CN110063933B - Glucan-based nanogel and preparation method and application thereof - Google Patents
Glucan-based nanogel and preparation method and application thereof Download PDFInfo
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- 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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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- 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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- 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
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- C08J3/075—Macromolecular gels
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- 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
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- 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 glucan-based nanogel which is prepared by taking glucan as a framework material, modifying vinyl ether acrylate to obtain a glucan derivative I, then respectively modifying sulfydryl and carboxyl on the basis of the glucan derivative I to obtain a glucan derivative II and a glucan derivative III, and mixing the glucan derivative II and the glucan derivative III for Michael addition reaction. Compared with the prior art, the dextran-based nanogel contains an acid-sensitive acetal group, realizes targeted release by utilizing the high permeability and retention Effect (EPR) of solid tumors and the microenvironment of tumor site partial acid, combines two completely different anti-tumor strategies of chemotherapy and photodynamic therapy, improves the anti-tumor effect through synergistic effect, and effectively reduces the toxic and side effects of chemotherapeutic drugs.
Description
Technical Field
The invention belongs to the technical field of antitumor drug carriers, and particularly relates to glucan-based nanogel and a preparation method and application thereof.
Background
The clinical effects of traditional small molecule chemotherapeutic drugs are limited by their indifferent damage to normal tissues and their multidrug resistance by tumors. Doxorubicin hydrochloride is a clinically classical broad-spectrum anti-tumor drug, which causes abnormal cell growth and eventual death mainly by interfering with DNA function, often in combination with other chemotherapeutic drugs. The common toxic and side effects comprise alopecia, bone marrow suppression, vomit, rash, stomatitis and the like, and the long-term use of the traditional Chinese medicine can cause serious cardiotoxicity, so that the clinical dose needs to be strictly controlled, and the application and the anti-tumor effect of the traditional Chinese medicine are limited. In order to overcome the defects, on one hand, a novel nano drug loading technology is introduced into a chemotherapy drug delivery system to improve the targeting of the drug to tumor tissues and improve the bioavailability of the drug, the doxorubicin coated by the liposome is used clinically, the anticancer effect of the drug is enhanced to a certain degree, the toxic and side effects can be reduced, and the problem of drug resistance of tumors cannot be solved. On the other hand, combination therapy combining multiple anticancer means is developed, such as combination of chemotherapy and immunotherapy, combination of chemotherapy and photodynamic therapy, combination of chemotherapy and gene therapy and the like, so as to make up for deficiencies of each other, reduce dosage of single drug, relieve toxic and side effects, inhibit growth and reproduction of tumor cells through different mechanisms, and inhibit generation of tumor drug resistance.
The nanogel is a three-dimensional reticular hydrogel particle with a nano size, has good structural stability, high drug entrapment efficiency and strong stability due to the porosity, has large surface area, is easy to carry out surface chemical modification, and can realize biodegradation through different response factors such as temperature, pH value, photo-thermal and the like to release the entrapped drug. Compared with other nano particles, the nano gel has the outstanding advantages of good biocompatibility and rich pores in a cross-linked structure, and the pore size can be effectively regulated and controlled by controlling the cross-linking degree so as to entrap therapeutic agents with different molecular weights, including small molecular drugs, proteins, nucleic acids and the like, prolong the in vivo circulation time and ensure that the therapeutic agents can smoothly reach tumor parts. The functional groups on the surface of the nanometer gel skeleton are modified, so that drugs with different physical and chemical properties can be effectively entrapped, the nanometer gel can be used as a carrier for co-entrapping multiple drugs to be applied to targeted therapy, and the research direction has application prospect, for example, the Chinese patent application publication specification with the publication number of 105968372B discloses the preparation and the application of the autofluorescent nanometer gel, the nanometer gel has strong selectivity by adopting a light-operated click chemical crosslinking method, the entrapped drugs and cells do not react, the effects of the drugs, proteins and cells can be well maintained, the complete and controllable release can be realized, and the nanometer gel can be used as an excellent slow release carrier of the proteins and the drugs.
Dextran, also known as dextran, is a natural source of homopolysaccharide in which glucose is used as a monomer and connected by glycosidic bonds, is non-toxic and has good biocompatibility, is an excellent drug carrier material, has abundant industrial and medical applications, and is commonly used as plasma substitute products and anticoagulant therapy in clinic. The carrier has good water solubility, no toxicity, no harm, excellent biocompatibility and easy structural modification, and is an excellent carrier of insoluble drugs, so the carrier is widely applied to the research and development of novel drug carriers. The dextran molecular structure contains a large amount of hydrophilic hydroxyl groups, can be easily modified through esterification and other modes, is connected with functional ligands, realizes targeted delivery of drugs, or changes the surface charge of materials, and improves the drug loading efficiency and stability. The dextran-based nanogel is a nano material with important medical value. The Chinese patent application publication specification with the publication number of 106902383A discloses a glycerol methacrylate modified glucose nanogel hemostatic material, which has excellent hemostatic effects on traumatic hemorrhage and internal organ hemorrhage such as liver, is a good novel hemostatic agent, has good application prospect in clinical trauma, and has no in-vivo bioresponse degradation. The Chinese patent application publication specification with the publication number of 109161036A discloses a pH/redox dual-response type glucan hydrogel and a preparation method thereof, wherein glucan is oxidized by sodium periodate to obtain aldehyde-based glucan, and a cross-linking agent simultaneously containing amino and a disulfide bond is added to the aldehyde-based glucan to perform a cross-linking reaction under an alkaline condition to obtain the glucan hydrogel, and the glucan hydrogel can be used as an environment-responsive drug carrier and applied to the field of biological materials.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides glucan-based nanogel and a preparation method and application thereof.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the glucan-based nanogel is prepared by taking glucan as a framework material, modifying vinyl ether acrylate to obtain a glucan derivative I, then respectively modifying sulfydryl and carboxyl on the basis of the glucan derivative I to obtain a glucan derivative II and a glucan derivative III, and mixing the glucan derivative II and the glucan derivative III for Michael addition reaction.
The dextran derivative I comprises an acid-sensitive acetal group, and the structure of the dextran derivative I is shown as follows:
the chemical structural formulas of the glucan derivative II and the glucan derivative III are shown as follows:
preferably, the method comprises the following steps:
the molecular weight of the framework material glucan is in the range of 10000-50000.
The sulfydryl modification is to adopt sulfydryl at one end of a sulfydryl reagent molecule to react with a carbon-carbon double bond on a glucan derivative I molecule, and the carboxyl modification is to adopt an anhydride molecule to react with hydroxyl on a glucan derivative I molecular chain; the Michael addition reaction is that the mercapto group on the molecule of the glucan derivative II and the carbon-carbon double bond on the molecule of the glucan derivative III generate the Michael addition reaction to form a gel network.
The sulfhydrylation reagent is selected from 2,2' - (1, 2-ethanediylbis oxo) bisethanethiol, ethanedithiol or bismercaptoethyl sulfide, and the anhydride is selected from succinic anhydride, glutaric anhydride or methylsuccinic anhydride.
The preparation method of the glucan-based nanogel comprises the following steps:
(1) preparation of dextran derivative I: dissolving glucan in an organic solvent, and carrying out condensation reaction on hydroxyl on glucan chains and vinyl at one end of vinyl ether acrylate by using p-toluenesulfonic acid as a catalyst to generate a vinyl ether acrylate modified glucan derivative I; preferably, the vinyl ether acrylate is fed in a molar amount of 20 to 30% based on the molar amount of the hydroxyl groups on the glucan sugar chain.
(2) Preparation of dextran derivative II: on the basis of the glucan derivative I, triethylamine is used as a catalyst, and a sulfydryl group at one end of a sulfhydrylation reagent molecule reacts with a carbon-carbon double bond at the tail end of an acrylate on the glucan derivative I molecule to generate a sulfydryl group modified glucan derivative molecule II; preferably, when the thiolating agent is selected from 2,2' - (1, 2-ethanediylbis-oxo) bisethanethiol, the molar amount of the thiolating agent to be added is in large excess of the amount of the carbon-carbon double bonds in the dextran derivative I chain, and is about 70 to 100 times the molar amount of the double bonds.
(3) Preparation of dextran derivative III: on the basis of the glucan derivative I, triethylamine is used as a catalyst, and anhydride molecules are reacted with hydroxyl on a molecular chain of the glucan derivative I to generate a carboxyl modified glucan derivative III; preferably, the acid anhydride is chosen from succinic anhydride and is dosed in a molar amount of about 4.8 to 9.7% of the molar amount of hydroxyl groups on the dextran sugar chains.
(4) Preparation of dextran-based nanogels: respectively dissolving the glucan derivative II and the glucan derivative III in pure water, quickly adding the mixture into an organic solvent for uniform dispersion after mixing at low temperature, carrying out Michael addition reaction on the whole system, and crosslinking glucan chain segments to form nanogel. Preferably, the two dextran derivative solutions are prepared in concentrations of 5-10mg/mL, such that the concentration of the nanogel particles in the organic solvent is 0.1-1 mg/mL.
As other preferences:
the organic solvent in the step (1) is selected from dimethyl sulfoxide or N, N-dimethylformamide; the organic solvent in the step (4) is selected from acetone, methanol or diethyl ether.
In the step (4), the mass ratio of the glucan derivative III to the glucan derivative II is 1 (1-3).
Referring to the glucan-based nanogel and the preparation method thereof, the invention also provides a glucan-based nanogel drug, which is prepared by taking glucan as a framework material, modifying with vinyl ether acrylate to obtain a glucan derivative I, then respectively modifying with sulfydryl and carboxyl on the basis of the glucan derivative I to obtain a glucan derivative II and a glucan derivative III, mixing the glucan derivative II and the glucan derivative III with a drug, carrying out Michael addition reaction between the glucan derivative II and the glucan derivative III, and loading the drug in gel.
Preferably, the method comprises the following steps:
the anti-tumor drug is selected from adriamycin, methotrexate or paclitaxel.
The mass ratio of the glucan derivative III to the glucan derivative II to the medicine is 1 (1-3) to 0.05-0.5.
The specific method for loading the medicine in the gel is as follows: dissolving the dextran derivative III, the dextran derivative II and the drug respectively, mixing the three components according to a certain proportion at low temperature, quickly adding the three components into an organic solvent for uniform dispersion, adding a proper amount of triethylamine to catalyze the whole system to generate Michael addition reaction, crosslinking the dextran chain segments to form nano gel, and loading the micromolecular chemotherapeutic drug into gel pores to obtain the chemotherapeutic drug-loaded dextran-based nano gel drug.
Finally, the invention provides the application of the glucan-based nanogel or the glucan-based nanogel medicament in preparing an anti-tumor medicament.
Preferably, the dextran-based nanogel can simultaneously encapsulate the anti-tumor drug and the photosensitizer by the following method:
and transferring the obtained glucan-based nanogel drug into a water phase, quickly adding a photosensitizer in a dissolved state according to a certain proportion, and allowing photosensitizer molecules to enter gel pores to obtain the glucan-based nanogel loaded with the chemotherapeutic drug and the photosensitizer together.
Further preferably:
the photosensitizer is selected from indocyanine green.
The mass ratio of the glucan-based nanogel drug to the photosensitizer is 1 (0.05-0.5).
Photodynamic therapy is a novel anticancer strategy which is gradually applied to clinic in recent years, and has the outstanding characteristics of no wound and easy repeated treatment. Different from the mechanism of traditional chemotherapy, the near infrared light with strong penetrating power irradiates and stimulates the photosensitizer to convert common oxygen molecules into singlet oxygen, which is active oxygen with cell killing power, and the cell killing effect can be generated by accumulating a certain concentration. Indocyanine green is an FDA-approved fluorescent dye for common clinical diagnosis, and is used for determining cardiac output, diagnosing liver function, and angiography of liver, stomach, ophthalmology, and the like. After entering blood through intravenous injection, the medicine can be quickly combined with plasma protein to be distributed to the whole body, has good biocompatibility, is nontoxic and harmless, but has short half-life in vivo, and is quickly eliminated by liver, then excreted to bile and finally discharged out of the body. The indocyanine green can absorb energy of near-infrared light with the wavelength of about 800nm, laser with the wavelength can penetrate through tissues to generate a photothermal effect to kill cells, and meanwhile, the absorbed energy can release active oxygen with cytotoxicity such as singlet oxygen and the like to kill cells at the irradiated parts in a targeted mode. Photodynamic therapy based on the photosensitizer indocyanine green can assist the use of chemotherapeutic drugs, and the protection of the nanogel can prolong the half-life period in vivo and protect the nanogel to smoothly target tumor parts.
The dextran-based nanogel which is degraded by etodolac in response to response can be combined with two completely different anti-tumor strategies of chemotherapy and photodynamic therapy, so that the advantages of the two strategies can be gained, the defects of the two strategies can be overcome, the anti-tumor effect can be effectively improved, the toxic and side effects and the tumor recurrence rate can be reduced, and meanwhile, the materials can be completely degraded and absorbed in vivo and are non-toxic and harmless.
Therefore, the invention aims at the defects of the traditional anti-tumor therapy, and can further expand a new idea of combining chemotherapy and photodynamic therapy on the basis of providing a novel glucan-based nanogel. The invention firstly prepares a novel glucan-based Nanogel (NG), which is nontoxic and harmless, has good biocompatibility, has the capability of responding and degrading in a tumor metaacid microenvironment, realizes targeted release of the entrapped medicament and degrades the entrapped medicament into glucose which can be absorbed and utilized by human bodies, has excellent biological safety, and improves the medicament carrying stability and medicament carrying efficiency of positively charged medicaments by utilizing electrostatic adsorption effect of carboxyl in the structure. Secondly, the invention designs and prepares a glucan-based nanogel drug (NG @ DOX-ICG) carrying chemotherapeutic drugs and photosensitizers together, on one hand, the EPR effect of nanoparticles and the microenvironment of tumor metaacid are utilized to lead the system to target tumor tissues to degrade and release drugs, thereby reducing the toxic and side effects of small-molecule chemotherapeutic drugs on normal tissues, simultaneously prolonging the half-life period of photosensitizers in vivo and leading the photosensitizers not to be eliminated in advance under the protection of nanogel, on the other hand, the chemotherapeutic drugs and active oxygen simultaneously play a role in killing cells and improving the antitumor curative effect.
The technical effects are as follows: compared with the prior art, the dextran-based nanogel contains an acid-sensitive acetal group, realizes targeted release by utilizing the high permeability and retention Effect (EPR) of solid tumors and the microenvironment of tumor site partial acid, combines two completely different anti-tumor strategies of chemotherapy and photodynamic therapy, improves the anti-tumor effect through synergistic effect, and effectively reduces the toxic and side effects of chemotherapeutic drugs.
Drawings
FIG. 1 is a schematic diagram of a molecular synthesis route of dextran derivatives.
FIG. 2 is a graph of particle size and morphology of the prepared 1.0mg/mL nanogel particle under an electron microscope, wherein: a is a particle size distribution diagram of the dextran-based nanogel and the entrapped adriamycin dextran-based nanogel; and the figure b is a morphology image of the glucan-based nanogel under a transmission electron microscope.
Fig. 3 shows the drug release behavior of drug loaded nanogels in different pH buffers, where: a is a trend graph of the change of the particle size of the glucan-based nanogel in buffers with different pH values along with time; and the graph b is a graph of the cumulative release rate of the drug in the doxorubicin-loaded glucan-based nanogel in buffers with different pH values.
Fig. 4 is the biocompatibility (MTT) results for the nanogels.
Figure 5 is the results of cellular uptake of drug-loaded nanogels.
Fig. 6 shows the results of cell killing by drug loaded nanogels.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
Example 1
(1) Synthesis of vinyl ether acrylate modified dextran derivative I
Dimethyl sulfoxide is distilled in advance to remove water for standby. Dissolving dextran with molecular weight of 20000 with ultrapure water, and freeze drying. 1.25g of lyophilized dextran was weighed, added with 65mL of anhydrous dimethyl sulfoxide and dissolved by heating appropriately, evacuated for 10 minutes to remove water and oxygen, and added with 125mg of p-toluenesulfonic acid (PSTA) and 1mL of vinyl ether acrylate while maintaining the nitrogen protection. The whole system is magnetically stirred and reacts for 6 hours under the anhydrous and anaerobic normal temperature state, and then 2-3 drops of triethylamine are added for quenching reaction. And transferring the reaction solution into methanol for dialysis overnight, removing the methanol by rotary evaporation, dropwise adding the rest reaction solution into enough glacial ethyl ether for precipitation, washing the precipitated white solid particles for multiple times by using the glacial ethyl ether, finally volatilizing the ethyl ether, adding purified water to dissolve the solid, and freeze-drying to obtain the vinyl ether acrylate modified glucan derivative I.
(2) Synthesis of thiol-modified Glucan derivatives II based on Glucan derivatives I
The dimethyl sulfoxide is distilled in advance and nitrogen is fully introduced to remove oxygen for standby. Weighing 300mg of glucan derivative I, dissolving the glucan derivative I in 6mL of dried dimethyl sulfoxide, weighing 4.78mL of 2,2' - (1, 2-ethanediylbis oxo) bisethanethiol, dissolving in 5mL of treated dimethyl sulfoxide, slowly dropwise adding the glucan derivative I solution under the protection of nitrogen, ensuring that sulfydryl is greatly excessive, dropwise adding a proper amount of triethylamine to be used as a catalyst, reacting overnight at normal temperature, dropwise adding a reaction solution into a mixed solvent (5:1) of ether and methanol to precipitate, centrifuging to remove an upper-layer solvent, washing a lower-layer solid by using ether for multiple times, finally volatilizing the ether, adding purified water to dissolve, and freeze-drying to obtain the glucan derivative II.
(3) Synthesis of carboxyl-modified Glucan derivative III based on Glucan derivative I
The dimethyl sulfoxide is distilled in advance and nitrogen is fully introduced to remove oxygen for standby. Weighing 200mg of glucan derivative I solid, dissolving the glucan derivative I solid in 6mL of dried dimethyl sulfoxide, adding 9mg of succinic anhydride solid into the whole system under the protection of nitrogen, dropwise adding a proper amount of triethylamine for catalytic reaction, reacting at normal temperature overnight under an anhydrous and anaerobic state, transferring the reaction solution into methanol for dialysis overnight, removing the methanol by rotary evaporation, dropwise adding the residual liquid into glacial ethyl ether for precipitation, centrifugally removing the upper layer of ethyl ether, washing the precipitated white solid for multiple times, finally volatilizing the ethyl ether, dissolving with purified water, and freeze-drying to obtain the carboxyl-modified glucan derivative III.
(4) Preparation of dextran-based nanogels based on michael addition reaction
A10 mg/mL solution of the carboxyl-modified dextran derivative III and a 10mg/mL solution of the thiol-modified dextran derivative II were prepared with purified water. And (3) mixing 100 mu L of each solution in a vortex mode, quickly dispersing the mixture into 10mL of acetone, dropwise adding 5 mu L of triethylamine serving as a catalyst, standing for reacting overnight, adding 2mL of purified water, removing the acetone by rotary evaporation, and concentrating and quantifying to obtain 1mg/mL of nanogel aqueous solution. The particle size and the electron microscope morphology of the nanogel particles are shown in figure 2.
(5) Preparation of drug-loaded dextran-based nanogel
Preparing 10mg/mL adriamycin solution and indocyanine green solution by using purified water, and storing the adriamycin solution and the indocyanine green solution in dark for later use. The preparation method of the drug-loaded nanogel is the same as that of the no-load nanogel, two cross-linking materials are mixed, adriamycin solution with a specific proportion (mass fraction: 5%, 10% and 20%) is mixed, then the mixture is rapidly dispersed into acetone, 5 mu L of triethylamine is dropwise added to be used as a catalyst, after standing and reacting overnight, 2mL of purified water is added, the acetone is removed by rotary evaporation, dialysis is carried out in the purified water for 3 hours in a dark place to remove free adriamycin molecules, and the concentration and quantification are carried out to obtain the 1.0mg/mL of adriamycin-encapsulated nanogel (NG @ DOX), wherein the particle size distribution diagram of the nanogel. And preparing nanogel simultaneously carrying the adriamycin and the indocyanine green, and mixing a certain amount (mass fraction: 20%) of indocyanine green solution into the adriamycin-carrying nanogel solution in a vortex manner to obtain 1.0mg/mL of nanogel (NG @ DOX-ICG) carrying the adriamycin and the indocyanine green together.
Example 2 acid degradation behavior validation of nanogels
2mL of dextran-based nanogel prepared in the same batch is divided into two parts, 1mL of the dextran-based nanogel is placed in a dialysis bag with the molecular weight cutoff of 3500, the dialysis bags are respectively placed in 20mL of buffer media with different pH values, the dialysis bags are respectively sampled and detected at the temperature of 37 +/-0.5 ℃ for 0h, 1h, 3h, 7h, 24h, 48h and 72h, and the result is shown in figure 3 (a).
EXAMPLE 3 Adriamycin in vitro acid-responsive Release assay
Taking the dextran-based nanogel (feeding amount 0.05:1) coated with adriamycin as an example, the in vitro acid response release behavior of the dextran-based nanogel is researched. 2mL of 1mg/mL doxorubicin-loaded dextran-based nanogel prepared in the same batch (the entrapment efficiency is 35.56% by an ultraviolet standard curve method) is divided into two parts, each 1mL solution is placed in a dialysis bag with the molecular weight cutoff of 3500, the dialysis bags are respectively placed in dissolution containers filled with 20mL dissolution media, and the doxorubicin in vitro release experiment is carried out in the dark at the medium temperature of 37 +/-0.5 ℃. Taking out 2mL of release liquid outside the dialysis bag at set time intervals of 0h, 0.5h, 1h, 2h, 3h, 4h, 6h, 17h and 24h, supplementing blank dissolution medium with the same volume, and measuring the content of adriamycin in the release liquid by fluorescence. The fluorescence excitation wavelength was 480nm and the emission wavelength was 557nm, and the results are shown in FIG. 3 (b). The experimental results show that 75.29% of the entrapped drug is released after 24 hours in the dissolution medium with pH 5.0, whereas only 30.0% of the drug is released after 24 hours in the system with pH 7.4, and the acid-responsive release of the nanogel is confirmed.
EXAMPLE 4 Nanogel cytotoxicity assay (MTT)
Human cervical cancer cells HeLa were used for the experiment, and cultured in a DMEM medium containing 10% fetal bovine serum and 3% antibiotics in an incubator at 37 ℃ and 5% carbon dioxide, and the cells were passaged every other day to select cells in an exponential growth phase in a good growth state. Inoculation of 10 in 96 well cell culture plates4One/ml (48 hours) or 5X 104Cells at a concentration of one cell/ml (24 hours) were cultured overnight at 37 ℃ in an incubator with 5% carbon dioxide. Preparing four nanogel aqueous solutions with different concentrations, adding 10 mu L of 5mg/mL MTT aqueous solution into each well respectively after incubating and culturing cells for 48 hours, adding 10 mu L of 5mg/mL MTT aqueous solution into each well respectively, continuously culturing for four hours, carefully removing a culture medium, adding 100 mu L of DMSO solution into each well respectively, placing the mixture on a multifunctional microplate reader after the blue-violet crystals are completely dissolved, detecting the absorbance, wherein the detection wavelength is 490nm, and the result is shown in figure 4, and the nanogel aqueous solution has a survival rate of more than 80 percent after culturing the cells for 48 hours under the highest nanogel concentration of 400 mu g/mL, thereby proving that the nanogel has almost no cytotoxicity and good biocompatibility.
Example 5 cell uptake experiments with drug loaded nanogels
Human cervical cancer cells HeLa were used for the experiment, and cultured in a DMEM medium containing 10% fetal bovine serum and 3% antibiotics in an incubator at 37 ℃ and 5% carbon dioxide, and the cells were passaged every other day to select cells in an exponential growth phase in a good growth state. Seeding of 500. mu.L concentration in 24-well cell culture plates 5X 104The cells were cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. Preparing 1mg/mL doxorubicin-loaded glucan-based nanogel, determining that the final concentration of the doxorubicin loaded in the nanogel is 36.7 mug/mL, and preparing a doxorubicin aqueous solution with the same concentration for later use. Adding 50 mu L of gel or free drug solution into each well at different time points for incubation, and performing a cell flow experiment after 12 hours at most to find that the fluorescence intensity of the cells has a trend of obviously increasing along with time, as shown in figure 5, proving that the drug-loaded gel or free drug is coatedAnd (4) taking up by cells.
Example 6 verification of cell killing efficacy of drug-loaded nanogels in combination with Near Infrared (NIR) irradiation
Human cervical cancer cells HeLa were used for the experiment, and cultured in a DMEM medium containing 10% fetal bovine serum and 3% antibiotics in an incubator at 37 ℃ and 5% carbon dioxide, and the cells were passaged every other day to select cells in an exponential growth phase in a good growth state. 100. mu.L of inoculum was inoculated in 96-well cell culture plates at a concentration of 5X 104The cells were cultured overnight at 37 ℃ in a 5% carbon dioxide incubator. Preparing the doxorubicin-entrapped dextran-based nanogel and the co-entrapped doxorubicin and indocyanine green dextran-based nanogel, concentrating and quantifying to obtain final doxorubicin concentrations of 2 mug/mL, 4 mug/mL and 8 mug/mL respectively, and preparing a free doxorubicin hydrochloride aqueous solution with the same concentration for later use, wherein the final indocyanine green concentration is 200 mug/mL. Adding 10 mu L of sample into each well respectively, adding 10 mu L of 5mg/mL MTT aqueous solution into each well after culturing for 24 hours, continuing culturing for 4 hours, carefully removing the culture medium, adding 100 mu L of DMSO solution into each well, placing on a multifunctional microplate reader to detect absorbance after the blue-violet crystals are completely dissolved, wherein the detection wavelength is 490nm, the result is shown in figure 6, the survival rate of the cells in 48 hours is less than 20% under the concentration of 8 mu g/mL, and the effective killing of the drug-loaded nanogel on tumor cells is shown.
Claims (9)
1. A glucan-based nanogel is characterized in that glucan is used as a framework material, vinyl ether acrylate modification is carried out to obtain a glucan derivative I, then sulfhydryl modification and carboxyl modification are respectively carried out to obtain a glucan derivative II and a glucan derivative III on the basis of the glucan derivative I, the glucan derivative II and the glucan derivative III are mixed to carry out Michael addition reaction to obtain the glucan-based nanogel, the glucan-based nanogel is hydrolyzed in weak acid,
the dextran derivative I comprises an acid-sensitive acetal group, and the structure of the dextran derivative I is shown as follows:
the chemical structural formulas of the glucan derivative II and the glucan derivative III are shown as follows:
II III。
2. the dextran-based nanogel according to claim 1, wherein the molecular weight of said backbone material dextran is in the range of 10000 to 50000.
3. The glucan-based nanogel according to claim 1, wherein said michael addition reaction is a michael addition reaction between a thiol group on the molecule of glucan derivative II and a carbon-carbon double bond on the molecule of glucan derivative III to form a gel network.
4. The method of preparing a dextran-based nanogel according to any of claims 1 to 3, comprising the steps of:
(1) preparation of dextran derivative I: dissolving glucan in an organic solvent, and carrying out condensation reaction on hydroxyl on glucan chains and vinyl at one end of vinyl ether acrylate by using p-toluenesulfonic acid as a catalyst to generate a vinyl ether acrylate modified glucan derivative I;
(2) preparation of dextran derivative II: on the basis of the glucan derivative I, triethylamine is used as a catalyst, and a sulfydryl group at one end of a sulfhydrylation reagent molecule reacts with a carbon-carbon double bond at the tail end of an acrylate on the glucan derivative I molecule to generate a sulfydryl group modified glucan derivative molecule II;
(3) preparation of dextran derivative III: on the basis of the glucan derivative I, triethylamine is used as a catalyst, and anhydride molecules are reacted with hydroxyl on a molecular chain of the glucan derivative I to generate a carboxyl modified glucan derivative III;
(4) preparation of dextran-based nanogels: respectively dissolving the glucan derivative II and the glucan derivative III in pure water, quickly adding the mixture into an organic solvent for uniform dispersion after mixing at low temperature, carrying out Michael addition reaction on the whole system, and crosslinking glucan chain segments to form nanogel.
5. The method for preparing dextran-based nanogel according to claim 4, wherein said organic solvent in step (1) is selected from the group consisting of dimethyl sulfoxide or N, N-dimethylformamide; the organic solvent in the step (4) is selected from acetone, methanol or ethanol.
6. The method for preparing a dextran-based nanogel according to claim 4, wherein in the step (4), the mass ratio of the dextran derivative III and the dextran derivative II is 1: (1-3).
7. A glucan-based nanogel drug is characterized in that glucan is used as a framework material, vinyl ether acrylate modification is carried out to obtain a glucan derivative I, then sulfydryl modification and carboxyl modification are respectively carried out on the basis of the glucan derivative I to obtain a glucan derivative II and a glucan derivative III, the glucan derivative II and the glucan derivative III are mixed with a drug, a Michael addition reaction is carried out between the glucan derivative II and the glucan derivative III, and the drug is loaded in gel to obtain the glucan-based nanogel drug;
the dextran derivative I comprises an acid-sensitive acetal group, and the structure of the dextran derivative I is shown as follows:
the chemical structural formulas of the glucan derivative II and the glucan derivative III are shown as follows:
II III。
8. the glucan-based nanogel drug according to claim 7, wherein said drug is selected from the group consisting of doxorubicin, methotrexate and paclitaxel.
9. Use of the dextran-based nanogel according to any one of claims 1 to 3 or the dextran-based nanogel drug according to claim 7 for the preparation of an anti-tumor drug.
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