CN105968372B - A kind of autofluorescence nanogel and the preparation method and application thereof - Google Patents
A kind of autofluorescence nanogel and the preparation method and application thereof Download PDFInfo
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- CN105968372B CN105968372B CN201610536475.1A CN201610536475A CN105968372B CN 105968372 B CN105968372 B CN 105968372B CN 201610536475 A CN201610536475 A CN 201610536475A CN 105968372 B CN105968372 B CN 105968372B
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- nanogel
- autofluorescence
- hyaluronic acid
- polyethylene glycol
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
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- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0073—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form semi-solid, gel, hydrogel, ointment
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- C08J2387/00—Characterised by the use of unspecified macromolecular compounds, obtained otherwise than by polymerisation reactions only involving unsaturated carbon-to-carbon bonds
Abstract
The invention discloses nanogels of a kind of autofluorescence and the preparation method and application thereof.The present invention prepares nanogel by combination reverse phase nanoprecipitation method and light-operated " tetrazolium-alkene " click chemistry method, " tetrazolium-alkene " the click chemistry cross-linking method used have many advantages, such as specificity it is strong, efficiently quickly, without catalyst, this can effectively maintain the bioactivity of the drug contained and protein, have a good application prospect in the fields such as drug controlled release carrier.The cross-linking method of nanogel disclosed by the invention has very strong selectivity, with the drug contained, especially pharmaceutical grade protein and cell does not react, the effect of capable of keeping drug, protein and cell well, complete, controllable release is realized, so as to the excellent slow-released carrier as protein and drug；Reach affected area, nanogel slow releasing pharmaceutical, the problem of reaching effective therapeutic effect, not will cause drug waste in the prior art.
Description
Technical field
The present invention relates to a kind of preparation method and applications of nanogel, and in particular to one kind is derivative based on polymer tetrazolium
Nanogel preparation method and the nanogel the answering in field of medicaments of object and the crosslinking agent containing methacrylate group
With.
Background technique
In the past few decades, the pharmaceutical grade protein based on antibody, cell factor, enzyme and transcription factor is widely used
In efficient treatment (Vermonden T, the Censi R, Hennink of the diseases such as diabetes, cardiovascular disease and malignant tumour
WE. Chem. Rev. 2012, 112, 2853-2888; Walsh G. Nat. Biotech. 2000, 18, 831-
833).Compared with the chemotherapeutics with higher toxic side effect, pharmaceutical grade protein specificity usually with higher is preferably controlled
Therapeutic effect and lower toxic side effect have clinically shown superior disease treatment effect.But the egg of these clinical uses
White matter drug is all extracellularly to work.Although there are many pharmaceutical grade protein to work in the cell, but without one into
Enter clinical use, the plasma half-life that this is primarily due to these protein drugs is short, internal degradation is fast, cell endocytic low efficiency,
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 hydrophily or amphipathy macromolecule chain by physics or chemical crosslinking
The hydrogel fines of shape structure, have high-moisture, good biocompatibility and porous 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 usual
Mild condition will not obviously damage the activity for containing drug；But there are stability, and poor, release package drug lacks faster
Point.Chemical crosslinking includes free radical polymerization, Michael addition reaction, amidation process, enzymic catalytic reaction, copper catalysis click chemistry
Reaction etc..But these chemical crosslink reactions do not have specificity usually, this may make drug participate in cross-linking reaction, cause to be denaturalized.
Moreover, being less able to realize the targeting of therapeutic agent in vivo although existing many reports convey drug using nanogel
Efficiently conveying.So need to develop specificity it is strong, efficiently quickly, without the cross-linking reaction method of catalysis, be used to prepare with target
The Biodegradable nano gel carrier of tropism, makes it be preferably applied for the fields such as drug controlled release carrier.
Summary of the invention
That the object of the present invention is to provide a species specificities is strong, efficiently quickly, without light-operated " tetrazolium-alkene " clickization of catalysis
The method that method prepares nanogel, the nanogel prepared with this method is in drug controlled release carrier and organizational project branch
It is had a good application prospect in the fields such as frame material.
To achieve the above object of the invention, the technical solution adopted by the present invention is that: a kind of preparation of autofluorescence nanogel
Method, comprising the following steps: water is added in polymer terazole derivatives and the crosslinking agent containing methacrylate group or is delayed
Mixed liquor is obtained in fliud flushing；Then mixed liquor is injected into organic solvent, obtains suspension；Then illumination reaction is carried out to obtain
Autofluorescence nanogel；
The polymer terazole derivatives have 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, glucan, chitosan, glue
Former albumen, polyethylene glycol or polyethylene glycol ester；The polyethylene glycol is linear or multi-arm polyethylene glycol, is expressed as
PEG-x-OH, x=2,4,6 or 8；The polyester is polylactide, poly- (lactide-co-glycolide), polycaprolactone or poly- carbon
Acid esters；The degree of polymerization of the polyester is 1~20；
The crosslinking agent containing methacrylate group has Formula II structure:
Formula II；
Wherein m >=2；
CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, glucan, glue
Former albumen, polyethylene glycol, polyethylene glycol ester, butanediamine, hexamethylene diamine, cystamine, cystine or lysine.
In above-mentioned technical proposal, the polyethylene glycol is linear or multi-arm polyethylene glycol, is expressed as PEG-x-OH, x=2,
4,6 or 8；The polyester is polylactide, poly- (lactide-co-glycolide), polycaprolactone or polycarbonate；The polyester
The degree of polymerization be 1~20；The molecular weight of the polyethylene glycol is 2~100 kg/mol.
In above-mentioned technical proposal, the molar ratio of methacrylate group and tetrazol group is 1: 1；In mixed liquor, polymerization
Object terazole derivatives concentration is 0.5~10mg/mL.
In above-mentioned technical proposal, the illumination reaction is ultraviolet lighting reaction；The wavelength of the ultraviolet light is 302-390
Nm, intensity are 0.8~100 mW/cm2, the time is 90~180s.
In above-mentioned technical proposal, the buffer includes phosphate (PB) buffer, 4-(2- ethoxy) -1- piperazine
Half sodium salt of sulfonic acid (HEPES) buffer solution, trishydroxymethylaminomethane (Tris) buffer solution, 2-morpholine ethane sulfonic acid (MES) are slow
Rush solution etc.；It is preferred that the PB buffer that pH is 7.4；The organic solvent includes acetone, acetonitrile, ethyl alcohol etc.；It is preferred that acetone.
The invention discloses the autofluorescence nanogels being prepared according to the above method；It is properly termed as light-operated " tetrazolium-
The autofluorescence nanogel of alkene " click chemistry preparation.
In the present invention, using polymer as main chain, tetrazol group is connect polymer terazole derivatives at random by O or NH
On main polymer chain end or side chain；In Formulas I structure, P indicates polymer, and n >=2 refer to that tetrazol group connects in main polymer chain
Quantity on end or side chain is a plurality of, such as in the embodiment of the present invention one, and polymer is hyaluronic acid lysine compound,
Multiple tetrazol groups are connected on its repetitive unit.
In the present invention, the crosslinking agent containing methacrylate group can may be small molecule, m >=2 for macromolecular
Refer to that the quantity of methacrylate group in crosslinking agent is a plurality of；In Formula II structure, when CL is small molecule, methacrylic acid
Ester group connects at small molecule both ends, such as the structure of the embodiment of the present invention three；When CL is polymer, methacrylate group passes through
O or NH connects 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 is connected to multiple methacrylate groups on repetitive unit.
In the present invention, the preparation method of polymer terazole derivatives is that condensing agent and catalysis are first added into tetrazolium solution
The tetrazolium solution activated is reacted in agent；Then aqueous solutions of polymers is added drop-wise in the tetrazolium solution of activation, is reacted at room temperature
To polymer terazole derivatives；The polymer is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine chemical combination
Object, glucan, chitosan, collagen, polyethylene glycol or polyethylene glycol ester.
In above-mentioned technical proposal, the solvent of tetrazolium solution is preferably that the mixing of dimethyl sulfoxide, dimethyl sulfoxide and water is molten
Liquid, methylene chloride or chloroform；Polymer is the water-soluble polymer containing amino or hydroxyl, preferably hyaluronic acid, hyalomitome
Sour lysine compound, hyaluronic acid cystamine compound, chitosan, glucan, polyethylene glycol or polyethylene glycol-oligomerization ester；
Wherein the polyethylene glycol is linear or multi-arm polyethylene glycol；The polyester is polylactide, poly- (lactide-co- second is handed over
Ester), polycaprolactone or polycarbonate.
In above-mentioned technical proposal, the molar ratio of hydroxyl or amido and tetrazolium in water-soluble polymer is preferably 1: 0.1
~2；The molar ratio of tetrazolium and condensing agent, catalyst is preferably 1: 2: 0.1.
Such as: first addition condensing agent dicyclohexylcarbodiimide (DCC) into the DMSO solution of tetrazolium small molecule (Tet),
4-dimethylaminopyridine (DMAP), reaction obtain the tetrazolium solution of activated carboxylic overnight；Then poly- containing amino or hydroxyl
It closes object aqueous solution to be added drop-wise to dropwise in the tetrazolium solution of above-mentioned activation, then reaction is stirred at room temperature 18~28 hours, obtain institute
Polymer terazole derivatives (the P-Tet statedn)；Specific reaction process is as follows:
Wherein R=H, Cl, Br, Me, NH2、NMe2、NO2Or OMe.
The present invention further discloses above-mentioned autofluorescence nanogels to prepare the application in tissue engineering bracket；It is above-mentioned
Autofluorescence nanogel is preparing the application in pharmaceutical grade protein；Above-mentioned autofluorescence nanogel is as slow releasing carrier of medication
Application.
The invention also discloses a kind of anti-tumor drugs, including above-mentioned autofluorescence nanogel and pharmaceutical grade protein.
Nanogel disclosed by the invention and the drug contained, especially pharmaceutical grade protein and cell do not react, and can keep medicine well
The effect of object, protein and cell, realizes complete, controllable release, so as to carry as the excellent sustained release of the drugs such as protein
Body；Affected area is reached, nanogel slow releasing pharmaceutical reaches effective therapeutic effect, not will cause drug in the prior art
The problem of waste.
Due to the above technical solutions, the present invention has the following advantages over the prior art:
1. the preparation method of nanogel disclosed by the invention have " click chemistry " strong specificity, rapidly and efficiently and
The mild feature of reaction condition, while without toxicity catalyst such as mantoquitas；Particularly, the present invention by design precursor material with
And injection, UV reactive are combined, obtained nanogel stability is strong, when for containing protein, stablizing in conjunction with drug, protecting
Card drug recycles without interruption in vivo.
2. the cross-linking method of nanogel disclosed by the invention has a very strong selectivity, and the drug contained, especially
The effect of pharmaceutical grade protein and cell do not react, and can keep drug, protein and cell well realizes complete, controllable release
It puts, so as to the excellent slow-released carrier as drugs such as protein；Reach affected area, nanogel slow releasing pharmaceutical reaches practical
Effective therapeutic effect, the problem of not will cause drug waste in the prior art.
3. nanogel prepared by the present invention has autofluorescence performance, observation nanogel carrier can be used in body
Outer endocytosis enters cell and penetrates the behavior of lesion tissue in vivo, provides advantageous item for ingestion of medicines monitoring, conveying observation
Part overcomes the defect that the prior art also needs separately to add fluorescer monitoring drug conveying.
4. nanogel persursor material disclosed by the invention has good biocompatibility and biological degradability, and
And it is from a wealth of sources, preparation is simple, cost is relatively low；Preparation process is controllable, is not necessarily to catalyst, product is made while economizing on resources more
It is pure, it is a kind of suitable for industrialized preparation method.
Detailed description of the invention
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 fundamental property characterization of nanogel in example IV；
Fig. 5 is the preparation of the hybridized hydrogel of the nanogel containing autofluorescence in embodiment six；
Fig. 6 is the behavior for controlling release pharmaceutical grade protein in embodiment seven outside nano gel, and the albumen released
The characterization of biological activity figure of matter drug；
Fig. 7 is the cell experiment phenogram of the nanogel of nanogel and load pharmaceutical grade protein in embodiment eight；
Fig. 8 is that protein nano gel is carried in embodiment nine to the treatment phenogram of the subcutaneous transplanted human breast carcinoma of mouse；
Fig. 9 is that protein nano gel is carried in embodiment ten to the treatment phenogram of mouse original position transplantable lung cancer；
Figure 10 is the histologic analysis figure that protein nano gel for treating mouse original position transplantable lung cancer is carried in embodiment ten.
Specific embodiment
With reference to the accompanying drawing and embodiment the invention will be further described:
The synthesis of one hyaluronic acid lysine terazole derivatives (HA-Lys-Tet) of embodiment
Under the conditions of nitrogen protection, tetrazolium (608 mg) is added in 50 mL, two neck bottle, dimethyl sulfoxide 10mL, DCC (120
Mg it) stirs 24 hours, HA-Lys-NH2(M n=35 K, 0.4 g) is dissolved in 30 mL formamides, and tetrazolium solution is added after dissolution
In, 10 min are stirred, are added DMAP (80 mg), are reacted 48 hours, filtering, filtrate water and dimethyl sulfoxide mixed solvent are saturating
It changes pure water dialysis after analysis into, 69 % of product HA-Lys-Tet(yield is obtained after freeze-drying)；HA-Lys-Tet nuclear-magnetism characterization is shown in attached
Fig. 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 arm polyethylene glycol terazole derivatives (PEG-Tet4), chitosan terazole derivatives (Chit-Tet) replaceable polymerization
Object is prepared, and structural formula is as follows:
。
The synthesis of two hyaluronic acid cystamine methacrylate derivative (HA-Cy-MA) of embodiment
HA-Cy-MA is synthesized in two steps, first methacrylic acid derivative (the MA-Cy-NH of cystamine2) by Boc-Cy-NH2With
It takes off Boc protection after methacrylic chloride reaction to obtain, then, under the conditions of nitrogen protection, by MA-Cy-NH2 (14.5 mg,
66 μm of ol) solution be added to HA (50 mg, 1.43 μm of ol) by EDC (75.9 mg, 0.396 mmol) and NHS
In the 5 mL secondary waters of (22.8 mg, 0.198 mmol) activation, it is placed in 40 DEG C of oil baths and is protected from light 24 hours, then use
Water dialysis, freeze-drying, 92 % of yield；HA-Cy-MA nuclear-magnetism characterization is shown in attached 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 three Cystine methacrylamide derivatives (MA-Cys-MA) of embodiment
Under the conditions of ice-water bath, the NaOH(1.5 M, 10 mL of cystine (1.2 g, 5.0 mmol)) solution is added drop-wise to
The DCM(10 mL of methacrylic chloride (2.0 mL, 20.6 mmol)) in, 4 h are reacted under the conditions of ice-water bath, are used during reaction
It is 9.0 that NaOH solution, which regulates and controls pH,.After reaction, water layer is separated with separatory funnel, then about 3 mL HCl(2 is added dropwise to it
M), filter, vacuum drying obtains 1.72 g of white powder, yield 91%.MA-Cys-MA nuclear-magnetism characterization is shown in attached 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).
Example IV macromolecules cross-linking prepares hyaluronic acid nanometer gel
Reverse phase nanoprecipitation method is comprehensively utilized with macromolecules cross-linking agent and light-operated " tetrazolium-alkene " cross-linking method prepares hyaluronic acid
Nanogel, prepare as follows: HA-Lys-Tet and HA-Cy-MA prepared by embodiment one and embodiment two is with molar ratio 1:1
7.4,10 mM of PB(pH being dissolved in) in, the polymer solution that concentration is 1.25 mg/mL is prepared, then mixed solution is infused
It is mapped in 100mL acetone, with ultraviolet light (wavelength 320-390 nm, 60 mW/cm of light intensity2) 3 min of radiation, revolving removing acetone
It is dialysed afterwards with PB, freeze-drying obtains nanogel.Referring to attached drawing 4, the partial size of nanogel can be surveyed with dynamic light scattering (DLS)
Amount, the nanogel size tunable (150-343 nm) obtained by such method, PDI very little (0.10-0.17) (Fig. 4 a), together
When nanogel pattern can be observed and obtain with transmission electron microscope (TEM), the nanogel of method preparation is spherical (Fig. 4 a)；Separately
Outside, the nanogel being prepared has stronger green fluorescence (figure in the case where the UV of 405nm excites light radiation at 450 nm
4b)；The cell endocytic and distribution situation in vivo of nanogel, the nanometer are observed using the autofluorescence performance of nanogel
Gel also shows good stability, is placed on 7.4,10 mM of PB(pH) and 10% FBS analogue body in the equal energy of blood environment
Partial size is kept to stablize (Fig. 4 c)；But nanogel is placed in the PB (7.4,10 mM of pH) of 10 mM GSH 4 hours
It can observe significant change of size, increase with time, partial size starts quickly to become larger, and shows that such nanogel has quickly
Reduction response property (Fig. 4 d).This can be used to realize the responsiveness quick release of drug in the cell, greatly increase packet
Carry the therapeutic effect of drug.
Five small molecule of embodiment crosslinking preparation hyaluronic acid nanometer gel
Reverse phase nanoprecipitation method is comprehensively utilized with small molecule crosslinking agent and light-operated " tetrazolium-alkene " cross-linking method prepares hyaluronic acid
Nanogel, by 8.5,10 mM of PB(pH of 1 mL HA-OEG-Tet(1 mg/mL) and MA-Cys-MA) solution (Tet and MA
The molar ratio of group is controlled in 1:1) be injected into 20 mL acetonitriles, then by the solution be placed in ultraviolet camera bellows (320-390 nm,
37.5 mW/cm2) 90 s of illumination.Rotary evaporation removes acetone, with 7.4,10 mM of PB(pH) dialysis, 12 h(MWCO 7000
Da), nanogel is obtained.Dynamic light scattering (DLS) the result shows that the method preparation nanogel 165 nm of average grain diameter, and
Unimodal Distribution is presented.The TEM caption nanogel has spherical structure, and most nano-particles size is about
The partial size that the partial size ratio DLS of nanogel in 100 nm, TEM picture is measured is small, probably due in TEM sample making course, nanometer
Moisture evaporates in gel, nanogel shrinkage, and the partial size taken is made to want small.
Light-operated " tetrazolium-alkene " the click chemistry nanogel of embodiment six contains and composite hydrogel for growth factor
Preparation
For containing vascular endothelial growth factor (VEGF), VEGF, HA-OEG-Tet and HA-Cy-MA are dissolved first
Into 7.4,10 mM of PB(pH) buffer solution, make 1.25 mg/mL of total polymer concentration.Then the solution is injected into third
In ketone, then with the extremely low portable uv analyzer of power (302 nm, 0.88 mW/cm2) illumination 180 seconds.Last rotary evaporation
Acetone is removed, with water 12 h of dialysis, obtains nanogel (VEGF-NGs) solution for containing VEGF.DLS is contained as the result is shown
Unimodal Distribution is presented in the nanogel (VEGF-NGs) of VEGF, and its average grain diameter is 173 nm.Tem observation shows VEGF-NGs
Diameter with spherical structure, and nanogel is about 100 nm.The nanogel for containing VEGF has preferable injection
Property, can direct injection using or prepare hydrogel composites in conjunction with hydrogel.
The preparation of hydrogel composites is first by mercapto-functionalized collagen (Col-SH) and the poly- second of oligo-ester carbonate-
Glycol-oligo-ester carbonate (OAC-PEG-OAC) is dissolved in 7.4,100 mM of PB(pH of 100 μ L respectively) in, until completely dissolved,
Two kinds of solution and VEGF-NGs solution are uniformly mixed at room temperature, then is placed in 37 DEG C of shaking tables and reacts to form composite hydrogel.It should
The forming process that the slave solution state of composite hydrogel becomes hydrogel is shown in Fig. 5 a.Meanwhile storage modulus (the G of composite hydrogel
`) change with time with loss modulus (G``) available rheometer measurement.The result shows that the modulus of the composite hydrogel is reachable
2000 Pa, gel time are about 3 minutes (Fig. 5 b).This contains the composite hydrogel of VEGF growth factor as organizational project branch
Frame has wide practical use in ischemic myocardium reparation.
Light-operated " tetrazolium-alkene " the click chemistry nanogel of embodiment seven is released for containing and controlling in vitro for pharmaceutical grade protein
It puts
For containing cromoci (CC), 10%) a certain amount of CC(theory drugloading rate is added to polymer always dense
Degree is 7.4,10 mM of PB(pH of the HA-OEG-Tet and HA-Cy-MA of 1.25 mg/mL) in solution, then the solution is injected
Into acetone, then with ultraviolet (320-390 nm, 50 mW/cm2) illumination 90 seconds.Last rotary evaporation removes acetone, is dialysed with water
12 h obtain nanogel (CC-NGs) solution for containing CC.It may be implemented with similar method to treatment albumen fruit granzyme B
(GrB) efficient package obtains the nanogel (GrB-NGs) for containing GrB.The release experiment of protein C C is in 37 DEG C at two kinds
Carried out in different dissolution mediums, i.e. PB(pH 7.4,10 mM) and 10 mM GSH 7.4,10 mM of PB(pH) solution.Take 1
ML contains CC-NGs sample in protein delivery bag (350 K of MWCO), is placed in the corresponding PB dissolution medium of 25 mL.Every
A sampling time point takes out 5 mL dissolution mediums, and supplements corresponding fresh medium.The sample freeze-drying that each time point is taken out,
It redissolves, is measured with ultraviolet (CC UV absorption wavelength is 410 nm).Every group of release test carries out three times, finally showing result in parallel
To test averaging of income value ± standard variance.Fig. 6 is the behavior that release pharmaceutical grade protein is controlled outside above-mentioned nano gel, and
The characterization of biological activity figure of the pharmaceutical grade protein released；The extracorporeal releasing experiment of protein shows (pH in physiological conditions
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
The CC more than 80% has been released under the reducing condition of mM GSH, after 10 h from nanogel.This illustrates CC-NGs in cytoplasm
Reducing environment under can quick release go out wrap up pharmaceutical grade protein.
The active test of the electronics transfer of protein is to be obtained by detecting it to the ABTS catalytic efficiency for being changed into ABTS+
's.It is 0.004 mg/mL that the CC released first, which is diluted to concentration with PBS solution,.Simultaneously configure same concentrations, without any
The CC of processing takes two kinds of solution of equal amount to be put into quartz sample pool, is added into two kinds of solution same amount of containing 10 μ L's
The PBS solution of the ABTS of 1 mg/mL of the hydrogenperoxide steam generator of 0.045 M and 100 μ L.Inversion is allowed to mix and use at once
UV spectrophotometer reads the absorption value at 410 nm, and as zero point, surveys within each 15 seconds primary.Each time point is corresponding
Ultraviolet absorption value subtracts the absorption value of first point to the variation (A) for the value that is absorbed, and being plotted against time with A indicates it
Activity change changes with time.Attached drawing 6b is the above-mentioned protein active detection figure released, as the result is shown in nanogel
The CC released still can be catalyzed the oxidation of ABTS quickly, and catalytic rate and the CC's without any processing is close, it was demonstrated that
The protein released from nanogel remains to preferably keep activity.
The cell experiment of embodiment eight empty nanogel and the nanogel for containing pharmaceutical grade protein
By taking the nanogel of the preparation in example IV as an example, the cell compatibility of empty nanogel is tested.It will be at fiber
It is thin that cell (L929), breast cancer cell (MCF-7), brain glioblastoma cell (U87) and lung carcinoma cell (A549) are layered on 96 holes respectively
On born of the same parents' culture plate, about 5000, each hole cell is added containing 10 % calf serums, 1% glutamate, antibiotic mould
The DMEM culture medium of element (100 IU/mL) and streptomysin (100 μ g/mL), then 37 DEG C are placed in, it is cultivated under 5% carbon dioxide conditions
12 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) into every hole, then 48 h are cultivated under 37 DEG C, 5% carbon dioxide conditions.Then, it is added to every hole
3-(4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) PBS solution (15 μ L, 5 mg/mL), and be put into
Continue to cultivate in incubator.After 4 h, remove the culture solution 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 is generated, and UV absorption of each hole at 492 nm is measured with microplate reader (Bio Tek).Cell
By obtaining compared with the control wells of only blanc cell are in the absorption at 492 nm, every group of empirical average carries out relative survival rate
4 times.Fig. 7 is the cell experiment phenogram of the nanogel of above-mentioned nanogel and load pharmaceutical grade protein.
Attached drawing 7a is cell survival rate figure, it can be seen that the survival rate that nanogel cell is added after 48 hours reaches
90 % or more illustrate that hyaluronic acid nanometer gel does not have toxicity.
The cytotoxicity of CC-NGs and GrB-NGs is also to be measured by mtt assay.The nanogel for containing protein drug is added
Enter breast cancer cell (expression of MCF-7, CD44 receptor height), lung carcinoma cell (expression of A549, CD44 receptor height) and glioma are thin
In born of the same parents' (U87, CD44 receptor low expression), and cultivate 4 h.Then the culture medium for carrying the nanogel of albumen is siphoned away, is added
Fresh cultured is based on 37 DEG C, continues to cultivate 92 h under 5% carbon dioxide conditions.10 μ L MTT are added into every hole after terminating for culture
PBS solution (5 mg/mL) and be put into incubator, continue cultivate 4 h act on MTT sufficiently with living cells.It then removes and contains
There is a culture solution of MTT, and 150 μ L DMSO are added and are used to dissolve the purple crystal first a ceremonial jade-ladle, used in libation that living cells and MTT are generated, and with enzyme mark
Instrument (Bio Tek) measures UV absorption of each hole at 492 nm.Comparative survival rate of cells calculation method is as above.Enclosed experiment
It is first then to add the nanogel for containing protein with HA(5 mg/mL) and 4 h of MCF-7 cell incubation.As a result table
Bright CC-NGs has higher anti-tumor activity to MCF-7 cell, and the 503nhibiting concentration (IC50) of cell is 0.52 μM of (figure
7b).Even if on the contrary, free CC when at concentrations up to 6.2 μM still without apparent cytotoxicity, this be primarily due to CC endocytosis into
Enter the poor ability of cell.It lives in addition, CC-NGs goes out the apoptosis being obviously reduced to the U87 cells show of CD44 receptor low expression
Property, and CC-NGs is substantially reduced the anti-tumor activity of the MCF-7 cell of preparatory closing CD44 receptor, these result explanations
CC-NGs is to enter cell by CD44 receptoe mediated endocytosis mechanism.Meanwhile GrB-NGs MCF-7 highly expressed to CD44 receptor
Higher anti tumor activity in vitro is all shown with A549 cell, is respectively 3.0 nM and 8.1 nM(Fig. 7 c).
Embodiment nine carries treatment of the protein nano gel to the subcutaneous transplanted human breast carcinoma of mouse
First using subcutaneous injection MCF-7 cell (1 × 107) PBS solution (50 μ L) to nude mice right lateral side establish
Human breast carcinoma subcutaneous tumor model.When the volume of tumour reaches 30 mm3Afterwards, nude mice is randomized into 4 groups, every group 5.So
Afterwards, GrB-NGs (25 μ g GrB equiv./kg), GrB- are injected to every group of lotus knurl mouse respectively by intravenous methods
NGs (100 μ g GrB equiv./kg), empty nanogel and PBS buffer solution, are administered once every three days, four are administered in total
It is secondary.Gross tumor volume and nude mice weight every other day measure once.The calculation formula of gross tumor volume: volume=* a*b*c, a be
Tumour longest edge, b are tumour most broadsides, and c is the height of tumour.Attached drawing 8 is the treatment of subcutaneous breast cancer, it can be found that PBS group
It is quick with the tumor volume growth of empty nanogel group.And GrB-NGs is 25 μ g GrB equiv./kg and 100 μ g in dosage
The growth of tumour can be effectively inhibited when GrB equiv./kg, and the higher inhibitory effect to tumour growth of dosage is more obvious.
Meanwhile GrB-NGs group is similar to PBS group, all reduces without result in the weight of animals, illustrates that GrB-NGs makees that apparent poison is not secondary
With (referring to Fig. 8).
Embodiment ten carries treatment of the protein nano gel to mouse original position human lung cancer transplantable tumor
Pass through A549 cell (1 × 10 of the injection with luciferase first7) PBS solution (50 μ L) arrive nude mice
Lung establishes human lung cancer situ tumor model.When the fluorescent value of tumour cell reaches 20000 p/s/cm2When/sr, nude mice quilt
It is randomly divided into 3 groups, every group 6.Then, GrB-NGs is injected to every group of lotus knurl mouse respectively by tail vein injection every three days
(150 μ g GrB equiv./kg GrB), blank nanogel and PBS, are administered four times in total.The fluorescence and nude mice that tumour goes out
Weight records once every three days, and Fig. 9 is to carry protein nano gel to the treatment phenogram of mouse original position transplantable lung cancer.Attached drawing
9a-c is lung cancer situ treatment figure, and PBS group is remarkably reinforced at any time with fluorescence intensity at the tumour of empty nanogel group, shows to swell
Tumor is in fast-growth.And the tumour fluorescence intensity in GrB-NGs treatment is weaker, this illustrates that GrB-NGs can effectively inhibit nude mice
The growth of lung cancer.Meanwhile the mouse weight variation of GrB-NGs group is not significant (Fig. 9 d), this aspect shows GrB-NGs without bright
On the other hand thin toxic side effect also illustrates that GrB-NGs can effectively inhibit the growth of mouse original position lung cancer, makes mouse growth shape
Condition is good.On the contrary, the mouse weight of PBS and blank nanogel control group significantly reduces, this is because mouse is with original position
The fast-growth of lung cancer, physical condition sharply decline.The survival assay of mouse also indicates that the mouse of GrB-NGs treatment group entire
During observation (40 days), there is no death；And the mouse of PBS and blank nanogel control group treat observation after,
All death (Fig. 9 e).H&E dyeing is it can be found that GrB-NGs is free from side effects to internal main organs (heart, liver, kidney etc.)
(Figure 10), this has further demonstrated that GrB-NGs will not cause apparent toxic side effect.
Claims (7)
1. a kind of preparation method of autofluorescence nanogel, which comprises the following steps: polymer tetrazolium is derivative
Object and crosslinking agent containing methacrylate group, which are added in water or buffer, obtains mixed liquor；Then mixed liquor is injected into
In organic solvent, suspension is obtained；Then it carries out illumination reaction and obtains autofluorescence nanogel；
The polymer terazole derivatives have Formulas I structure:
Formula I；
Wherein n >=2；
R is H, NH2、NMe2、OMe、NO2、Cl、Br、Me、CO2Me PhNHBoc group；
P is hyaluronic acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucan, chitosan, collagen egg
White, polyethylene glycol or polyethylene glycol ester；
The crosslinking agent containing methacrylate group has Formula II structure:
Formula II；
Wherein m >=2；
CL is hyaluronic acid, hyaluronic acid cystamine compound, hyaluronic acid lysine compound, chitosan, glucan, collagen egg
White, polyethylene glycol, polyethylene glycol ester, butanediamine, hexamethylene diamine, cystamine, cystine or lysine；
Methacrylate group and the molar ratio of tetrazol group are 1: 1；In mixed liquor, polymer terazole derivatives concentration is
0.5~10 mg/mL；
The buffer includes phosphate buffer, 4-(2- ethoxy) half sodium salt buffer solution of -1- piperazine ethanesulfonic acid, three hydroxyls
Aminomethane buffer solution or 2-morpholine ethane sulfonic acid buffer solution；The organic solvent includes acetone, acetonitrile or second
Alcohol；
The illumination reaction is ultraviolet lighting reaction；The wavelength of the ultraviolet light is 302~390 nm, and intensity is 0.8~100
mW/cm2, the reaction time is 90~180s.
2. the preparation method of autofluorescence nanogel according to claim 1, it is characterised in that: the polyethylene glycol is line
Property polyethylene glycol or multi-arm polyethylene glycol；The polyester be polylactide, poly- (lactide-co-glycolide), polycaprolactone or
Person's polycarbonate.
3. the preparation method of autofluorescence nanogel according to claim 1, it is characterised in that: polymer terazole derivatives
Preparation method be that condensing agent and catalyst are first added into tetrazolium solution, react the tetrazolium solution activated；Then poly-
It closes object aqueous solution to be added drop-wise in the tetrazolium solution of activation, room temperature reaction obtains polymer terazole derivatives；The polymer is
Bright matter acid, hyaluronic acid lysine compound, hyaluronic acid cystamine compound, glucan, chitosan, collagen, poly- second two
Alcohol or polyethylene glycol ester.
4. the autofluorescence nanogel of any one preparation method preparation according to claims 1 to 3.
5. a kind of anti-tumor drug, including autofluorescence nanogel and pharmaceutical grade protein described in claim 4.
6. autofluorescence nanogel described in claim 4 is preparing the application in pharmaceutical grade protein or tissue engineering bracket.
7. application of the autofluorescence nanogel as slow releasing carrier of medication described in claim 4.
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| CN103396554A (en) * | 2013-07-02 | 2013-11-20 | 苏州大学 | Hydrogel, preparation method thereof and applications |
| CN103694479A (en) * | 2013-11-21 | 2014-04-02 | 中国科学院长春应用化学研究所 | Polymer, glucose-sensitive nanogel, glucose-sensitive drug-loading nanogel and their preparation methods |
| CN104940138A (en) * | 2015-06-16 | 2015-09-30 | 江南大学 | Preparation method of stimulation-sensitive hyaluronic acid in-situ gel |
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| CN103396554A (en) * | 2013-07-02 | 2013-11-20 | 苏州大学 | Hydrogel, preparation method thereof and applications |
| CN103694479A (en) * | 2013-11-21 | 2014-04-02 | 中国科学院长春应用化学研究所 | Polymer, glucose-sensitive nanogel, glucose-sensitive drug-loading nanogel and their preparation methods |
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