CN113372465A - Modified sodium hyaluronate, preparation method and microspheres prepared by using modified sodium hyaluronate - Google Patents
Modified sodium hyaluronate, preparation method and microspheres prepared by using modified sodium hyaluronate Download PDFInfo
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- CN113372465A CN113372465A CN202110708434.7A CN202110708434A CN113372465A CN 113372465 A CN113372465 A CN 113372465A CN 202110708434 A CN202110708434 A CN 202110708434A CN 113372465 A CN113372465 A CN 113372465A
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- sodium hyaluronate
- modified sodium
- water
- mixture
- hana
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- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical class [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 title claims abstract description 50
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- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
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- Medicinal Preparation (AREA)
Abstract
The invention discloses a preparation method of modified sodium hyaluronate, which comprises the following steps: (1) weighing a certain amount of HANa into a three-neck flask, adding a sodium hydroxide aqueous solution, and stirring to dissolve the HANa to prepare a first mixture; (2) weighing a certain amount of first molecules, dissolving the first molecules in N, N-Dimethylformamide (DMF), and reacting the first molecules with the first mixture in the three-neck flask to prepare a second mixture; (3) and purifying the second mixture to obtain the modified sodium hyaluronate. And preparing microspheres by using the modified sodium hyaluronate, forming a water-in-oil emulsion by using a microfluidic device, and carrying out polymerization reaction under ultraviolet illumination. The microspheres can quickly and efficiently adsorb hydrophilic drugs, namely, the hydrophilic drugs can be adsorbed in a short time, and the microspheres have higher encapsulation efficiency and drug-loading capacity. In addition, the HANa-GMA wet ball has certain elasticity.
Description
FIELD
The invention relates to the technical field of biological medicines, and particularly relates to modified sodium hyaluronate, a preparation method and microspheres prepared by applying the modified sodium hyaluronate.
Background
Hyaluronic Acid (HA) is a naturally occurring glycosaminoglycan composed of N-acetyl-D-glucosamine and D-glucuronic acid. This linear polysaccharide is widely present in all tissues and is a major component of the extracellular matrix (ECM) of animal tissues. It has important structural and biological functions including cell proliferation, differentiation, morphogenesis, wound healing, etc. It also has the ability to specifically interact with most mammalian cell membrane receptors. However, pure HA HAs some disadvantages, such as poor mechanical properties, too high possibility of being absorbed by tissues, and not being retained in tissues for a long time, and various disadvantages limit the application of pure HA in biomaterials. For this reason, modification studies of HA molecules have been initiated by a large number of scholars.
The hydroxyl groups of HA can be reacted with glutaraldehyde (ADH), divinyl sulfone (DVS), methacrylate, and the like to give modified HA. Due to the multiple modifiable chemical sites of HA, hydrogels suitable for different fields can be manufactured through different modification sites and modes.
Generally, the physical and chemical properties of HA can be changed to a certain extent by chemical modification of HA, and the modifier Glycidyl Methacrylate (GMA) is utilized to modify the HA through ester exchange and epoxy ring-opening reaction, so that a carbon-carbon double bond capable of being polymerized by illumination can be introduced to the HA. However, the GMA modified HA usually requires complex ion exchange in dimethyl sulfoxide (DMSO), which is an organic solvent, and the cost is high. And the molecular weight of the HANa is high (hundreds of thousands to millions), the solution viscosity is high, the modification can be generally carried out only at low concentration (about 1 wt.%), and the efficiency is low. For the same reason, the microspheres are also usually prepared only at low concentrations.
SUMMARY
In a first aspect, the present disclosure relates to a method for preparing modified sodium hyaluronate, comprising grafting a first molecule to sodium hyaluronate HANa in an aqueous solution by a one-pot synthesis method to obtain the modified sodium hyaluronate;
wherein: the structural formula of the first molecule is one or more of the following structural formulas:
in a second aspect, the present disclosure is directed to a modified sodium hyaluronate made by the steps of,
(1) weighing a certain amount of HANa into a three-neck flask, adding a sodium hydroxide aqueous solution, and stirring to dissolve the HANa to prepare a first mixture;
(2) weighing a certain amount of first molecules, dissolving the first molecules in N, N-Dimethylformamide (DMF), and reacting the first molecules with the first mixture in the three-neck flask to prepare a second mixture;
(3) and purifying the second mixture to obtain the modified sodium hyaluronate.
In a third aspect, the disclosure relates to a preparation method of modified sodium hyaluronate microspheres, which comprises the steps of preparing a water-in-oil emulsion and carrying out polymerization reaction under ultraviolet illumination.
In a fourth aspect, the present disclosure relates to a modified sodium hyaluronate microsphere for preparing a water-in-oil emulsion using a microfluidic device, which is prepared by the following steps,
(1) manufacturing a single emulsion capillary microfluidic device by a micromachining technology; the adopted specific means comprises the steps of stretching, needle forging and polishing of a circular capillary tube, and assembling the circular capillary tube with a corresponding square capillary tube;
(2) dissolving the modified sodium hyaluronate and the water-soluble photoinitiator in deionized water to obtain a solution as a dispersion phase;
(3) dissolving a water-in-oil emulsifier and an oil-soluble photoinitiator in liquid paraffin to obtain a solution as a continuous phase;
(4) injecting the dispersed phase and the continuous phase into an inner phase channel and an outer phase channel of the micro-fluidic device from corresponding inlets respectively, controlling the flow rate, and forming single emulsion droplets in the micro-channels;
(5) and (3) carrying out ultraviolet irradiation on the prepared single emulsion liquid drop to initiate liquid drop polymerization, and purifying to obtain the microsphere.
In a fifth aspect, the disclosure relates to the use of the modified sodium hyaluronate microspheres in hydrophilic drugs.
Brief description of the drawings
FIG. 1 shows an enlarged view of the NMR hydrogen spectra of HANa, GMA, and HANa-GMA and the carbon-carbon double bond positions of HANa-GMA in example 1 of the present disclosure;
fig. 2 shows a schematic diagram of a capillary microfluidic device of the present disclosure for making single emulsion droplets;
FIG. 3 shows an optical micrograph of microspheres from example 3 of the present disclosure;
FIG. 4 shows an optical micrograph of microspheres from example 4 of the present disclosure;
FIG. 5 shows an optical micrograph of microspheres from example 5 of the present disclosure;
FIG. 6 shows a chart of HANa-GMA dry bulb drug loading process in example 7 of the present disclosure;
FIG. 7 shows the maximum drug loading of HANa-GMA dry and wet spheres in example 7 of the present invention;
FIG. 8 shows the HANa-GMA wet bulb compression process of example 8 of the present disclosure.
Detailed description of the invention
In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.
Unless otherwise required by the disclosure, throughout the specification and the appended claims, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".
Reference throughout the specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that a particular reference element, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment, and furthermore, particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.
Definition of
In the present disclosure, the term "microfluidic technology" is a technology for precisely controlling and processing microfluid, and hydrogel microspheres prepared by using the technology have the advantages of good monodispersity, high stability, high yield and the like. The photochemical reaction has the advantages of non-physical contact, adjustable dosage, clean energy, no toxic by-products and the like, and can realize the precise control of time and space, thereby being widely applied to the construction of medical hydrogel.
In the present disclosure, the term "photoinitiator" is also called photosensitizer or light curing agent, and is a compound that can absorb energy with a certain wavelength in an ultraviolet region (250-420 nm) or a visible light region (400-800 nm) to generate free radicals, cations, etc., thereby initiating polymerization, crosslinking, and curing of monomers.
By "water-soluble photoinitiator" is meant a photoinitiator that is soluble in water.
The term "oil-soluble photoinitiator" refers to a photoinitiator that is compatible with oily substances.
In the present disclosure, the term "crosslinking agent" refers to a substance capable of causing crosslinking of a polymer.
In this disclosure, the term "sodium hyaluronate" has the chemical formula (C)14H20NO11Na) N, which is an inherent component in human body, is disaccharide glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, has no species specificity, and is widely present in tissues such as placenta, amniotic fluid, crystalline lens, articular cartilage, skin dermis and the like; device for cleaning the skinIt is distributed in cytoplasm and intercellular substance, and has lubricating and nourishing effects on cells and cell organs contained therein.
In the present disclosure, the term "glycidyl methacrylate" is an ester compound of formula C7H10O3Also called methacrylic acid-2, 3-epoxypropyl ester, abbreviated as GMA. A colorless transparent liquid. Is insoluble in water and soluble in most common organic solvents. Because GMA has two functional groups of active vinyl and ionic reactive epoxy in the molecule, the GMA can be polymerized in a functional group mode and also can be polymerized in an ionic reaction mode.
In the present disclosure, the term "water-in-oil emulsifier" refers to an emulsifier capable of forming a water-in-oil (W/O) emulsion, in which a water-soluble substance is dispersed in an oil-soluble solvent in the form of droplets (of micron order), water being an internal phase, and oil being a continuous external phase, preventing the droplets from aggregating with each other, maintaining a uniform emulsion state.
In the present disclosure, the term "doxorubicin hydrochloride" is an antitumor antibiotic, which has a broad antitumor spectrum and can kill various tumor cells by inhibiting the synthesis of genetic material nucleic acid of the cancer cells. Molecular formula C27H29NO11HCI, an orange-red loose cake or powder, readily soluble in water, DMSO, tetrahydrofuran, alcohol, and insoluble in acetone, chloroform, benzene, diethyl ether.
In the present disclosure, the term "irinotecan hydrochloride" is used for the treatment of advanced colorectal cancer patients. Treating patients with advanced colorectal cancer who have not received chemotherapy before with 5-fluorouracil and folinic acid; patients who failed treatment with a regimen containing 5-fluorouracil were treated as single agents.
In the present disclosure, the term "epirubicin hydrochloride" is a cell cycle non-specific drug that is effective against a variety of transplanted tumors and can be used to treat lung and ovarian cancer. Compared to doxorubicin, the therapeutic effect was equal or slightly higher, but the toxicity to the heart was less.
Detailed Description
In a first aspect, the present disclosure relates to a method for preparing modified sodium hyaluronate, comprising grafting a first molecule to sodium hyaluronate HANa in an aqueous solution by a one-pot synthesis method to obtain the modified sodium hyaluronate;
wherein: the structural formula of the first molecule is one or more of the following structural formulas:
in certain embodiments, the first molecule is Glycidyl Methacrylate (GMA), which is of the formula
In certain embodiments, the molecular weight of the sodium hyaluronate is 3000 to 10000 g/mol.
In certain embodiments, the molecular weight of the sodium hyaluronate is 3000 g/mol.
Wherein: the sodium hyaluronate has the characteristics of low molecular weight and low viscosity.
In certain embodiments, the method of making comprises the steps of:
(1) weighing a certain amount of HANa into a three-neck flask, adding a sodium hydroxide aqueous solution, and stirring to dissolve the HANa to prepare a first mixture;
(2) weighing a certain amount of first molecules, dissolving the first molecules in N, N-Dimethylformamide (DMF), and reacting the first molecules with the first mixture in the three-neck flask to prepare a second mixture;
(3) and purifying the second mixture to obtain the modified sodium hyaluronate.
In certain embodiments, the concentration of aqueous sodium hydroxide solution in step (1) is 1 wt.%; the reaction temperature in the step (2) is 50 to 80 ℃, and the reaction time is 6 to 10 hours.
In certain embodiments, the purification treatment step in step (3) is specifically: and (3) firstly dripping absolute ethyl alcohol into the second mixture for precipitation, then soaking and ultrasonically cleaning the mixture by using 95% ethyl alcohol, finally soaking and cleaning the mixture by using the absolute ethyl alcohol, and drying the precipitate at the temperature of 35-45 ℃ to obtain the modified sodium hyaluronate.
In certain embodiments, the degree of substitution of the modified sodium hyaluronate can be adjusted by the amount of the first molecule.
In certain embodiments, the sodium hyaluronate to first molecule is 1: 1 (w: v).
Furthermore, the invention provides HANa-GMA with the end group capable of crosslinking carbon-carbon double bonds by illumination, the preparation method is simple, and the HANa-GMA can be prepared in high concentration in aqueous solution by a one-pot method.
In a second aspect, the present disclosure relates to a modified sodium hyaluronate made by the steps of:
(1) weighing a certain amount of HANa into a three-neck flask, adding a sodium hydroxide aqueous solution, and stirring to dissolve the HANa to prepare a first mixture;
(2) weighing a certain amount of first molecules, dissolving the first molecules in N, N-Dimethylformamide (DMF), and reacting the first molecules with the first mixture in the three-neck flask to prepare a second mixture;
(3) and purifying the second mixture to obtain the modified sodium hyaluronate.
In a third aspect, the disclosure relates to a preparation method of modified sodium hyaluronate microspheres, which comprises the steps of preparing a water-in-oil emulsion and carrying out polymerization reaction under ultraviolet illumination.
In certain embodiments, a "water-in-oil" type emulsion is prepared using a microfluidic device, comprising the steps of:
(1) manufacturing a single emulsion capillary microfluidic device by a micromachining technology;
(2) dissolving the modified sodium hyaluronate of claim 5 and a water-soluble photoinitiator in deionized water to obtain a solution as a dispersed phase;
(3) dissolving a water-in-oil emulsifier and an oil-soluble photoinitiator in liquid paraffin to obtain a solution as a continuous phase;
(4) injecting the dispersed phase and the continuous phase into an inner phase channel and an outer phase channel of the micro-fluidic device from corresponding inlets respectively, controlling the flow rate, and forming single emulsion droplets in the micro-channels;
(5) and (3) carrying out ultraviolet irradiation on the prepared single emulsion liquid drop to initiate liquid drop polymerization, and purifying to obtain the microsphere.
Wherein: the present disclosure can provide a monodisperse high elastic hydrogel microsphere by preparing the microsphere in a microfluidic device. The microspheres prepared by the microfluidic device are significantly superior to those prepared in a reaction kettle. The microspheres prepared by the reaction kettle have non-uniform particle size distribution, and the particle size distribution can be very wide, from several micrometers to thousands of micrometers. Therefore, it is necessary to obtain microspheres of a target particle size by sieving. In comparison, the method for preparing the catalyst by using the reaction kettle has the advantages of more working procedures, low yield, high cost and more generated three wastes.
In certain embodiments, the nozzle internal diameter of the inner phase microchannel of the microfluidic device is from 10 to 200 μm.
In certain embodiments, the modified sodium hyaluronate of step (2) is present in an amount of 1 to 20% by weight.
In certain embodiments, the modified sodium hyaluronate of step (2) is present in an amount of 1 to 10% by weight.
In certain embodiments, the purification step in step (5) comprises: collecting wet microspheres initiated to be subjected to liquid drop polymerization by ultraviolet irradiation in a mixed solution of isopropanol and deionized water;
then fully washing with isopropanol, soaking with normal saline, soaking with isopropanol for water storage, and drying to obtain HANa-GMA dry spheres;
and soaking the HANa-GMA dry ball in normal saline to swell and balance to obtain a HANa-GMA wet ball.
In certain embodiments, the water-soluble photoinitiator includes, but is not limited to, one or more of 2-ketoglutaric acid, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, and 2, 2-azobis (2-methylpropylamidine) dihydrochloride.
In certain embodiments, the water-soluble photoinitiator is selected from 2-oxoglutarate.
In certain embodiments, the water-soluble photoinitiator is present in an amount of 1 to 5% by weight.
In certain embodiments, the water-soluble photoinitiator is present at 1% by weight.
In certain embodiments, the water-in-oil emulsifier is selected from one or more of ABIL EM 90, Span 80, Tween 60.
In certain embodiments, the water-in-oil emulsifier is selected from ABIL EM 90.
In certain embodiments, the water-in-oil emulsifier is present in an amount of 3 to 10% by weight.
In certain embodiments, the water-in-oil emulsifier is present in an amount of 10% by weight.
In certain embodiments, the oil-soluble photoinitiator includes, but is not limited to, one or more of 2-hydroxy-2-methylpropanone, 2-methyl-1- (4-methylthiophenyl) -2-morpholin-1-one, 1-hydroxycyclohexyl phenyl ketone, benzil dimethyl ether.
In certain embodiments, the oil-soluble photoinitiator is selected from 2-hydroxy-2-methylpropenone.
In certain embodiments, the oil-soluble photoinitiator is present in an amount of 1 to 5% by weight.
In certain embodiments, the oil-soluble photoinitiator is present at 2% by weight.
In certain embodiments, the flow rate of the dispersed phase is from 0.05 to 2mL/h and the flow rate of the continuous phase is from 1 to 10 mL/h.
In certain embodiments, isopropanol to deionized water is 4: 1 (v: v)
Wherein: the preparation process of the microsphere is carried out at room temperature.
In a fourth aspect, the present disclosure relates to a modified sodium hyaluronate microsphere prepared by the steps of,
(1) manufacturing a single emulsion capillary microfluidic device by a micromachining technology;
(2) dissolving the modified sodium hyaluronate and the water-soluble photoinitiator in deionized water to obtain a solution as a dispersion phase;
(3) dissolving a water-in-oil emulsifier and an oil-soluble photoinitiator in liquid paraffin to obtain a solution as a continuous phase;
(4) injecting the dispersed phase and the continuous phase into an inner phase channel and an outer phase channel of the micro-fluidic device from corresponding inlets respectively, controlling the flow rate, and forming single emulsion droplets in the micro-channels;
(5) and (3) carrying out ultraviolet irradiation on the prepared single emulsion liquid drop to initiate liquid drop polymerization, and purifying to obtain the microsphere.
In a fifth aspect, the disclosure relates to the use of the modified sodium hyaluronate microspheres in hydrophilic drugs.
Wherein: hydrophilic drugs include: doxorubicin hydrochloride, irinotecan hydrochloride, epirubicin hydrochloride.
The modified sodium hyaluronate microspheres can be used for loading the hydrophilic drugs.
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 1 preparation of HANa-GMA with photocrosslinkable carbon-carbon double bond as end group
(1) An appropriate amount of sodium hydroxide was weighed and dissolved in deionized water to prepare an aqueous solution having a concentration of 1 wt.%. Then, 5g of HANa having a molecular weight of 3000g/mol was weighed into a three-necked flask, and 10g of the above-mentioned aqueous sodium hydroxide solution was added thereto, and the mixture was stirred on a magnetic stirrer to be dissolved.
(2) 5mL of GMA is dissolved in N, N-Dimethylformamide (DMF), added into the three-neck flask, and placed in a 60 ℃ oil bath to be stirred and reacted for 10 hours.
(3) And (3) firstly dripping the solution obtained by the reaction into absolute ethyl alcohol for precipitation, then soaking and ultrasonically cleaning the solution by using 95% ethyl alcohol, finally soaking and cleaning the solution by using the absolute ethyl alcohol, and drying the precipitate in a vacuum oven at 40 ℃ to obtain the final product, namely HANa-GMA.
FIGS. 1a, b, c and d are enlarged views of the GMA, HANa-GMA and HANa-GMA double bonds, respectively, and d corresponds to chemical shifts of 5.7ppm and 6.1ppm of the double bond in a; in addition, the proportion of the chemical shift of the position H of the double bond in the nuclear magnetic spectrum of the product is 1: 5.8 after the integration of the chemical shift of 5.7ppm and the chemical shift of 4.5ppm of H on a sugar ring-OH, and the degree of substitution is about 17.24 percent.
Example 2
Modified sodium hyaluronate was prepared according to the method of example 1, except that GMA was changed to the structural formula
The first molecule of (1).
Example 3 microfluidic preparation method of HANa-GMA monodisperse hydrogel microspheres with end groups of photo-crosslinkable carbon-carbon double bonds
Firstly, a glass capillary circular tube is thinned and burned off by a needle drawing instrument, the tip diameter of the capillary tube is cut to the diameter of a nozzle required by an experiment by a needle burning instrument, the tip inner diameter is cut to the required diameter, and an inlet capillary tube, a square tube, a glass collecting tube and a needle head are coaxially arranged on a glass slide for assembly. The HANa-GMA prepared in example 1 and the water-soluble photoinitiator 2-oxoglutaric acid were dissolved in deionized water simultaneously, and the mass percentage of the HANa-GMA in the solution was 10 wt.%, and the mass percentage of the photoinitiator was 1 wt.%. The above materials are mixed uniformly to form a dispersion phase. The mass percent of the water-in-oil emulsifier ABIL EM 90 in the continuous phase is 10 wt.%, and the mass percent of the oil-soluble photoinitiator 2-hydroxy-2-methyl propyl phenyl ketone is 2 wt.%. Injecting the dispersed phase and the continuous phase into a channel of a microfluidic device, wherein the flow rates are 0.1mL/h and 5mL/h respectively, the inner diameter of a nozzle is 30 micrometers, obtaining single emulsion droplets by utilizing the shearing force and the interfacial tension of the continuous phase relative to the dispersed phase, finally obtaining HANa-GMA hydrogel microspheres by ultraviolet irradiation polymerization, and collecting the HANa-GMA hydrogel microspheres in a mixed solution of isopropanol and deionized water.
The invention utilizes a capillary microfluidic device to prepare single emulsion droplets (the schematic diagram of the principle is shown in figure 2), namely, oil phase fluid is used as a continuous phase to shear a water phase so as to be dispersed in the oil phase to form micro droplets. The invention utilizes the self-made glass capillary tube design to manufacture the microfluidic device suitable for generating the hydrogel microspheres. The preparation method is simple, and the produced hydrogel microspheres have the advantages of controllable size and good monodispersity.
Example 4
The preparation of HANa-GMA hydrogel microspheres was carried out as in example 3, except that the flow rate of the continuous phase was 4 mL/h.
Example 5
The preparation of HANa-GMA hydrogel microspheres was carried out as in example 3, except that the flow rate of the continuous phase was 3 mL/h.
Example 6
As shown in fig. 3 to 5, it can be seen that the particle size of the hydrogel microspheres can be controlled by controlling the flow rates of the dispersed phase and the mobile phase during the preparation of the HANa-GMA hydrogel microspheres. The HANa-GMA hydrogel microspheres prepared by the method have uniform particle size.
Example 7
And (3) fully washing the obtained microspheres with isopropanol, soaking the microspheres in normal saline, then soaking the microspheres in isopropanol to remove water, and drying to obtain the HANa-GMA dry spheres. Taking a proper amount of dry spheres on a glass slide, and dropwise adding a doxorubicin hydrochloride aqueous solution of 5mg/g, as can be seen from fig. 6, the color of the solution is obviously lightened at 3min, which indicates that a large amount of doxorubicin hydrochloride is adsorbed by the microspheres; when the time is 5min, the microspheres almost completely adsorb the doxorubicin hydrochloride in the solution, and the solution is nearly colorless;
as can be seen from FIG. 7, the drug loading until the final wet bulb reached 89.72 mg/g.
Example 8
The microspheres which are swelled and balanced in normal saline and dyed are fired into glass capillaries with certain diameters, and whether the microspheres can smoothly pass through the capillaries and rapidly recover the appearance is observed. As can be seen from FIG. 8, the HANa-GMA wet ball with a diameter of 400 μm can smoothly pass through a glass capillary with an inner diameter of 150 μm, and can rapidly recover the morphology without the phenomenon of microsphere breakage and the like.
In summary, the following steps: the invention has at least the following advantages:
(1) the invention provides HANa-GMA with an end group capable of photo-crosslinking carbon-carbon double bonds, which has a simple preparation method, can be prepared in a water solution at high concentration by a one-pot method, and solves the problems that in the prior art, the modification is complex, and the microsphere preparation can only be carried out at low concentration, so that the efficiency is low;
(2) the invention provides a method for preparing HANa-GMA with an end group capable of photocrosslinking carbon-carbon double bonds and a water-soluble photoinitiator which are simultaneously dissolved in deionized water to serve as a dispersed phase, wherein the concentration of the HANa-GMA can be up to 1-20 wt.%. Dissolving a water-in-oil emulsifier and an oil-soluble photoinitiator in liquid paraffin as a continuous phase, obtaining single emulsion droplets by using a microfluidic technology, and initiating the droplets by ultraviolet irradiation to perform simple free radical polymerization to obtain hydrogel microspheres;
(3) the microspheres in the invention can quickly and efficiently adsorb hydrophilic drugs, namely, the hydrophilic drugs can be adsorbed in a short time, and a new path is opened for efficient preparation and loading of hydrophilic drugs based on degradable hydrogel microspheres. Meanwhile, the coating has higher encapsulation efficiency and drug loading capacity. In addition, the microspheres have certain elasticity.
From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure, and that such modifications or improvements are intended to be within the scope of the appended claims.
Claims (10)
1. A preparation method of modified sodium hyaluronate is characterized in that a first molecule is grafted to sodium hyaluronate (HANa) in an aqueous solution by adopting a one-pot synthesis method to prepare the modified sodium hyaluronate;
wherein: the structural formula of the first molecule is one or more of the following structural formulas:
2. the method of claim 1, wherein the first molecule is Glycidyl Methacrylate (GMA) having the formula
3. The method of manufacturing according to claim 1 or 2, comprising the steps of:
(1) weighing a certain amount of HANa into a three-neck flask, adding a sodium hydroxide aqueous solution, and stirring to dissolve the HANa to prepare a first mixture;
(2) weighing a certain amount of first molecules, dissolving the first molecules in N, N-Dimethylformamide (DMF), and reacting the first molecules with the first mixture in the three-neck flask to prepare a second mixture;
(3) and purifying the second mixture to obtain the modified sodium hyaluronate.
4. The method according to claim 3, wherein the concentration of the aqueous sodium hydroxide solution in step (1) is 1 wt.%; the reaction temperature in the step (2) is 50 to 80 ℃, and the reaction time is 6 to 10 hours; the purification treatment step in the step (3) is specifically as follows: and (3) firstly dripping absolute ethyl alcohol into the second mixture for precipitation, then soaking and ultrasonically cleaning the mixture by using 95% ethyl alcohol, finally soaking and cleaning the mixture by using the absolute ethyl alcohol, and drying the precipitate at the temperature of 35-45 ℃ to obtain the modified sodium hyaluronate.
5. Modified sodium hyaluronate prepared by the process according to any one of claims 1 to 4.
6. A preparation method of modified sodium hyaluronate microspheres is characterized in that water-in-oil emulsion is prepared, and polymerization reaction is carried out under ultraviolet illumination.
7. The preparation method according to claim 6, wherein the preparation of the "water-in-oil" emulsion by means of a microfluidic device comprises the following steps:
(1) manufacturing a single emulsion capillary microfluidic device by a micromachining technology;
(2) dissolving the modified sodium hyaluronate of claim 5 and a water-soluble photoinitiator in deionized water to obtain a solution as a dispersed phase;
(3) dissolving a water-in-oil emulsifier and an oil-soluble photoinitiator in liquid paraffin to obtain a solution as a continuous phase;
(4) injecting the dispersed phase and the continuous phase into an inner phase channel and an outer phase channel of the micro-fluidic device from corresponding inlets respectively, controlling the flow rate, and forming single emulsion droplets in the micro-channels;
(5) and (3) carrying out ultraviolet irradiation on the prepared single emulsion liquid drop to initiate liquid drop polymerization, and purifying to obtain the microsphere.
8. The preparation method according to claim 7, wherein the water-soluble photoinitiator comprises one or more of but not limited to 2-ketoglutaric acid, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone, 2-azobis (2-methylpropylamidine) dihydrochloride, preferably 2-ketoglutaric acid; the mass percentage content of the water-soluble photoinitiator is 1 to 5 percent, and the mass percentage content of the water-soluble photoinitiator is preferably 1 percent; the water-in-oil emulsifier is selected from one or more of ABIL EM 90, Span 80, Tween 80 and Tween 60, and is preferably ABIL EM 90; the weight percentage content of the water-in-oil emulsifier is 3 to 10 percent, and is preferably 10 percent; the oil-soluble photoinitiator comprises but is not limited to one or more of 2-hydroxy-2-methyl propyl ketone, 2-methyl-1- (4-methylthiophenyl) -2-morpholine-1-acetone, 1-hydroxycyclohexyl phenyl ketone and benzil dimethyl ether, and preferably 2-hydroxy-2-methyl propyl ketone; the mass percentage content of the oil-soluble photoinitiator is 1 to 5 percent, and preferably 2 percent; the flow rate of the dispersed phase is 0.05 to 2mL/h and the flow rate of the continuous phase is 1 to 10 mL/h.
9. The modified sodium hyaluronate microspheres prepared by the preparation method of claim 7.
10. The use of the modified sodium hyaluronate microspheres of claim 9 in a hydrophilic drug.
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