阅读说明：本技术 一种微反应器制备海藻酸钙微球的装置及方法和应用 (Device and method for preparing calcium alginate microspheres by using microreactor and application of device ) 是由 满佳 张善国 张松 李建勇 李剑峰 王晓杰 于 2019-09-29 设计创作，主要内容包括：本发明涉及海藻酸钙微球制备技术领域,尤其涉及一种基于微反应器制备海藻酸钙微球的装置及方法和应用。所述方法包括：1)先将外相流体通入第二点胶针头中,然后进入外相玻璃管,待外相流体流出其出口时,通过第一点胶针头和内相玻璃管通入内相流体；2)由于外相流体剪切力的作用,内相流体在内相玻璃管的出口处形成由外相流体所包覆的液滴,随外相流体流入收集培养皿；3)收集时不断移动芯片的位置,使在培养皿中收集的液滴彼此之间隔开,完成后对得到的液滴静置,除去多余外相流体,即得。发明基于微反应器,并且利用油水两相制备的海藻酸钙微球具有粒径均一、球形度好、溶胀比可控的特点,能够更好地满足载药、催化、检测方面的应用。(The invention relates to the technical field of preparation of calcium alginate microspheres, in particular to a device and a method for preparing calcium alginate microspheres based on a microreactor and application of the device and the method. The method comprises the following steps: 1) introducing the external phase fluid into the second dispensing needle head, then introducing the external phase fluid into the external phase glass tube, and introducing the internal phase fluid through the first dispensing needle head and the internal phase glass tube when the external phase fluid flows out of an outlet of the external phase glass tube; 2) the outer phase fluid forms liquid drops coated by the outer phase fluid at the outlet of the inner phase glass tube under the action of the shearing force of the outer phase fluid, and the liquid drops flow into the collection culture dish along with the outer phase fluid; 3) continuously moving the position of the chip during collection to separate the droplets collected in the culture dish, standing the obtained droplets after the collection is finished, and removing redundant external phase fluid to obtain the culture dish. The calcium alginate microspheres prepared by oil-water two-phase are based on the microreactor, have the characteristics of uniform particle size, good sphericity and controllable swelling ratio, and can better meet the application in the aspects of drug loading, catalysis and detection.)

1. The utility model provides a device based on micro-reactor preparation calcium alginate microballon which characterized in that includes:

the first dispensing needle head and the second dispensing needle head are fixed on the bottom plate in parallel;

one port of the inner glass tube is communicated with the cavity of the first dispensing needle head;

one port of the outer phase glass tube is communicated with the cavity of the second dispensing needle head;

the other port of the inner phase glass tube penetrates through the cavity of the second dispensing needle head and then extends into the outer phase glass tube;

the other port of the outer phase glass tube is positioned in the collection culture dish, and the central axis of the outer phase glass tube is vertical to the collection culture dish.

2. The apparatus for preparing calcium alginate microspheres according to claim 1, further comprising syringes for injecting the inner phase fluid and the outer phase fluid, respectively, wherein the first dispensing needle and the second dispensing needle are connected to one syringe, respectively, and the respective syringes are driven by a flow pump.

3. The apparatus for preparing calcium alginate microspheres of claim 1, wherein the bottom plate is a 7101 model glass slide, the inner phase glass tube and the outer phase glass tube are capillary glass tubes with a length of 100-140mm, the inner diameter of the inner phase glass tube is 0.1-0.15mm, and the inner diameter of the outer phase glass tube is 0.5-0.7 mm; preferably, the inner phase glass tube and the outer phase glass tube are fixed together with the glass slide by means of gluing.

4. A method for preparing calcium alginate microspheres, which is performed by using the device of any one of claims 1 to 3, and which comprises the following steps:

(1) introducing the external phase fluid into the second dispensing needle head, then introducing the external phase fluid into the external phase glass tube, and introducing the internal phase fluid through the first dispensing needle head and the internal phase glass tube when the external phase fluid flows out of an outlet of the external phase glass tube;

(2) the outer phase fluid forms liquid drops coated by the outer phase fluid at the outlet of the inner phase glass tube under the action of the shearing force of the outer phase fluid, and the liquid drops flow into the collection culture dish along with the outer phase fluid;

(3) continuously moving the position of the chip during collection to separate the droplets collected in the culture dish, standing the droplets coated by the obtained outer phase fluid after the collection is finished, completely reacting the inner phase droplets with the outer phase fluid to obtain aged calcium alginate microspheres, and cleaning the aged calcium alginate microspheres to remove redundant outer phase fluid to obtain the calcium alginate microspheres;

in the step (1), the inner phase fluid is a sodium alginate solution; the external phase fluid is an undecanol solution of calcium iodide.

5. The method of preparing calcium alginate microspheres of claim 4, wherein the concentration of alginate ions in the inner phase fluid is 1.0 wt%.

6. The method for preparing calcium alginate microspheres according to claim 4, wherein the concentration of calcium ions in the external phase fluid is 0.001 wt% to 1.97 wt%, more preferably 0.001 wt% to 0.115 wt%; further preferably from 0.08% to 0.115% by weight.

7. The method for preparing calcium alginate microspheres according to claim 4, wherein the method for preparing the sodium alginate solution comprises the following steps: weighing sodium alginate powder, adding deionized water, and dissolving in a water bath magnetic stirrer at 75-90 deg.C for 1.5-3 hr;

preferably, the preparation method of the undecanol solution of calcium iodide is as follows: adding calcium iodide into undecanol solution, dissolving, and standing for 1-7 days.

8. The method for preparing calcium alginate microspheres of claim 4, wherein in the step (1), the flow rate of the fixed internal phase fluid is 0.5 mL/h; the flow rate of the external phase fluid is 1-12 mL/h; preferably the ratio of the flow rates of the inner phase fluid to the outer phase fluid is 0.5: 4;

preferably, in the step (3), the position of the chip is continuously moved during the collection, so that the distance between adjacent calcium alginate microspheres collected in the culture dish is not less than 1.5 mm;

preferably, in the step (3), the aging time is not less than 6 hours, so that the inner phase liquid droplets and the outer phase liquid are sufficiently reacted.

9. The method for preparing calcium alginate microspheres according to claim 4, wherein in the step (3), the aged microspheres are placed in ethanol for centrifugal washing to remove excessive external phase fluid; the method specifically comprises the following steps: the centrifugal rotation speed is 180 plus 260r/min, firstly, the calcium alginate microspheres in the oil phase are gathered to the center of a collection culture dish under the centrifugal action, the oil is sucked out from the edge of the collection culture dish by a suction pipe, then, absolute ethyl alcohol is added to clean the calcium alginate microspheres, the operation is repeated, and then, the microspheres are stored in the absolute ethyl alcohol.

10. Use of the device for preparing calcium alginate microspheres based on a microreactor as defined in any one of claims 1-3 and/or the calcium alginate microspheres prepared by the method as defined in any one of claims 4-9 in the fields of food, pharmaceutical and medical testing.

Technical Field

The invention relates to the technical field of preparation of calcium alginate microspheres, in particular to a device and a method for preparing calcium alginate microspheres based on a microfluidic chip and application of the device.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Alginate is a natural polysaccharide extracted from brown algae, and calcium alginate microspheres can be obtained after reaction of alginate and calcium ions. The alginate microspheres have good biocompatibility and biodegradability, and are widely applied to the fields of food, pharmacy, medical detection and the like. The microspheres produced by the traditional process (such as a spray drying method) have uneven particle size distribution and poor sphericity; the existing technology for preparing calcium alginate microspheres by a microfluidic method utilizes a water-oil two-phase gel reaction, so that raindrop-shaped particles with poor sphericity are easily formed, and the swelling effect is not obvious; in addition, patent document 201810255265.4 discloses a method for preparing micro-fluidic double-aqueous-phase monodisperse calcium alginate microspheres, which comprises the following steps: taking a polyethylene glycol aqueous solution as an external aqueous phase, and taking a glucan and sodium alginate aqueous solution as an internal aqueous phase; generating double-aqueous-phase sodium alginate liquid drops at the conical tip of the coaxial capillary device; wherein the external water phase is continuously fed, and the internal water phase is periodically and intermittently fed; the double-aqueous-phase sodium alginate droplets are introduced into a calcium chloride aqueous solution to generate the required monodisperse calcium alginate microspheres, and the research of the inventor finds that the surface tension generated by the external aqueous phase and the internal aqueous phase in the method is unstable, and the internal aqueous phase is easy to form a jet line under the action of weak shear force and is not easy to form balls.

In addition, the article of the Linghai eagle for preparing monodisperse calcium alginate gel microspheres in microfluidic chips introduces that the microfluidic chips are prepared by adopting a micromachining technology taking soft lithography as a main means and are applied to preparing monodisperse calcium alginate microspheres; the test result shows that: the diameter of the calcium alginate micro-gel beads can be controlled within the range of 50 mu m to hundreds of mu m, and the monodispersity is good. However, the inventor researches and discovers that: the method is essentially characterized in that water phase calcium ions react with water phase sodium alginate, and when the method is used for reaction in an S-shaped channel, liquid drops need to flow through pipelines at different angles, so that the flow velocity in the S-shaped channel is uneven, adjacent liquid drops are easy to adsorb each other, and larger liquid drops are formed by fusion; in all the methods, calcium ions and sodium alginate are dissolved in aqueous solution and are essentially aqueous two-phase reaction; in the existing method, no technical means for respectively dissolving calcium ions and sodium alginate in oil and water for reaction exists; in order to solve the above problems, a preparation method and a formula of calcium alginate microspheres which are stably formed into spheres through oil-water two-phase reaction are needed.

Disclosure of Invention

Aiming at the technical problems, the invention aims to provide a device for preparing calcium alginate microspheres based on a microfluidic chip, a method and application thereof. The method comprises the steps of respectively introducing an oil phase in which calcium ions are dissolved and a water phase in which sodium alginate is dissolved into an outer-phase glass tube and an inner-phase glass tube in a microfluidic chip, forming water-in-oil droplets at the outlet of the inner-phase glass tube under the action of shearing force of the outer oil phase, carrying out gel reaction at an oil-water interface, and aging to obtain the calcium alginate microspheres with the characteristics of uniform particle size distribution, good sphericity and controllable swelling ratio.

The first object of the present invention: provides a device for preparing calcium alginate microspheres based on a microreactor.

The second object of the present invention: provides a method for preparing calcium alginate microspheres based on a microreactor.

The third object of the present invention: provides a formula for preparing calcium alginate microspheres based on a microreactor.

The fourth object of the present invention: provides the application of the method for preparing the calcium alginate microspheres based on the microreactor.

In order to realize the purpose, the invention discloses the following technical scheme:

firstly, the invention discloses a device for preparing calcium alginate microspheres based on a microreactor, which comprises the following components: the device comprises a bottom plate, a first dispensing needle head, a second dispensing needle head, an inner phase glass tube, an outer phase glass tube and a collection culture dish; the first dispensing needle head and the second dispensing needle head are fixed on the bottom plate side by side, one port of the inner phase glass tube is communicated with the cavity of the first dispensing needle head, one port of the outer phase glass tube is communicated with the cavity of the second dispensing needle head, and the other port of the inner phase glass tube penetrates through the cavity of the second dispensing needle head and then extends into the outer phase glass tube; the other port of the outer phase glass tube is positioned in the collection culture dish, and the central axis of the outer phase glass tube is vertical to the collection culture dish.

Secondly, the invention discloses a method for preparing calcium alginate microspheres based on a microreactor, which comprises the following steps:

(1) introducing the external phase fluid into the second dispensing needle head, then introducing the external phase fluid into the external phase glass tube, and introducing the internal phase fluid through the first dispensing needle head and the internal phase glass tube when the external phase fluid flows out of an outlet of the external phase glass tube;

(2) the outer phase fluid forms liquid drops coated by the outer phase fluid at the outlet of the inner phase glass tube under the action of the shearing force of the outer phase fluid, and the liquid drops flow into the collection culture dish along with the outer phase fluid;

(3) continuously moving the position of the chip during collection to separate the droplets collected in the culture dish, standing the droplets coated by the obtained outer phase fluid after the collection is finished, completely reacting the inner phase droplets with the outer phase fluid to obtain aged calcium alginate microspheres, and cleaning the aged calcium alginate microspheres to remove redundant outer phase fluid to obtain the calcium alginate microspheres.

Compared with the prior art, the preparation method and the formula of the calcium alginate microspheres have the following beneficial effects in the aspect of performance:

(1) the calcium alginate microspheres prepared by oil-water two-phase based on the microreactor have the characteristics of uniform particle size, good sphericity and controllable swelling ratio, so that the application in the aspects of drug loading, catalysis and detection can be better met.

(2) The device and the method can be used for preparing water-in-oil droplets, so that the reaction area of the internal and external phase fluids is large and uniform, and microspheres with good sphericity and uniform particle size can be obtained after aging.

(3) The invention utilizes the oil phase containing calcium ions as the external phase and the collecting phase, thereby avoiding the defects of microsphere fusion, difficult separation of microspheres after oil-water mixing, microsphere dehydration and the like which are generated by dissolving calcium ions and sodium alginate in the water phase in the traditional method.

(4) According to the invention, calcium ions are dissolved in the oil phase, water-in-oil droplets can be stably formed in the microreactor, and the characteristic that the shearing force of oil-water two phases is large and the droplets are easy to form is retained.

(5) According to the invention, by adjusting the ratio of the concentration of calcium ions in the oil phase to the concentration of sodium alginate in the water phase, a preparation formula with high sphericity and controllable swelling ratio is preferably selected, and the formula has a guiding effect on the application of calcium alginate microspheres in the industries of medicine and the like.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

Fig. 1 is a schematic structural diagram of an apparatus for preparing calcium alginate microspheres in example 1 of the present invention.

FIG. 2 is a schematic view of the process for preparing calcium alginate microspheres in example 2 of the present invention

FIG. 3 is an optical microscopic image of calcium alginate microspheres obtained under different flow rate ratios of inner and outer phase fluids in example 5 of the present invention, wherein Q_ORepresents the flow rate of the external phase, the flow rate of the internal phase Q_iThe internal phase flow rate is indicated. Fixed internal phase flow rate Q_iThe flow rate of the external phase is adjusted to be between 1ml/h and 12ml/h so as to control the size of the microspheres, wherein the flow rate of the external phase is 0.5 ml/h.

FIG. 4 is an optical microscope photograph of droplets before the ungelled reaction of sodium alginate-in-oil solutions prepared at different calcium ion concentrations in the external phase of example 6 of the present invention.

FIG. 5 is an optical microscope photograph of the sodium alginate-in-oil solution microspheres prepared under different calcium ion concentrations in the external phase of example 6 of the present invention after complete gelation reaction and aging.

FIG. 6 is an optical microscope photograph of sodium alginate microspheres prepared under the condition that the flow rate ratio of the external fluid in example 7 is 8, after swelling in different concentrations of the external phase fluid. a4, b4 and c4 are images of microspheres a3, b3 and c3 after gel reaction and swelling after soaking in deionized water for 2 hours.

Fig. 7 is an optical microscope picture of sodium alginate microspheres with a preferred concentration of 0.1 wt% in external phase fluid prepared in example 7 of the present invention after loading with drugs.

FIG. 8 is an optical microscope photograph of sodium alginate microspheres prepared in comparative example 1 of the present invention.

FIG. 9 is an optical microscope photograph of sodium alginate microspheres prepared in comparative example 3 of the present invention.

Wherein the symbols represent: 1-bottom plate, 2-first dispensing needle, 3-second dispensing needle, 4-internal phase glass tube, 5-external phase glass tube and 6-collection culture dish.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms also are intended to include the plural forms as well, unless the context clearly indicates otherwise, and it should be further understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.

As described above, some of the microspheres prepared by the prior art methods have uneven particle size distribution, poor sphericity, and insignificant swelling effect. Therefore, the invention provides a device and a method for preparing calcium alginate microspheres based on a microreactor.

In some typical embodiments, in step (1), the internal phase fluid is a sodium alginate solution; the external phase fluid is an undecanol solution of calcium iodide; optionally, the concentration of alginate ions in the inner phase fluid is fixed at 1 wt%; the concentration of calcium ions in the external phase fluid is 0.001-1.97 wt%, preferably 0.001-0.115 wt%, calcium ions with different concentrations have great influence on the sphericity and surface appearance of the prepared calcium alginate microspheres, and the low concentration can cause poor swelling capacity of the microspheres; too high a concentration results in too low a sphericity.

Further, the concentration of calcium ions is preferably 0.08-0.115 wt%, the obtained calcium alginate microspheres have the best sphericity and good controllability of swelling ratio, and the optimal formula of the calcium alginate microspheres suitable for medical use can be obtained.

Specifically, the preparation method of the sodium alginate solution comprises the following steps: weighing sodium alginate powder, adding deionized water, and dissolving in a water bath magnetic stirrer at 80 deg.C for 3 hr.

Specifically, the preparation method of the undecanol solution of calcium iodide comprises the following steps: weighing calcium iodide with different masses, placing in undecanol solution, and standing for 1-5 days according to different prepared concentrations; to obtain calcium iodide solution of 0.001 wt% -1.97 wt% of undecanol.

The oil phase (undecanol solution of calcium iodide) prepared by the invention is one of the key technologies, and in the existing method for preparing the calcium alginate microspheres based on the oil-water two-phase used in microfluidics, the oil phase only plays a role in coating water-phase droplets, and no solute exists; the calcium iodide is dissolved in the oil phase undecanol solution, and after the aqueous phase liquid drops are stably generated, calcium ions in the oil phase can continuously react with sodium alginate liquid drops in the internal aqueous phase to form balls.

In some typical embodiments, in step (1), the fixed internal phase fluid flow rate is 0.5 mL/h; the flow rate of the external phase fluid is 1-12mL/h, and the difference of the flow rate of the external phase can cause the particle size and the coefficient of variation of the microspheres obtained after aging to be different, thus causing the different physicochemical characteristics of the calcium alginate microspheres. Therefore, the invention obtains the high sphericity microspheres with different sizes by controlling the flow rate ratio of the internal phase and the external phase. Preferably, the ratio of the flow rate of the inner phase fluid to the flow rate of the outer phase fluid is 0.5:4, and the physicochemical properties of the microspheres obtained under these conditions are better.

In some exemplary embodiments, in step (3), the position of the chip is continuously moved during the collection, so that the distance between adjacent calcium alginate microspheres collected in the culture dish is not less than 1.5mm, in order to allow the droplets to individually perform the gelation reaction, and this operation avoids the use of surfactant, so that the prepared microspheres have better biocompatibility.

In some typical embodiments, in step (3), the aging time is not less than 6h, so that the inner phase liquid droplets and the outer phase liquid are fully reacted.

In some exemplary embodiments, in step (3), the aged microspheres are placed in ethanol for centrifugal washing to remove excess external phase fluid; the method specifically comprises the following steps: the centrifugal rotation speed is 180 plus 260r/min, firstly, the calcium alginate microspheres in the oil phase are gathered to the center of a collection culture dish under the centrifugal action, the oil is sucked out from the edge of the collection culture dish by a suction pipe, then, absolute ethyl alcohol is added to clean the calcium alginate microspheres, the operation is repeated, and then, the microspheres are stored in the absolute ethyl alcohol.

In some typical embodiments, the calcium alginate microspheres prepared by the method and the device for preparing the calcium alginate microspheres based on the microreactor are used in the fields of food, pharmacy, medical detection and the like.

The invention will now be further described with reference to the accompanying drawings and detailed description.