containing methacrylic acid ester group or buffer obtain
To carrier solution；The concentration of described polymer terazole derivatives is 0.5～10 mg/mL；
(2) pharmaceutical grade protein is added in the carrier solution of step (1), obtain mixed liquor；
(3) mixed liquor of step (2) is expelled in organic solvent, obtains suspension；Then carry out ultraviolet lighting reaction to obtain
Antitumor drug；
Described polymer terazole derivatives has a Formulas I structure:
Formula I；
Wherein n >=2；
R is H, NH2、NMe2、OMe、NO2、Cl、Br、Me、CO2Me or PhNHBoc；
P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucosan, chitosan, collagen egg
In vain, Polyethylene Glycol or polyethylene glycol ester；
The described cross-linking agent containing methacrylic acid ester group has a Formula II structure:
Formula II；
Wherein m >=2；
CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, glucosan, collagen egg
In vain, Polyethylene Glycol, polyethylene glycol ester, butanediamine, hexamethylene diamine, cystamine, cystine or lysine.
In technique scheme, described Polyethylene Glycol is linear or multi-arm polyethylene glycol, is expressed as PEG-x-OH, x=2,
4,6 or 8；Described polyester is polylactide, poly-(lactide-co-glycolide), polycaprolactone or Merlon；Described polyester
The degree of polymerization be 1～20；The molecular weight of described Polyethylene Glycol is 2～100 kg/mol.
In technique scheme, in step (1), methacrylic acid ester group is 1: 1 with the mol ratio of tetrazol group；Described
Buffer includes phosphate buffer, 4-(2-ethoxy)-1-piperazine ethanesulfonic acid half sodium salt buffer solution, trihydroxy methyl amino
Methane buffer solution or MES buffer solution；Described organic solvent includes acetone, acetonitrile or ethanol；Described poly-
Ethylene glycol is linear polyethylene glycol or multi-arm polyethylene glycol；Described polyester be polylactide, poly-(lactide-co-glycolide),
Polycaprolactone or Merlon.
In technique scheme, the wavelength of described ultraviolet lighting reaction is 302～390 nm, and intensity is 0.8～100 mW/
cm2, the time is 90～180s.
In technique scheme, after the reaction of step (3) ultraviolet lighting, rotary evaporation removes organic solvent, then saturating with water
Analysis obtains antitumor drug.
In the present invention, polymer terazole derivatives is connected on by O or NH using polymer at random as main chain, tetrazol group
On main polymer chain end or side chain；In Formulas I structure, P represents that polymer, n >=2 refer to that tetrazol group is connected on main polymer chain
Quantity on end or side chain is a plurality of, and in the such as embodiment of the present invention one, polymer is hyaluronic acid lysine compound,
Multiple tetrazol group it is connected on its repetitive.
In the present invention, the cross-linking agent containing methacrylic acid ester group can be that macromole can also be for little molecule, m >=2
Refer to that in cross-linking agent, the quantity of methacrylic acid ester group is a plurality of；In Formula II structure, when CL is little molecule, methacrylic acid
Ester group is connected on little molecule two ends, such as the structure of the embodiment of the present invention three；When CL is polymer, methacrylic acid ester group passes through
O or NH is connected on main polymer chain end or side chain, and such as the structure of the embodiment of the present invention two, polymer is hyaluronic acid cystamine
Compound, its repetitive is connected to multiple methacrylic acid ester group.
In the present invention, the preparation method of polymer terazole derivatives is, first adds condensing agent and catalysis in tetrazolium solution
Agent, reaction obtains the tetrazolium solution of activation；Then aqueous solutions of polymers being added drop-wise in the tetrazolium solution of activation, room temperature reaction obtains
To polymer terazole derivatives；Described polymer is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine chemical combination
Thing, glucosan, chitosan, collagen protein, Polyethylene Glycol or polyethylene glycol ester.
In technique scheme, it is molten with the mixing of water that the solvent of tetrazolium solution is preferably dimethyl sulfoxide, dimethyl sulfoxide
Liquid, dichloromethane or chloroform；Polymer is the water-soluble polymer containing amino or hydroxyl, preferably hyaluronic acid, hyalomitome
Acid lysine compound, hyaluronic acid cystamine compound, chitosan, glucosan, Polyethylene Glycol or Polyethylene Glycol-oligomerization ester；
Wherein said Polyethylene Glycol is linear or multi-arm polyethylene glycol；Described polyester is polylactide, poly-(lactide-co-second is handed over
Ester), polycaprolactone or Merlon.
In technique scheme, hydroxyl or the mol ratio of amido and tetrazolium in water-soluble polymer are preferably 1: 0.1
～2；Tetrazolium and condensing agent, the mol ratio of catalyst are preferably 1: 2: 0.1.
Such as: first addition condensing agent dicyclohexylcarbodiimide (DCC) in the DMSO solution of the little molecule of tetrazolium (Tet),
DMAP (DMAP), reaction overnight obtains the tetrazolium solution of activated carboxylic；Then poly-containing amino or hydroxyl
Compound aqueous solution is dropwise added drop-wise in the tetrazolium solution of above-mentioned activation, then is stirred at room temperature reaction 18～28 hours, obtains institute
Polymer terazole derivatives (the P-Tet statedn)；Concrete course of reaction is as follows:
Wherein R=H, Cl, Br, Me, NH2、NMe2、NO2Or OMe.
Antitumor drug prepared by the present invention, including nanogel and pharmaceutical grade protein, carrier and pharmaceutical grade protein and
Cell does not reacts, and can well keep effect of medicine, protein and cell, it is achieved complete, controlled release, reaches conscientiously to have
The therapeutic effect of effect, the problem not resulting in the waste of prior art Chinese medicine.
Owing to technique scheme is used, the present invention compared with prior art has the advantage that
The preparation method of antitumor drug the most disclosed by the invention has the strong specificity, rapidly and efficiently and instead of " click chemistry "
Answer the feature of mild condition, simultaneously without toxicity catalyst such as mantoquitas；Particularly, the present invention by design precursor material and
In conjunction with injection, UV reactive, the nanogel stability obtained is strong, and bag is combined stable with medicine after carrying protein, it is ensured that medicine
Circulation is interference-free in vivo.
The preparation method of antitumor drug the most disclosed by the invention has the strongest selectivity, carrier and the medicine wrapping load,
Especially pharmaceutical grade protein and cell does not reacts, and can well keep effect of medicine, protein and cell, it is achieved completely, can
The release of control, arrives affected area, nanogel slow releasing pharmaceutical, reaches effective therapeutic effect, do not result in prior art
The problem of Chinese medicine waste.
Accompanying drawing explanation
Fig. 1 is the hydrogen nuclear magnetic spectrogram of hyaluronic acid lysine terazole derivatives in embodiment one；
Fig. 2 is the hydrogen nuclear magnetic spectrogram of hyaluronic acid cystamine methacrylate derivative in embodiment two；
Fig. 3 is the hydrogen nuclear magnetic spectrogram of cystine methacrylate derivative in embodiment three；
Fig. 4 is the behavior of nano gel outer control release pharmaceutical grade protein in embodiment four, and the protein medicine discharged
The characterization of biological activity figure of thing；
Fig. 5 is the cell experiment phenogram of the nanogel of nanogel and load pharmaceutical grade protein in embodiment five；
Fig. 6 is the treatment phenogram carrying protein nano gel transplanted human breast carcinoma subcutaneous to mice in embodiment six；
Fig. 7 is the treatment phenogram carrying protein nano gel in embodiment seven to mice transplantable lung cancer in situ；
Fig. 8 is the histologic analysis figure carrying protein nano gel for treating mice transplantable lung cancer in situ in embodiment seven.
Detailed description of the invention
Below in conjunction with the accompanying drawings and embodiment the invention will be further described:
The synthesis of embodiment one hyaluronic acid lysine terazole derivatives (HA-Lys-Tet)
Under nitrogen protective condition, 50 mL two neck bottles add tetrazolium (608 mg), dimethyl sulfoxide 10mL, DCC (120 mg)
Stir 24 hours, HA-Lys-NH2(M n=35 K, 0.4 g) is dissolved in 30 mL Methanamides, adds in tetrazolium solution after dissolving,
Stir 10 min, add DMAP (80 mg), react 48 hours, filter, after filtrate water and the dialysis of dimethyl sulfoxide mixed solvent
Change pure water dialysis into, after lyophilization, obtain product HA-Lys-Tet(productivity 69 %)；HA-Lys-Tet nuclear-magnetism characterizes sees accompanying drawing 1,1H NMR (D2O/DMSO-d6): HA: δ 1.82, 2.70–3.68, and 4.23–4.38; Lys: δ 0.92,
1.06, 1.52, 2.97, 3.61 and 3.95; Tet: δ 7.91,7.92 and 6.79, 6.80。
Four arms Polyethylene Glycol terazole derivatives (PEG-Tet4), chitosan terazole derivatives (Chit-Tet) replaceable polymerization
Thing prepares, and structural formula is as follows:
。
The synthesis of embodiment two hyaluronic acid cystamine methacrylate derivative (HA-Cy-MA)
HA-Cy-MA synthesizes in two steps, first methacrylic acid derivative (the MA-Cy-NH of cystamine2) by Boc-Cy-NH2And first
After the reaction of base acryloyl chloride, de-Boc protection obtains, then, under nitrogen protective condition, by MA-Cy-NH2 (14.5 mg, 66
μm ol) solution join HA (50 mg, 1.43 μm ol) by EDC (75.9 mg, 0.396 mmol) and NHS
In bis-water of 5 mL that (22.8 mg, 0.198 mmol) activates, it is placed in lucifuge in 40 DEG C of oil baths and reacts 24 hours, then use
Water is dialysed, lyophilization, productivity 92 %；HA-Cy-MA nuclear-magnetism characterizes sees accompanying drawing 2,1H NMR (D2O): HA: δ 2.00,
2.86–3.88, and 4.44–4.52; Cys: δ 2.70, 3.11–3.15 and 3.56; MA: δ 1.92, 5.45
and 5.69。
The synthesis of embodiment three cystine methacrylamide derivatives (MA-Cys-MA)
Under the conditions of ice-water bath, by the NaOH(1.5 M, 10 mL of cystine (1.2 g, 5.0 mmol)) solution is added drop-wise to methyl
The DCM(10 mL of acryloyl chloride (2.0 mL, 20.6 mmol)) in, under the conditions of ice-water bath, react 4 h, during reaction, use NaOH
Solution regulation and control pH is 9.0.After reaction terminates, separate water layer with separatory funnel, then be added dropwise over about 3 mL HCl(2 M to it),
Filter, vacuum drying, obtain white powder 1.72 g, productivity 91%.MA-Cys-MA nuclear-magnetism characterizes sees accompanying drawing 3,1H NMR(400
MHz, DMSO-d6): MA (δ 5.72,5.39 and 1.85), Cys (δ 12.92,8.24,4.53,3.18 and 3.03).
Embodiment four light-operated " tetrazolium-alkene " click chemistry nanogel is released for bag load and the external control of pharmaceutical grade protein
Put
Carry as a example by cytochrome C (CC) by bag, be 10% by a certain amount of CC(theory drug loading) join total polymer concentration and be
PB(pH 7.4,10 mM of HA-OEG-Tet and HA-Cy-MA of 1.25 mg/mL) in solution, then this solution is expelled to third
In ketone, then with ultraviolet (320-390 nm, 50 mW/cm2) illumination 90 seconds.Last rotary evaporation removes acetone, with water dialysis 12
H, obtains bag and carries nanogel (CC-NGs) solution of CC.Can realize treatment albumen fruit grain enzyme B(GrB by similar method)
Efficient parcel, obtain bag and carry the nanogel (GrB-NGs) of GrB.The release experiment of protein C C in 37 DEG C two kinds of differences
Release medium in carry out, i.e. PB(pH 7.4,10 mM) and PB(pH 7.4,10 mM of 10 mM GSH) solution.Take 1 mL
Bag load CC-NGs sample, in protein delivery bag (MWCO 350 K), is placed in 25 mL corresponding PB release medium.Each
Sampling time point, takes out 5 mL release medium, and supplements corresponding fresh medium.The sample lyophilizing that each time point is taken out, multiple
Molten, measure by ultraviolet (CC uv absorption wavelength is 410 nm).Often release test is parallel carries out three times for group, and final display result is
Experiment averaging of income value ± standard variance.Fig. 4 is the behavior of above-mentioned nano gel outer control release pharmaceutical grade protein, and releases
The characterization of biological activity figure of the pharmaceutical grade protein released；The extracorporeal releasing experiment of protein show in physiological conditions (pH 7.4,
37 DEG C) CC can be wrapped in HA-NGs well, after 48 h, burst size about 30%(Fig. 4 a).On the contrary, containing 10 mM
Under the reducing condition of GSH, from nanogel, after 10 h, discharge the CC more than 80%.CC-NGs is cytoplasmic in this explanation
The pharmaceutical grade protein of parcel can be quickly discharged under reducing environment.
The test of the electron transfer activity of protein is to obtain by detecting its catalytic efficiency that ABTS is changed into ABTS+
's.First it is 0.004 mg/mL that the CC PBS solution discharged is diluted to concentration.Simultaneously configuration same concentrations, without any
The CC processed, takes two kinds of solution of equal amount and puts in quartz sample pool, add same amount of containing 10 μ L's in two kinds of solution
The PBS solution of the ABTS of the hydrogenperoxide steam generator of 0.045 M and 1 mg/mL of 100 μ L.Inversion is allowed to mix and use at once
The absorption value at 410 nm read by UV spectrophotometer, and as zero point, within each 15 seconds, surveys once.Each time point is corresponding
Ultraviolet absorption value deducts the absorption value of first point thus the change (A) of the value that is absorbed, and is plotted against time with A and represents it
Activity change is over time.Accompanying drawing 4b is the above-mentioned protein active detection figure discharged, and result shows from nanogel
The CC discharged still can be catalyzed the oxidation of ABTS quickly, catalytic rate and CC close without any process, it was demonstrated that
The protein discharged from nanogel remains to preferably keep activity.
The empty nanogel of embodiment five and bag are loaded with the cell experiment of the nanogel of pharmaceutical grade protein
Comprehensively utilize anti-phase nanoprecipitation method with macromolecules cross-linking agent and light-operated " tetrazolium-alkene " cross-linking method prepares hyaluronic acid nanometer
Gel, prepares as follows: be dissolved in mol ratio 1:1 by HA-Lys-Tet and HA-Cy-MA prepared by embodiment one and embodiment two
PB(pH 7.4,10 mM) in, prepare the polymer solution that concentration is 1.25 mg/mL, then mixed solution is expelled to
In 100mL acetone, with ultraviolet light (wavelength 320-390 nm, light intensity 60 mW/cm2) radiating 3 min, rotation is used after acetone is evaporated off
PB dialyses, and lyophilization obtains nanogel.
The cell compatibility of the empty nanogel of test.By fibroblast (L929), breast cancer cell (MCF-7), brain glue
Matter oncocyte (U87) and lung carcinoma cell (A549) are layered on 96 porocyte culture plates respectively, about 5000, each hole cell, then
Add containing 10 % calf serums, 1% glutamate, Glu, antibiotics penicillin (100 IU/mL) and streptomycin (100 μ g/mL)
DMEM culture medium, then it is placed in 37 DEG C, cultivate 12 h under 5% carbon dioxide conditions.Then, the PB(10 of 20 μ L nanogels is added
MM, pH 7.4) solution (concentration of final nanogel is 0.2,0.4,0.6,0.8 and 1.0 mg/mL) is in every hole, then 37
DEG C, cultivate 48 h under 5% carbon dioxide conditions.Subsequently, add 3-(4,5-dimethylthiazole-2 to every hole)-2,5-diphenyl four
The PBS solution (15 μ L, 5 mg/mL) of nitrogen azoles bromide (MTT), and put into continuation cultivation in incubator.After 4 h, remove containing
The culture fluid of MTT, adds 150 μ L DMSO for dissolving the purple crystal first a ceremonial jade-ladle, used in libation that living cells and MTT generate, and uses microplate reader
(Bio Tek) measures each hole uv absorption at 492 nm.Right by with only blanc cell of comparative survival rate of cells
Comparing according to hole absorption at 492 nm and obtain, often group empirical average carries out 4 times.Fig. 5 is above-mentioned nanogel and carries protein
The cell experiment phenogram of the nanogel of medicine.
Accompanying drawing 5a is cell survival rate figure, it can be seen that the survival rate adding nanogel cell after 48 hours all reaches
90 more than %, illustrate that hyaluronic acid nanometer gel does not has toxicity.
The cytotoxicity of CC-NGs and GrB-NGs is also to be measured by mtt assay.The nanogel that bag carries protein drug is added
Entering breast cancer cell (MCF-7, CD44 receptor high expressed), lung carcinoma cell (A549, CD44 receptor high expressed) and cerebral glioma are thin
In born of the same parents' (low expression of U87, CD44 receptor), and cultivate 4 h.Then the culture medium carrying the nanogel of albumen is siphoned away, add
Fresh cultured, based on 37 DEG C, continues under 5% carbon dioxide conditions to cultivate 92 h.Cultivate and terminate backward every hole adds 10 μ L MTT
PBS solution (5 mg/mL) and put in incubator, continue cultivate 4 h makes MTT fully act on living cells.Remove subsequently and contain
There is a culture fluid of MTT, and add 150 μ L DMSO for dissolving the purple crystal first a ceremonial jade-ladle, used in libation that living cells generates with MTT, and use enzyme mark
Instrument (Bio Tek) measures each hole uv absorption at 492 nm.Comparative survival rate of cells computational methods are as above.Enclosed experiment
It is first to use HA(5 mg/mL) and MCF-7 cell incubation 4 h, then add bag and carry the nanogel of bag load protein.Result table
Bright CC-NGs has higher anti-tumor activity to MCF-7 cell, and the 503nhibiting concentration (IC50) of its cell is 0.52 M(figure
5b).On the contrary, even if free CC when at concentrations up to 6.2 M still without obvious cytotoxicity, this is primarily due to CC endocytosis and enters
Enter the poor ability of cell.Live it addition, the U87 cells show of CC-NGs expression low to CD44 receptor goes out the apoptosis being obviously reduced
Property, and the anti-tumor activity of the MCF-7 cell that CC-NGs is to closing CD44 receptor in advance substantially reduces, these result explanations
CC-NGs is to enter cell by CD44 receptoe mediated endocytosis mechanism.Meanwhile, the GrB-NGs MCF-7 to CD44 receptor high expressed
All showing higher anti tumor activity in vitro with A549 cell, it is respectively 3.0 nM and 8.1 nM(Fig. 5 c).
Embodiment six carries the treatment of protein nano gel transplanted human breast carcinoma subcutaneous to mice
First by subcutaneous injection MCF-7 cell (1 × 107) PBS solution (50 μ L) establish people to the right lateral side of nude mice
Breast carcinoma subcutaneous tumor model.When the volume of tumor reaches 30 mm3After, nude mice is randomized into 4 groups, often group 5.Then, logical
Cross intravenous methods respectively to often organizing lotus tumor mouse injection GrB-NGs (25 g GrB equiv./kg), GrB-NGs
(100 g GrB equiv./kg), empty nanogel and PBS buffer solution, be administered once, be altogether administered four times for every three days.Swollen
Tumor volume and nude mice body weight are every other day measured once.The computing formula of gross tumor volume: volume=* a*b*c, a are tumors
Longest edge, b is tumor broadside, and c is the height of tumor.Accompanying drawing 8 is the treatment of subcutaneous breast carcinoma, it appeared that PBS group and sky
The tumor volume growth of nanogel group is quick.And GrB-NGs is 25 g GrB equiv./kg and 100 g GrB at dosage
Can effectively suppress the growth of tumor during equiv./kg, and the highest inhibition to tumor growth of dosage is the most obvious.With
Time, GrB-NGs group is similar to PBS group, all reduces without result in the weight of animals, illustrates that GrB-NGs does not has obvious toxic and side effects
(seeing Fig. 6).
Embodiment seven carries the treatment to mice people's transplantable lung cancer in situ of the protein nano gel
First pass through the A549 cell (1 × 10 of injection band luciferase7) PBS solution (50 μ L) to the pulmonary of nude mice
Establish people's pulmonary carcinoma situ tumor model.When the fluorescent value of tumor cell reaches 20000 p/s/cm2During/sr, nude mice is by random
It is divided into 3 groups, often group 6.Then, tail vein injection within every three days, is passed through respectively to often organizing lotus tumor mouse injection GrB-NGs(150 g
GrB equiv./kg GrB), blank nanogel and PBS, be altogether administered four times.Fluorescence that tumor goes out and nude mice body weight every three
Once, Fig. 7 is the treatment phenogram carrying protein nano gel to mice transplantable lung cancer in situ to it record.Accompanying drawing 7a-c is pulmonary carcinoma
Situ treatment figure, at the tumor of PBS group and empty nanogel group, fluorescence intensity is remarkably reinforced in time, shows that tumor is in fast fast-growing
Long.And tumor fluorescence intensity in GrB-NGs treatment is more weak, this explanation GrB-NGs can suppress the growth of lung cancer in nude mice effectively.
Meanwhile, the Mouse Weight change of GrB-NGs group is notable (Fig. 7 d), and on the one hand this show that GrB-NGs makees without the malicious secondary of detail
With, the most also explanation GrB-NGs can suppress the growth of mice pulmonary carcinoma in situ effectively, makes mouse growth in order.Phase
Instead, the Mice Body weight average of PBS and blank nanogel matched group significantly reduces, this is because quick along with original position pulmonary carcinoma of mice
Growth, health drastically declines.The survival assay of mice also indicates that the mice of GrB-NGs treatment group is during whole observation
(40 days), do not have dead generation；And the mice of PBS and blank nanogel matched group is after Ureteral Calculus terminates, all dead
(Fig. 7 e).It appeared that GrB-NGs is to internal main organs (heart, liver, kidney etc.), be free from side effects (Fig. 8) in H&E dyeing, this
Further demonstrate that GrB-NGs will not cause obvious toxic and side effects.
Claims (10)
1. a preparation method for antitumor drug, comprises the following steps:
(1) will polymer terazole derivatives and the addition water of the cross-linking agent containing methacrylic acid ester group or buffer be carried
Liquid solution；The concentration of described polymer terazole derivatives is 0.5～10 mg/mL；
(2) pharmaceutical grade protein is added in the carrier solution of step (1), obtain mixed liquor；
(3) mixed liquor of step (2) is expelled in organic solvent, obtains suspension；Then carry out ultraviolet lighting reaction to obtain
Antitumor drug；
Described polymer terazole derivatives has a Formulas I structure:
Formula I；
Wherein n >=2；
R is H, NH2、NMe2、OMe、NO2、Cl、Br、Me、CO2Me or PhNHBoc；
P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucosan, chitosan, collagen egg
In vain, Polyethylene Glycol or polyethylene glycol ester；
The described cross-linking agent containing methacrylic acid ester group has a Formula II structure:
Formula II；
Wherein m >=2；
CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, glucosan, collagen egg
In vain, Polyethylene Glycol, polyethylene glycol ester, butanediamine, hexamethylene diamine, cystamine, cystine or lysine.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: in step (1), methacrylate
Group is 1: 1 with the mol ratio of tetrazol group.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: described buffer includes that phosphate delays
Rush liquid, 4-(2-ethoxy)-1-piperazine ethanesulfonic acid half sodium salt buffer solution, trishydroxymethylaminomethane buffer solution or 2-
Morpholino b acid buffer solution.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: described organic solvent include acetone,
Acetonitrile or ethanol.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: described Polyethylene Glycol is linear poly-second
Glycol or multi-arm polyethylene glycol；Described polyester is polylactide, poly-(lactide-co-glycolide), polycaprolactone or poly-carbon
Acid esters.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: the preparation of polymer terazole derivatives
Method is, first adds condensing agent and catalyst in tetrazolium solution, and reaction obtains the tetrazolium solution of activation；Then polymer water
Solution is added drop-wise in the tetrazolium solution of activation, and room temperature reaction obtains polymer terazole derivatives；Described polymer be hyaluronic acid,
Glucosan, chitosan, collagen protein, Polyethylene Glycol or polyethylene glycol ester.
The preparation method of antitumor drug the most according to claim 6, it is characterised in that: described tetrazolium and condensing agent, catalysis
The mol ratio of agent is 1: 2: 0.1.
The preparation method of antitumor drug the most according to claim 6, it is characterised in that: described condensing agent is dicyclohexyl carbon
Diimine；Described catalyst is DMAP.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: the wavelength of described ultraviolet lighting reaction
Being 302～390 nm, intensity is 0.8～100 mW/cm2, the time is 90～180s.
The preparation method of antitumor drug the most according to claim 1, it is characterised in that: step (3) ultraviolet lighting reacts
After, rotary evaporation removes organic solvent, then obtains antitumor drug with water dialysis.
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