CN110063933A - A kind of glucan base nanogel and its preparation method and application - Google Patents
A kind of glucan base nanogel and its preparation method and application Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
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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
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0021—Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
- C08J3/246—Intercrosslinking of at least two polymers
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/02—Dextran; Derivatives thereof
Abstract
The invention discloses a kind of glucan base nanogels, using glucan as framework material, it carries out vinyl ether acrylate and modifies to obtain glucan derivative I, then based on glucan derivative I, sulfydryl modification and carboxyl modified are carried out respectively, glucan derivative II and glucan derivative III are obtained, the two is mixed and carries out Michael addition reaction to get the glucan base nanogel.Compared with the existing technology, gained glucan base nanogel of the invention contains the acetal groups of acid-sensitive, Targeting delivery is realized using the high-permeability and retention effect (EPR) of solid tumor and the microenvironment of tumor locus meta-acid, it has been combined chemotherapy and the entirely different antitumor strategy of two kinds of mechanism of optical dynamic therapy simultaneously, pass through synergistic effect, antitumous effect is improved, and toxic and side is effectively reduced.
Description
Technical field
The invention belongs to antineoplastic drug carrier technical fields, and in particular to a kind of glucan base nanogel and its preparation
Methods and applications.
Background technique
The indifference injury of traditional small molecule chemotherapeutic drug normal tissue and tumour generate multidrug resistance and limit it
Clinical effectiveness.Doxorubicin hydrochloride is clinically classical broad-spectrum anti-cancer drug, main by interfering the function of DNA to cause
Cell growth abnormity is simultaneously final dead, is often combined with other chemotherapeutics.Its common toxic side effect includes alopecia, marrow suppression
System, vomiting, fash and oral inflammation etc., long-time service will lead to serious cardiac toxic, thus the dosage of clinical use needs sternly
Lattice control, limits its application and antitumous effect.Many defects of classic chemotherapy promote the tumor therapy of other mechanism
Research and development, in order to overcome these drawbacks, one side novel nano drug delivery technologies are introduced in chemotherapeutics delivery system
In, drug is improved to the targeting of tumor tissues, improves its bioavilability, and the adriamycin of existing liposome entrapment is for facing
Bed, it is not only enhancing the antitumaous effect of drug to a certain degree, but also can mitigate toxic side effect, but still it is resistance to solve tumour generation
The problem of pharmacological property.On the other hand be developed in conjunction with the combined therapies of a variety of anticancer means, as chemotherapy and immunotherapy combination,
Combination, chemotherapy and combination of gene therapy of chemotherapy and photodynamic therapy etc. reduce single medicine medicament to learn from other's strong points to offset one's weaknesses
Amount alleviates toxic side effect, inhibits growth of tumour cell breeding by different mechanisms, while inhibiting the generation of drug resistance of tumor.
Nanogel is three-dimensional netted hydrogel particle under nano-scale, and structural stability is good, because containing hole therefore drug
Contain that high-efficient, stability is strong, surface area is big, is easy to carry out surface chemical modification, passes through different response factors such as temperature, pH
Value, photo-thermal etc. realize that biodegrade, release are contained drug.Compared to other nanoparticles, the outstanding advantage of nanogel exists
The hole abundant in its good biocompatibility and cross-linked structure can effectively regulate and control hole by controlling crosslinking degree
Size extends circulation time in vivo to contain therapeutic agent of different molecular weight, including small-molecule drug, protein, nucleic acid etc.,
Ensure that they smoothly reach tumor locus.Modification transformation is carried out by the functional group to nanogel skeleton surface, it can be effective
The drug for containing different physicochemical properties is applied to targeted therapy as the total carrier for carrying a variety of drugs, and being one has
The research direction of application prospect, as the Chinese invention patent application prospectus of Publication No. 105968372B discloses one kind
The preparation and application of the nanogel of autofluorescence, the nanogel have very strong selection using light-operated click chemistry cross-linking method
Property, the effect of drug and cell contained does not react, can keep drug, protein and cell well, realize completely,
Controllable release, so as to the excellent slow-released carrier as protein and drug.
Glucan, also known as dextran are the natural origin homotype polysaccharide being connected as monomer by glycosidic bond using glucose,
Nontoxic and biocompatibility is good, is excellent drug carrier material, has industry abundant and medical usage, is clinically commonly used for
Dextran product and anticoagulant therapy.Due to its good water solubility, nontoxic, biocompatibility is splendid, is easy to carry out structure and changes
It makes, is the excellent carrier of insoluble drug, thus be widely used in the research and development of new drug carrier.Dextran molecule structure
In contain a large amount of hydrophilic hydroxyl groups, can relatively easily be modified by modes such as esterifications, connect some functional ligands, realize
The targeting of drug conveys, or changes the surface charge of material, improves drug-loading efficiency and stability.Glucan base nanogel is one
Class has the nano material of important medical value.The Chinese invention patent application prospectus of Publication No. 106902383A discloses
A kind of gel, glucose nano gel hemostatic material of glyceral methacrylate modification, goes out in the internal organs such as traumatic hemorrhage and liver
Blood has excellent haemostatic effect, is a kind of good new type hemostat, has good application prospect in terms of clinical wound, but should
Nano material in vivo degrade by inanimate object responsiveness.The Chinese invention patent application prospectus of Publication No. 109161036A
A kind of pH/ redox double-response type dextran hydrogel and preparation method thereof is disclosed, glucan is through sodium periodate oxidation
Aldehyde radical glucan is obtained, generation cross-linking reaction under the crosslinking agent alkaline condition simultaneously containing amino and disulfide bond is added and obtains Portugal
Glycan hydrogel can be used as environment-responsive pharmaceutical carrier applied to technical field of biological material.
Summary of the invention
Goal of the invention: in view of the above technical problems, the present invention provides a kind of glucan base nanogel and its preparation sides
Method and application.
Technical solution: in order to achieve the above object of the invention, the technical solution adopted in the present invention is as follows:
A kind of glucan base nanogel carries out vinyl ether acrylate and modifies to obtain Portugal using glucan as framework material
Polysaccharid derivative I carries out sulfydryl modification and carboxyl modified respectively, obtains glucan and spread out then based on glucan derivative I
The two is mixed progress Michael addition reaction and coagulated to get the glucan base nanometer by biological II and glucan derivative III
Glue.
The glucan derivative I includes the acetal groups of acid-sensitive, and structure is as follows:
The chemical structural formula of the glucan derivative II and glucan derivative III is as follows:
As preferred:
The molecular weight of the framework material glucan is in 10000~50000 ranges.
The sulfydryl modification is the carbon carbon on sulfydryl and glucan derivative I molecule using sulfhydrylization reagent molecule one end
Double bond reaction, the carboxyl modified are to be reacted using anhydride molecule with the hydroxyl on glucan derivative I strand；The mikey
That addition reaction is that the carbon-carbon double bond on the sulfydryl and glucan derivative III molecule on glucan derivative II molecule occurs
Michael addition reaction forms gel network.
The sulfhydrylization reagent is selected from 2,2'- (1,2- second diyl dioxygen generation) double ethyl mercaptans, dithioglycol or double mercapto ethyls
Thioether, the acid anhydrides are selected from succinic anhydride, glutaric anhydride or methyl succinic acid anhydrides.
The preparation method of the glucan base nanogel, comprising the following steps:
(1) preparation of glucan derivative I: glucan is dissolved in organic solvent, using p-methyl benzenesulfonic acid as catalyst, benefit
Condensation reaction occurs with the vinyl of hydroxyl and vinyl ether acrylate one end on dextran chain, generates vinyl ethers propylene
The glucan derivative I of acid esters modification；Preferably, the mole that feeds intake of vinyl ether acrylate is hydroxyl on glucan sugar chain
The 20%~30% of group mole.
(2) preparation of glucan derivative II: on the basis of glucan derivative I, using triethylamine as catalyst, mercapto is utilized
The sulfydryl of base reagent molecule one end is reacted with the carbon-carbon double bond of acrylate end on glucan derivative I molecule, generates mercapto
The glucan derivative molecule II of base modification；Preferably, it is double to be selected from 2,2'- (1,2- second diyl dioxygen generation) for the sulfhydrylization reagent
When ethyl mercaptan, the mole that feeds intake need to be in excess in the carbon-carbon double bond amount on glucan derivative I chain, about double bond mole significantly
70~100 times.
(3) it the preparation of glucan derivative III: on the basis of glucan derivative I, using triethylamine as catalyst, utilizes
Anhydride molecule is reacted with the hydroxyl on glucan derivative I strand, generates the glucan derivative III of carboxyl modified；It is preferred that
, when the acid anhydrides is selected from succinic anhydride, the mole that feeds intake is about the 4.8- of hydroxyl group mole on glucan sugar chain
9.7%.
(4) preparation of glucan base nanogel: glucan derivative II and glucan derivative III are dissolved in respectively pure
In water, evenly dispersed in organic solvent, whole system generation Michael addition reaction is added rapidly to after mixing under low temperature, Portugal is poly-
Sugar chain section crosslinks to form nanogel.Preferably, two kinds of glucan derivative solution concentrations of preparation are 5-10mg/
ML makes the concentration 0.1-1mg/mL of nanogel particle in organic solvent.
Preferably as other:
Organic solvent described in step (1) is selected from dimethyl sulfoxide or N,N-dimethylformamide；Step has described in (4)
Solvent is selected from acetone, methanol or ether.
In step (4), the mass ratio of the glucan derivative III and glucan derivative II are 1:(1-3).
Referring to above-mentioned glucan base nanogel and preparation method thereof, it is solidifying that the present invention also provides a kind of glucan base nanometers
Glue drug carries out vinyl ether acrylate and modifies to obtain glucan derivative I using glucan as framework material, then poly- with Portugal
Based on sugar derivatives I, sulfydryl modification and carboxyl modified are carried out respectively, obtains glucan derivative II and glucan derivative
III mixes the two with drug, makes to carry out Michael addition reaction between glucan derivative II and glucan derivative III,
Drug is loaded in gel to get the glucan base nanogel drug.
As preferred:
The anti-tumor drug is selected from adriamycin, methotrexate (MTX) or taxol.
The mass ratio of the glucan derivative III, glucan derivative II and drug is 1:(1-3): (0.05-0.5).
It is described that drug is loaded in gel method particularly includes: it is derivative to dissolve glucan derivative III, glucan respectively
Object II and drug, the lower three kinds of components of low temperature rapidly join evenly dispersed in organic solvent after being mixed in a certain ratio, be added appropriate
Triethylamine is catalyzed whole system and Michael addition reaction occurs, and glucan segment is cross-linked to form nanogel simultaneously for small molecule
It treats drug encapsulation to enter in gel pore, obtains the glucan base nanogel drug for containing chemotherapeutics.
Finally, the present invention provides the glucan base nanogels or the glucan base nanogel drug to make
Application in standby anti-tumor drug.
Preferably, the glucan base nanogel can contain anti-tumor drug and photosensitizer simultaneously, the method is as follows:
Above-mentioned resulting glucan base nanogel drug is transferred to water phase, is rapidly joined under certain proportion dissolved state
Photosensitizer, photosensitizer molecule enters gel pore, obtains the total glucan base nanogel for carrying chemotherapeutics and photosensitizer.
It is further preferred:
The photosensitizer is selected from indocyanine green.
The mass ratio of the glucan base nanogel drug and photosensitizer is 1:(0.05-0.5).
Photodynamic therapy is gradually to be applied to clinical new type anticancer strategy in recent years, and outstanding feature is hurtless measure and easily
It is treated in being repeated several times.Different from the mechanism of classic chemotherapy, it mainly passes through the stronger near infrared light stimulation light of penetration power
Common oxygen molecules are converted singlet oxygen by quick dose, this is a kind of active oxygen for having Execution, accumulates a certain concentration
Killing functions of immunocytes is generated, this therapeutic strategy effectively prevents the toxic side effect of normal tissue, and treatment is repeated several times and may be used also
To inhibit tumor recurrence rate.Indocyanine green be FDA approval clinical common diagnosis fluorescent dye, for determine cardiac output,
Diagnose the angiographies such as liver function and liver, stomach, ophthalmology.It, can rapidly and plasma protein after its intravenous administration enters blood
In conjunction with and be distributed to whole body, biocompatibility is good, nontoxic, but its Half-life in vivo is short, removes heel row rapidly by liver
It lets out bile and finally excretes.The energy for the near infrared light that indocyanine green energy absorbing wavelength is about 800nm, this wavelength
Laser can penetrate tissue, generate photo-thermal effect and kill cell, while the energy absorbed can discharge singlet oxygen etc. with cell
The cell of irradiated site is killed in the active oxygen of toxicity, targeting.Optical dynamic therapy based on photosensitizer indocyanine green can auxiliary
The use of drug is treated, the protection of nanogel can extend its Half-life in vivo, protect its smooth target tumor position.
The glucan base nanogel combination chemotherapy and photodynamic therapy both mechanism for relying on acid response degradation are completely not
Same antitumor strategy, can learn from other's strong points to offset one's weaknesses, and effectively improve antitumous effect, reduce toxic side effect and tumor recurrence rate, simultaneously
Material itself can be realized degradable in vivo and be absorbed, nontoxic.
Therefore, the present invention is directed to many drawbacks of traditional anti-tumor therapy, and providing, a kind of novel glucan base nanometer is solidifying
On the basis of glue, it can further expand a kind of by new approaches associated with chemotherapy and photodynamic therapy.The method comprises the steps of firstly, preparing
A kind of novel glucan base nanogel (NG), itself nontoxic biocompatibility is good, and has micro- in tumour meta-acid
The ability of degradation is responded in environment, auto-degradation, which can be absorbed by the body, while realization contains the Targeting delivery of drug utilizes
Glucose, biological safety is splendid, and the carboxyl in structure improves it to positively charged drug using Electrostatic Absorption effect
Carry drug stabilisation and drug-loading efficiency.Secondly, present invention design is prepared for the total glucan base nanometer for carrying chemotherapeutics and photosensitizer
Gel medicine (NG@DOX-ICG), on the one hand makes the system target using the microenvironment of the EPR effect of nanoparticle and tumour meta-acid
Degradation drug release occurs to tumor tissues, mitigates the toxic side effect of small molecule chemotherapeutic drug normal tissue, while extending photosensitizer
Half-life in vivo, do not remove it in advance under the protection of nanogel, another aspect chemotherapeutics and active oxygen are simultaneously
Killing functions of immunocytes is played, antitumor curative effect is improved.
Technical effect: compared with the existing technology, present invention gained glucan base nanogel contains the acetal radical of acid-sensitive
Group realizes Targeting delivery using the high-permeability and retention effect (EPR) of solid tumor and the microenvironment of tumor locus meta-acid, simultaneously
It has been combined chemotherapy and the entirely different antitumor strategy of two kinds of mechanism of optical dynamic therapy, by synergistic effect, has improved antitumor effect
Fruit, and toxic and side is effectively reduced.
Detailed description of the invention
Fig. 1 is glucan derivative molecule synthesis route schematic diagram.
Fig. 2 is the partial size and Electronic Speculum shape appearance figure of the nanogel particle of preparation gained 1.0mg/mL, in which: a is glucan
Base nanogel and the grain size distribution for containing adriamycin glucan base nanogel；Scheming b is glucan base nanogel saturating
Shape appearance figure under radio mirror.
Fig. 3 is drug release behavior of the medicament-carried nano gel in different pH buffers, in which: a is glucan base nanogel
The tendency chart that partial size changes over time in different pH buffers；Figure b is to contain adriamycin glucan base nanogel in difference
The Accumulation dissolution figure of drug in pH buffer.
Fig. 4 is biocompatibility (MTT) result of nanogel.
Fig. 5 is the cellular uptake result of medicament-carried nano gel.
Fig. 6 is the cell killing result of medicament-carried nano gel.
Specific embodiment
With reference to the accompanying drawing and specific embodiment the invention will be further described.
Embodiment 1
(1) synthesis of the glucan derivative I of vinyl ether acrylate modification
It is spare that dimethyl sulfoxide distills water removal in advance.The glucan that molecular weight is 20000 freezes dry after being dissolved with ultrapure water
It is dry spare.Glucan after weighing 1.25g frozen dried, is added 65mL anhydrous dimethyl sulphoxide and suitably heating makes it dissolve,
10 minutes removing water and oxygen are vacuumized, maintains that 125mg p-methyl benzenesulfonic acid (PSTA) and 1mL ethylene are added under nitrogen protection state
Base ether acrylate.After magnetic agitation reaction 6 hours, 2~3 drop triethylamines are added under anhydrous and oxygen-free normal temperature state in whole system
Quenching reaction.Reaction solution is transferred to dialysed overnight in methanol, revolving removes methanol, residual reaction liquid is added drop-wise to enough ice second
Precipitation in ether repeatedly washs the white solid particle of precipitation with ice ether, finally flings to ether, adds purified water dissolution solid
Body, freeze-drying obtain the glucan derivative I of vinyl ether acrylate modification.
(2) mercapto-modified glucan derivative II is synthesized based on glucan derivative I
Dimethyl sulfoxide distills in advance and sufficiently logical nitrogen deoxygenation is spare.300mg glucan derivative I is weighed to be dissolved in
In 6mL dried dimethyl sulfoxide, measures 2,2'- (1,2- second diyl dioxygen generation) double ethyl mercaptan 4.78mL and be dissolved in 5mL processing
In the dimethyl sulfoxide crossed, glucan derivative I solution is slowly added dropwise thereto under nitrogen protection, it is ensured that sulfydryl is significantly excessive
And it is catalyst that appropriate triethylamine, which is added dropwise, reacts overnight under room temperature, reaction solution is added drop-wise to the mixed solvent (5:1) of ether methanol
Middle Precipitation is centrifuged off upper layer solvent and repeatedly washs lower layer's solid with ether, finally flings to ether, adds purifying water-soluble
Solution, freeze-drying obtain glucan derivative II.
(3) the glucan derivative III based on glucan derivative I synthesis carboxyl modified
Dimethyl sulfoxide distills in advance and sufficiently logical nitrogen deoxygenation is spare.Weigh the dissolution of 200mg glucan derivative I solid
In the dried dimethyl sulfoxide of 6mL, whole system is made to be in addition 9mg succinic anhydride solid under nitrogen protection, is added dropwise suitable
Triethylamine catalysis reaction is measured, normal-temperature reaction is stayed overnight under anhydrous and oxygen-free state, then reaction solution is transferred to dialysed overnight in methanol, is revolved
Methanol is evaporated off, remaining liq is added drop-wise to Precipitation in ice ether, be centrifuged off upper layer ether and repeatedly washs precipitation
White solid finally flings to ether, and purified water is added to dissolve, and freeze-drying obtains the glucan derivative III of carboxyl modified.
(4) preparation of the glucan base nanogel based on Michael addition reaction
The glucan derivative III solution of the carboxyl modified of 10mg/mL and the sulfydryl modification of 10mg/mL are prepared with purified water
Glucan derivative II solution.After above two solution respectively takes 100 μ L vortex mixeds, rapid dispersion into 10mL acetone, and
5 μ L triethylamines are added dropwise as catalyst, stand after reaction overnight, 2mL purified water is added and simultaneously rotates removing acetone, concentration quantifies
To the nanogel aqueous solution of 1mg/mL.The partial size and Electronic Speculum shape appearance figure of nanogel particle are as shown in Figure 2.
(5) preparation of medicine glucan base nanogel is carried
It is kept in dark place with the Doxorubicin solution of purified water preparation 10mg/mL and indocyanine green solution spare.Medicament-carried nano is solidifying
The preparation method of glue is mixed into special ratios (mass fraction: 5%, with above-mentioned unloaded gel while mixing two kinds of cross-linked materials
10%, 20%) Doxorubicin solution further plays rapid dispersion into acetone, and 5 μ L triethylamines are added dropwise as catalyst, stands anti-
After should staying overnight, 2mL purified water is added and rotates removing acetone, is protected from light in purified water dialysis 3 hours and removes free adriamycin
Molecule, concentration quantitatively obtain the nanogel (NG@DOX) for containing adriamycin of 1.0mg/mL, and grain size distribution is shown in Fig. 2 a.System
The standby nanogel for containing adriamycin and indocyanine green simultaneously, then contain adriamycin nano gel solution mesoscale eddies be mixed into it is quantitative
(mass fraction: indocyanine green solution 20%) obtains the nanogel of total the load adriamycin and indocyanine green of 1.0mg/mL
(NG@DOX-ICG)。
The acid degradation behavior of 2 nanogel of embodiment is verified
It takes with crowd 1.0mg/mL glucan base nanogel 2mL of preparation, is divided into two, each 1mL is placed in molecular cut off
To be put in the 20mL buffer medium of different pH respectively in 3500 bag filter, under 37 ± 0.5 DEG C of oscillations, in 0h, 1h, 3h,
7h distinguishes the partial size of the sample detection sample when 48h, 72h for 24 hours, as a result as shown in Fig. 3 (a), finds this glucan base nanometer
The partial size of gel remains unchanged in the buffer of pH 7.4,72 hours still stable homogeneous, and passes through in the buffer of pH 5.0
It is chaotic and degradation wadding collection occurs to cross 7 hours systems, this result proves that the acetal groups of acid response hydrolyze in weak acid, makes
Gel network degradation.
3 doxorubicin in vitro acid of embodiment responds release experiment
By taking the glucan base nanogel (inventory 0.05:1) for containing adriamycin as an example, it is solidifying to study the glucan base nanometer
The external acid response release behavior of glue.Take the same crowd of glucan base nanogel 2mL for containing adriamycin for preparing 1mg/mL (ultraviolet
Calibration curve method measure its contain efficiency be 35.56%), be divided into two, each 1mL solution be placed in molecular cut off be 3500 it is saturating
It analyses in bag, is put in the dissolution container for filling 20mL dissolution medium, is protected from light respectively, Ah mould is carried out under 37 ± 0.5 DEG C of medium temperatures
Plain extracorporeal releasing experiment.By setting time interval 0h, 0.5h, 1h, 2h, 3h, 4h, 6h, 17h, take out outside 2mL bag filter for 24 hours
Release liquid, and the blank dissolution medium of same volume is added, doxorubicin content in fluoremetry release liquid.Fluorescence exciting wavelength is
480nm, launch wavelength 557nm, as a result as shown in Fig. 3 (b).The experimental results showed that 24 is small in the dissolution medium of pH 5.0
When after have 75.29% the drug that contains be released, there was only 30.0% drug quilt and in the system of pH 7.4, after 24 hours
The acid response release of release, the nanogel is confirmed.
4 nanogel cell toxicity test (MTT) of embodiment
User's cervical cancer cell HeLa is tested, to contain the DMEM culture medium of 10% fetal calf serum and 3% antibiotic, 37
DEG C, cultivate in the incubator of 5% carbon dioxide, it is good, raw in index that cell every other day passes on primary selection growth conditions
Long-term cell is tested.10 are inoculated in 96 porocyte culture plates4A/milliliter (48 hours) or 5 × 104A/milliliter (24
Hour) cell of concentration, the overnight incubation in 37 DEG C, the incubator of 5% carbon dioxide.The nanometer for preparing four kinds of various concentrations is solidifying
Glue solution adds 10 μ L to every hole, and after cell incubation culture 48 hours, every hole adds the MTT aqueous solution of the 5mg/mL of 10 μ L,
Continue after cultivating four hours, medium removed with care, every hole adds the DMSO solution of 100 μ L, completely molten to bluish violet crystallization
Xie Hou is placed on multi-function microplate reader and detects absorbance, Detection wavelength 490nm, and result is as shown in figure 4, in 400 μ g/mL
Highest nanogel concentration under, still have 80% or more survival rate after cell culture 48 hours, it was demonstrated that this nanogel is almost
No cytotoxicity, biocompatibility are good.
The cellular uptake of 5 medicament-carried nano gel of embodiment is tested
User's cervical cancer cell HeLa is tested, to contain the DMEM culture medium of 10% fetal calf serum and 3% antibiotic, 37
DEG C, cultivate in the incubator of 5% carbon dioxide, it is good, raw in index that cell every other day passes on primary selection growth conditions
Long-term cell is tested.It is 5 × 10 that 500 μ L concentration are inoculated in 24 porocyte culture plates4The cell of a/milliliter, 37
DEG C, overnight incubation in the incubator of 5% carbon dioxide.Prepare 1mg/mL contains adriamycin glucan base nanogel, measurement
Its doxorubicin concentration finally contained is 36.7 μ g/mL, and preparation is stand-by with the adriamycin aqueous solution of concentration.In different time points to
The gel of 50 μ L is added in every hole or free drug solution is incubated for, and the experiment of cell stream formula is carried out after longest 12 hours, and discovery is thin
Born of the same parents' fluorescence intensity has the tendency that being remarkably reinforced at any time, as shown in Figure 5, it was demonstrated that carries medicinal gel or free drug by cellular uptake.
Embodiment 6 is combined the Cell killing efficacy of near-infrared (NIR) irradiation verifying medicament-carried nano gel
User's cervical cancer cell HeLa is tested, to contain the DMEM culture medium of 10% fetal calf serum and 3% antibiotic, 37
DEG C, cultivate in the incubator of 5% carbon dioxide, it is good, raw in index that cell every other day passes on primary selection growth conditions
Long-term cell is tested.It is 5 × 10 that 100 μ L concentration are inoculated in 96 porocyte culture plates4The cell of a/milliliter, 37
DEG C, overnight incubation in the incubator of 5% carbon dioxide.Preparation contain adriamycin glucan base nanogel and altogether carry adriamycin and
Indocyanine green glucan base nanogel, it is respectively 2 μ g/mL, 4 μ g/mL, 8 μ g/mL that its quantitative final doxorubicin concentration, which is concentrated,
Preparation is stand-by with the free doxorubicin hydrochloride aqueous solution of concentration, the final concentration of 200 μ g/mL of indocyanine green.It is separately added into every hole
10 μ L samples, every hole adds the MTT aqueous solution of the 5mg/mL of 10 μ L after culture 24 hours, continues culture 4 hours, careful to remove training
Base is supported, every hole adds the DMSO solution of 100 μ L, after bluish violet crystallization is completely dissolved, is placed in detect on multi-function microplate reader and inhale
Luminosity, Detection wavelength 490nm, as shown in fig. 6, under the concentration of 8 μ g/mL, 48 hours survival rates of cell are less than result
20%, it is shown that effective killing of the medicament-carried nano gel to tumour cell.
Claims (10)
1. a kind of glucan base nanogel, which is characterized in that using glucan as framework material, carry out vinyl ether acrylate
Glucan derivative I is modified to obtain, then based on glucan derivative I, sulfydryl modification and carboxyl modified is carried out respectively, obtains
The two is mixed and carries out Michael addition reaction to get the glucan base by glucan derivative II and glucan derivative III
Nanogel.
2. glucan base nanogel according to claim 1, which is characterized in that the molecule of the framework material glucan
Amount is in 10000~50000 ranges.
3. glucan base nanogel according to claim 1, which is characterized in that the sulfydryl modification is using sulfhydrylation
The sulfydryl of reagent molecule one end is reacted with the carbon-carbon double bond on glucan derivative I molecule, and the carboxyl modified is using acid anhydrides
Molecule is reacted with the hydroxyl on glucan derivative I strand；The Michael addition reaction is glucan derivative II molecule
On sulfydryl and glucan derivative III molecule on carbon-carbon double bond occur Michael addition reaction formed gel network.
4. glucan base nanogel according to claim 3, which is characterized in that the sulfhydrylization reagent is selected from 2,2'-
(1,2- second diyl dioxygen generation) double ethyl mercaptans, dithioglycol or double mercapto ethyl thioethers, the acid anhydrides are selected from succinic anhydride, glutaric acid
Acid anhydride or methyl succinic acid anhydrides.
5. the preparation method of the described in any item glucan base nanogels of claim 1-4, which is characterized in that including following step
It is rapid:
(1) preparation of glucan derivative I: glucan is dissolved in organic solvent, using p-methyl benzenesulfonic acid as catalyst, utilizes Portugal
Condensation reaction occurs for the vinyl of hydroxyl and vinyl ether acrylate one end in polysaccharide chains, generates vinyl ether acrylate
The glucan derivative I of modification；
(2) preparation of glucan derivative II: on the basis of glucan derivative I, using triethylamine as catalyst, sulfhydrylation is utilized
The sulfydryl of reagent molecule one end is reacted with the carbon-carbon double bond of acrylate end on glucan derivative I molecule, is generated sulfydryl and is repaired
The glucan derivative molecule II of decorations；
(3) preparation of glucan derivative III: on the basis of glucan derivative I, using triethylamine as catalyst, acid anhydrides is utilized
Molecule is reacted with the hydroxyl on glucan derivative I strand, generates the glucan derivative III of carboxyl modified；
(4) glucan derivative II and glucan derivative III the preparation of glucan base nanogel: are dissolved in pure water respectively
In, evenly dispersed in organic solvent, whole system generation Michael addition reaction, glucan are added rapidly to after mixing under low temperature
Segment crosslinks to form nanogel.
6. the preparation method of glucan base nanogel according to claim 5, which is characterized in that described in step (1)
Organic solvent is selected from dimethyl sulfoxide or N,N-dimethylformamide；Organic solvent described in step (4) be selected from acetone, methanol or
Ethyl alcohol.
7. the preparation method of glucan base nanogel according to claim 5, which is characterized in that described in step (4)
The mass ratio of glucan derivative III and glucan derivative II are 1:(1-3).
8. a kind of glucan base nanogel drug, which is characterized in that using glucan as framework material, carry out vinyl ethers propylene
Acid esters modifies to obtain glucan derivative I, then based on glucan derivative I, carries out sulfydryl modification and carboxyl modified respectively,
Glucan derivative II and glucan derivative III are obtained, the two is mixed with drug, makes glucan derivative II and glucan
Michael addition reaction is carried out between derivative III, and drug is loaded in gel to get the glucan base nanogel medicine
Object.
9. glucan base nanogel drug according to claim 8, which is characterized in that the anti-tumor drug be selected from Ah
Mycin, methotrexate (MTX) or taxol.
10. the described in any item glucan base nanogels of claim 1-4 or glucan Ji Na according to any one of claims 8
Rice gel medicine application in preparation of anti-tumor drugs.
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