CN105968372A - Self-fluorescence nanogel and preparation method and application thereof - Google Patents
Self-fluorescence nanogel and preparation method and application thereof Download PDFInfo
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- CN105968372A CN105968372A CN201610536475.1A CN201610536475A CN105968372A CN 105968372 A CN105968372 A CN 105968372A CN 201610536475 A CN201610536475 A CN 201610536475A CN 105968372 A CN105968372 A CN 105968372A
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- nanogel
- autofluorescence
- preparation
- polyethylene glycol
- hyaluronic acid
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Classifications
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
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- A—HUMAN NECESSITIES
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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Abstract
The invention discloses a self-fluorescence nanogel and a preparation method and an application thereof. The self-fluorescence nanogel is jointly prepared by a reverse-phase nanoprecipitation method and an optically-controlled tetrazole-alkene click chemistry method; the tetrazole-alkene click chemistry method has the advantages of strong specificity, high efficiency, rapidity, no catalyst and the like, so that the bioactivity of entrapped drugs and protein can be effectively maintained; the application prospect in the fields of releasing of carriers by drug control and the like is good. The self-fluorescence nanogel has the advantages that the crosslink method has strong selectivity; the self-fluorescence nanogel does not react with the entrapped drug, especially protein, drugs and cells, so that the functions of drugs, protein and cells can be well maintained, the complete and controllable release is realized, and the self-fluorescence nanogel can be used as a good slow-releasing carrier for the protein and drugs; in the disease part, the drug is slowly released by the nanogel, the feasible and effective treatment effect is reached, and the problem of waste in drug in prior art is solved.
Description
Technical field
The present invention relates to the preparation method and application of a kind of nanogel, be specifically related to a kind of derivative based on polymer tetrazolium
Thing and the nanogel preparation method containing methacrylic acid ester group cross-linking agent, and this nanogel answering at field of medicaments
With.
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 so that it is be preferably applied for the fields such as drug controlled release carrier.
Summary of the invention
It is an object of the invention to provide a species specificity strong, the quickest, without light-operated " tetrazolium-alkene " clickization being catalyzed
Method prepares the method for nanogel, and the nanogel prepared by the method props up at drug controlled release carrier and organizational project
The fields such as frame material have good application prospect.
To achieve the above object of the invention, the technical solution used in the present invention is: the preparation of a kind of autofluorescence nanogel
Method, comprises the following steps: polymer terazole derivatives and the cross-linking agent containing methacrylic acid ester group are added water or delays
Rush and liquid obtains mixed liquor；Then mixed liquor is expelled in organic solvent, obtains suspension；Then carry out illumination reaction to obtain
Autofluorescence nanogel；
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；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；Institute
The degree of polymerization stating polyester is 1～20；
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, methacrylic acid ester group is 1: 1 with the mol ratio of tetrazol group；In mixed liquor, polymerization
Thing terazole derivatives concentration is 0.5～10mg/mL.
In technique scheme, described illumination reaction is ultraviolet lighting reaction；The wavelength of described ultraviolet light is 302-390
Nm, intensity is 0.8～100 mW/cm2, the time is 90～180s.
In technique scheme, described buffer includes phosphate (PB) buffer, 4-(2-ethoxy)-1-piperazine
Sulfonic acid half sodium salt (HEPES) buffer solution, trishydroxymethylaminomethane (Tris) buffer solution, MES (MES) delays
Dissolved liquid etc.；Preferably pH is the PB buffer of 7.4；Described organic solvent includes acetone, acetonitrile, ethanol etc.；Preferably acetone.
The invention discloses the autofluorescence nanogel prepared according to said method；Be properly termed as light-operated " tetrazolium-
Alkene " the autofluorescence nanogel prepared of click chemistry.
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.
The present invention further discloses the application in preparing tissue engineering bracket of the above-mentioned autofluorescence nanogel；Above-mentioned
The application in preparing pharmaceutical grade protein of the autofluorescence nanogel；Above-mentioned autofluorescence nanogel is as slow releasing carrier of medication
Application.
The invention also discloses a kind of antitumor drug, including above-mentioned autofluorescence nanogel and pharmaceutical grade protein.
The medicine that nanogel disclosed by the invention carries with bag, especially pharmaceutical grade protein and cell does not reacts, and can well keep medicine
Effect of thing, protein and cell, it is achieved complete, controlled release, thus can carry as the excellent slow release of the medicines such as protein
Body；Arrive affected area, nanogel slow releasing pharmaceutical, reach effective therapeutic effect, do not result in prior art Chinese medicine
The problem of waste.
Owing to technique scheme is used, the present invention compared with prior art has the advantage that
The preparation method of nanogel the most disclosed by the invention have " click chemistry " strong specificity, rapidly and efficiently and reaction
The feature of mild condition, simultaneously without toxicity catalyst such as mantoquitas；Particularly, the present invention is by design precursor material and knot
Closing injection, UV reactive, the nanogel stability obtained is strong, when being used for wrapping load protein, is combined stable with medicine, it is ensured that medicine
Thing circulates in vivo without interruption.
The cross-linking method of nanogel the most disclosed by the invention has the strongest selectivity, the medicine carried with bag, especially
Pharmaceutical grade protein and cell do not react, and can well keep effect of medicine, protein and cell, it is achieved complete, controlled releases
Put, thus can be as the excellent slow-released carrier of the medicines such as protein；Reach affected area, nanogel slow releasing pharmaceutical, arrive conscientiously
Effective therapeutic effect, the problem not resulting in the waste of prior art Chinese medicine.
3. the nanogel that prepared by the present invention has autofluorescence performance, can be used to observe nanogel carrier at body
Outer endocytosis enters cell and penetrates the behavior of lesion tissue in vivo, observes, for ingestion of medicines monitoring, conveying, the bar providing favourable
Part, overcomes prior art and also needs to separately add the defect that fluorescent agent monitors that medicine carries.
Nanogel persursor material the most disclosed by the invention has good biocompatibility and biological degradability, and
And wide material sources, prepare simple, cost is relatively low；Preparation process is controlled, it is not necessary to catalyst, makes product more while economizing on resources
Pure, it is that one is applicable to industrialized preparation method.
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 that the fundamental property of nanogel in embodiment four characterizes；
Fig. 5 is the preparation of the hybridized hydrogel in embodiment six containing autofluorescence nanogel；
Fig. 6 is the behavior of nano gel outer control release pharmaceutical grade protein in embodiment seven, and the protein medicine discharged
The characterization of biological activity figure of thing；
Fig. 7 is the cell experiment phenogram of the nanogel of nanogel and load pharmaceutical grade protein in embodiment eight；
Fig. 8 is the treatment phenogram carrying protein nano gel transplanted human breast carcinoma subcutaneous to mice in embodiment nine；
Fig. 9 is the treatment phenogram carrying protein nano gel in embodiment ten to mice transplantable lung cancer in situ；
Figure 10 is the histologic analysis figure carrying protein nano gel for treating mice transplantable lung cancer in situ in embodiment ten.
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 macromolecules cross-linking prepares hyaluronic acid nanometer gel
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.Seeing accompanying drawing 4, the particle diameter of nanogel can use dynamic light scattering (DLS) to measure,
The nanogel size tunable (150-343 nm) obtained by this kind of method, PDI the least (0.10-0.17) (Fig. 4 a), simultaneously
The pattern of nanogel can be observed with transmission electron microscope (TEM) and obtain, and nanogel prepared by this method is spherical (Fig. 4 a)；Separately
Outward, the nanogel prepared, under the UV exciting light of 405nm radiates, has stronger green fluorescence (figure at 450 nm
4b)；Utilize the autofluorescence performance of nanogel to observe cell endocytic and distribution situation, this nanometer in vivo of nanogel
Gel also shows good stability, is placed on PB(pH 7.4,10 mM) and 10% FBS analogue body in the equal energy of blood environment
Keep particle diameter stable (Fig. 4 c)；But nanogel is placed in the PB (pH 7.4,10 mM) of 10 mM GSH 4 hours
Just it is observed that significant change of size, increasing in time, particle diameter starts quickly to become big, shows that this kind of nanogel has quickly
Reduction response property (Fig. 4 d).This can be used to realize medicine and quickly discharge in intracellular response, is greatly increased bag
The therapeutic effect of medicine carrying thing.
Embodiment five little molecule crosslinking preparation hyaluronic acid nanometer gel
Comprehensively utilize anti-phase nanoprecipitation method with little molecule cross-linking agent and light-operated " tetrazolium-alkene " cross-linking method prepares hyaluronic acid nanometer
Gel, by 1 mL HA-OEG-Tet(1 mg/mL) and PB(pH 8.5,10 mM of MA-Cys-MA) solution (Tet and MA group
Mol ratio control at 1:1) be expelled in 20 mL acetonitriles, then this solution is placed in ultraviolet camera bellows (320-390 nm, 37.5
MW/cm2) illumination 90 s.Rotary evaporation removes acetone, with PB(pH 7.4,10 mM) dialyse 12 h(MWCO 7000 Da),
To nanogel.Dynamic light scattering (DLS) result shows mean diameter 165 nm of nanogel prepared by this method, and presents list
Peak is distributed.This nanogel of TEM caption has spherical structure, and most nano-particles size is about 100 nm,
The particle diameter of the nanogel in TEM picture is less than the particle diameter that DLS measures, probably due in TEM sample making course, water in nanogel
Divide evaporation, nanogel shrinkage, and make the particle diameter taken little.
Embodiment six light-operated " tetrazolium-alkene " click chemistry nanogel carries and composite aquogel for the bag of somatomedin
Preparation
Carry as a example by VEGF (VEGF) by bag, first VEGF, HA-OEG-Tet and HA-Cy-MA are dissolved into PB
In (pH 7.4,10 mM) buffer solution, making total polymer concentration is 1.25 mg/mL.Then this solution is expelled in acetone,
Again with portable uv analyzer (302 nm, 0.88 mW/cm that power is extremely low2) illumination 180 seconds.Last rotary evaporation removes
Acetone, dialyses 12 h with water, obtains bag and carries nanogel (VEGF-NGs) solution of VEGF.DLS result display bag carries VEGF's
Nanogel (VEGF-NGs) presents Unimodal Distribution, and its mean diameter is 173 nm.Tem observation display VEGF-NGs has ball
Shape structure, and the diameter of nanogel is about 100 nm.This bag carries the nanogel of VEGF and has preferable injectivity, can be straight
Connect to inject to use or be combined with hydrogel and prepare hydrogel composites.
The preparation of hydrogel composites is first by mercapto-functionalized collagen protein (Col-SH) and oligo-ester carbonate-poly-second
Glycol-oligo-ester carbonate (OAC-PEG-OAC) is dissolved in PB(pH 7.4,100 mM of 100 L respectively) in, until completely dissolved,
Two kinds of solution and VEGF-NGs solution at room temperature mix homogeneously, then it is placed in 37 DEG C of shaking table reaction formation composite aquogels.Should
The becoming the forming process of hydrogel from solution state and see Fig. 5 a of composite aquogel.Meanwhile, the storage modulus (G of composite aquogel
`) rheometer measurement can be used over time with loss modulus (G``).Result show the modulus of this composite aquogel up to
2000 Pa, gel time is about 3 minutes (Fig. 5 b).This bag carries the composite aquogel of VEGF somatomedin and props up as organizational project
Frame has wide practical use in ischemic myocardium reparation.
Embodiment seven 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. 6 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. 6 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 6b 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 eight and bag are loaded with the cell experiment of the nanogel of pharmaceutical grade protein
As a example by the nanogel of the preparation in embodiment four, the cell compatibility of the empty nanogel of test.By fibroblast
(L929), breast cancer cell (MCF-7), brain glioblastoma cell (U87) and lung carcinoma cell (A549) are layered on 96 porocyte trainings respectively
Support on plate, about 5000, each hole cell, add containing 10 % calf serums, 1% glutamate, Glu, antibiotics penicillin
The DMEM culture medium of (100 IU/mL) and streptomycin (100 μ g/mL), then it is placed in 37 DEG C, cultivate 12 under 5% carbon dioxide conditions
h.Then, add the PB(10 mM, pH 7.4 of 20 μ L nanogels) solution (concentration of final nanogel is 0.2,0.4,
0.6,0.8 and 1.0 mg/mL) in every hole, then at 37 DEG C, under 5% carbon dioxide conditions, cultivate 48 h.Subsequently, add to every hole
3-(4,5-dimethylthiazole-2)-2, the PBS solution (15 μ L, 5 mg/mL) of 5-diphenyltetrazolium bromide bromide (MTT), and put into
Incubator continues cultivate.After 4 h, remove the culture fluid containing MTT, add 150 μ L DMSO for dissolve living cells with
The purple crystal first a ceremonial jade-ladle, used in libation that MTT generates, and measure each hole uv absorption at 492 nm by microplate reader (Bio Tek).Cell
Relative survival rate is by obtaining compared with the absorption at 492 nm of the control wells of only blanc cell, and often group empirical average is carried out
4 times.Fig. 7 is the cell experiment phenogram of above-mentioned nanogel and the nanogel carrying pharmaceutical grade protein.
Accompanying drawing 7a 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
7b).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. 7 c).
Embodiment nine 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. 8).
Embodiment ten 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. 9 is the treatment phenogram carrying protein nano gel to mice transplantable lung cancer in situ to it record.Accompanying drawing 9a-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. 9 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. 9 e).It appeared that GrB-NGs is to internal main organs (heart, liver, kidney etc.), be free from side effects (Figure 10) in H&E dyeing,
This has further demonstrated that GrB-NGs will not cause obvious toxic and side effects.
Claims (10)
1. the preparation method of an autofluorescence nanogel, it is characterised in that comprise the following steps: polymer tetrazolium is derived
Thing and the cross-linking agent containing methacrylic acid ester group add in water or buffer and obtain mixed liquor；Then mixed liquor is expelled to
In organic solvent, obtain suspension；Then carry out illumination reaction and obtain autofluorescence nanogel；
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 autofluorescence nanogel the most according to claim 1, it is characterised in that: methacrylic acid ester group
It is 1: 1 with the mol ratio of tetrazol group；In mixed liquor, polymer terazole derivatives concentration is 0.5～10 mg/mL.
The preparation method of autofluorescence nanogel the most according to claim 1, it is characterised in that: described buffer includes phosphorus
Phthalate buffer, 4-(2-ethoxy)-1-piperazine ethanesulfonic acid half sodium salt buffer solution, trishydroxymethylaminomethane buffer solution
Or MES buffer solution；Described organic solvent includes acetone, acetonitrile or ethanol.
The preparation method of autofluorescence nanogel the most according to claim 1, it is characterised in that: described illumination reaction is purple
Outer illumination reaction；The wavelength of described ultraviolet light is 302～390 nm, and intensity is 0.8～100 mW/cm2, the response time be 90～
180s。
The preparation method of autofluorescence nanogel the most according to claim 1, it is characterised in that: described Polyethylene Glycol is line
Property Polyethylene Glycol or multi-arm polyethylene glycol；Described polyester be polylactide, poly-(lactide-co-glycolide), polycaprolactone or
Person's Merlon.
The preparation method of autofluorescence nanogel the most according to claim 1, it is characterised in that: polymer terazole derivatives
Preparation method be in tetrazolium solution, first add condensing agent and catalyst, reaction obtains the tetrazolium solution of activation；Then poly-
Compound aqueous solution is added drop-wise in the tetrazolium solution of activation, and room temperature reaction obtains polymer terazole derivatives；Described polymer is
The acid of bright matter, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucosan, chitosan, collagen protein, poly-second two
Alcohol or polyethylene glycol ester.
Autofluorescence nanogel prepared by any one preparation method the most according to claims 1 to 6.
8. an antitumor drug, including autofluorescence nanogel and pharmaceutical grade protein described in claim 7.
9. autofluorescence nanogel application in preparing pharmaceutical grade protein or tissue engineering bracket described in claim 7.
10. autofluorescence nanogel described in claim 7 is as the application of slow releasing carrier of medication.
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| WO2018010053A1 (en) * | 2016-07-10 | 2018-01-18 | 苏州大学张家港工业研究院 | Method for preparing antitumor drug |
| CN110251456A (en) * | 2019-06-06 | 2019-09-20 | 苏州大学 | A kind of preparation method and application of nanogel |
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| CN112661929A (en) * | 2021-01-18 | 2021-04-16 | 丽水学院 | Polyurethane functionalized by click chemistry and preparation method thereof |
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