WO2019015636A1 - Method for continuously preparing gelatin nanoparticles based on microfluidic control chip device - Google Patents

Abstract

A method for continuously preparing gelatin nanoparticles based on a microfluidic control chip device. In the method, a micrometer-scale microfluidic control chip device capable of making a gelatin aqueous solution and a polar organic solvent form a concentric liquid is used, and compared with a traditional macroscopic preparation method, the specific surface area of a liquid in a microfluidic chip is increased, and substance exchange and heat diffusion are more uniform and quicker, which is beneficial to increasing the production rate of nanoparticles, controlling the size distribution of the particles, and reducing unnecessary additional products. In the method, the generation time of gelatin particles is within the range of 0.01-10 seconds; therefore, the reaction efficiency is higher.

Description

Continuous preparation method of gelatin nano particles based on microfluidic chip device Technical field

The invention belongs to the field of bioengineering and relates to a continuous preparation method of gelatin nanoparticles based on a microfluidic chip device.

Background technique

Nanomaterials have been widely used in microelectronics, chemistry, energy, life sciences and medicine. Especially in recent years, nanoparticle materials have shown more and more application value in the field of biomedicine, such as nanoparticles for medical imaging, nanocarrier materials for targeted drug controlled release. Nanomaterials currently available in the biomedical field include nanoemulsions, organic nanoparticles, dendrimer polymers, micelles, liposomes and polymers. The advantage of these nanomaterials in the biomedical field is that the high specific surface area of nanomaterials makes them highly soluble and highly permeable, which is important for many water-insoluble or poorly water-soluble drugs, which can increase the molecular weight of drugs. Release; at the same time, how to achieve long-term release and targeted release of nanomedicine has always been an important research direction in the field of nanobiomedical materials. Although nanomaterials have important application value in the field of biomedicine, how to realize the industrial scale preparation of organic or composite nanomaterials is still one of the bottlenecks restricting the application of nanobiomaterials.

Microfluidic technology is an emerging technology in the field of micromachining technology that is designed to construct channels of tens to hundreds of microns and manipulate tiny volumes of fluid (features range from 10 ^-9 to 10 ^-18 L). The key to this technology is to micro-scale the experimental techniques such as synthesis on the bench in a traditional laboratory to a microfluidic chip of a few centimeters. The main feature of this system is the miniaturization of the fluid environment, in which the microfluidic channel is about 100μm (about the diameter of human hair), and chemical reagents are sent to the chip through various injection pumps for synthesis, separation or analysis. In recent years, microfluidic technology has become one of the important tools in biopharmaceutical research. For example, the above microfluidic technology has been used to support complex chemical reaction processes or drug screening processes for studying cell-drug interactions for the production of micron-sized particles or droplets for drug control. Applications such as release or cell embedding. Although microfluidic technology has shown great potential for microscale material synthesis processing, how to apply this technology to nanotechnology Related research reports in the field of materials synthesis and preparation are still very few.

Protein (such as albumin, gelatin, collagen) nanoparticles have important application value in the field of biomedicine. Because of its non-toxicity, strong stability, no antigenicity, and large-scale production, it can be used as a drug controlled release carrier. Gelatin is a derivative of collagen. It is a polymer material composed of a large number of amino acids. It has good biocompatibility, biodegradability, non-toxicity, no immunogenicity, and can be chemically modified. A type of implantable biomaterial approved by the US Food and Drug Administration. Gelatin materials have been used extensively in tissue engineering and drug delivery. Recent studies have shown that gelatin nanoparticles have a significant effect on the release of biologically active factors. However, the processing of gelatin nanoparticles has been a difficult problem to promote its application.

Invention US 2008/0003292 discloses a process for preparing gelatin nanoparticles using a conventional reaction vessel having a maximum particle diameter of 350 nm, which can be used as a carrier system for a drug, which is prepared by adding acetone to a gelatin aqueous solution dropwise to prepare gelatin particles. . The method is based on the traditional laboratory preparation method and needs to be prepared in batches. Because the nanoparticles are very sensitive to the preparation parameters during the preparation process, the nanoparticle product parameters obtained between different preparation batches are difficult to maintain stable.

The invention CN 103841965 A discloses a continuous process for the preparation of gelatin nanoparticles in a reactor comprising a process line of a mixing unit. First, the invention uses a millimeter-scale fluid pipeline reactor. Due to the limited material exchange rate in a millimeter-scale pipeline, the patent requires the design of a special geometry for the rapid mixing of a gelatin aqueous solution with a poor solvent polar organic solvent. The channel structure enables physical blending of the two-phase fluid. In addition, the method combines two-phase fluids through a fluid channel to form a parallel flow laminar flow, and then blends the two phases by physical blending, the reaction takes a long time, and the reaction time of the two-phase solution to form gelatin particles on the chip is Seconds. In particular, the structural design and preparation of the microfluidic channel are complicated, and the preparation cost of such a reaction chip is increased. Also, the method is prepared under acidic conditions (pH 2-4), and the acidic environment limits the loading or specific application of certain specific drugs.

Due to the nucleation and growth process of nanoparticles, the parameters such as temperature, good solvent/poor solvent mixing ratio and stirring rate will affect the formation of nanoparticles. The traditional preparation method using agitation blending method often results in batches of nanoparticle products. The difference between the two is significant, and it is difficult to ensure the stability of the performance parameters (such as size and size distribution) of the nanoparticle products between batches.

In order to realize the loading of biomacromolecules in gelatin particles, the existing methods mainly use the preparation of gelatin particles to adsorb the drug molecules on the surface of the particles, or chemically graft the drug molecules on the gelatin particles. In the former, because macromolecules cannot enter the gelatin particles, they usually cause rapid release of the drug; the latter requires additional chemical reactions and is inefficient. If one-step preparation of a macromolecular drug can be achieved, the efficiency of preparation will be greatly improved, and the release cycle of the macromolecule will also be increased.

Summary of the invention

In view of the above-mentioned drawbacks in the prior art, the present invention provides a continuous preparation method of gelatin nanoparticles based on a microfluidic chip device. The invention designs a micro-scale microfluidic chip capable of forming a concentric fluid of a gelatin aqueous solution and a polar organic solvent as a reactor for synthesizing gelatin nanoparticles, and the material exchange rate of the fluid in the micrometer-scale channel is fast. The nucleation growth of the particles and the formation of the nanoparticles are greatly accelerated, and the reaction efficiency is improved.

The technical solution of the present invention is as follows:

A continuous preparation method for preparing gelatin nanoparticles based on a microfluidic chip device, comprising the following steps:

(1) Dissolving gelatin in deionized water to obtain an aqueous solution of gelatin as an internal phase, a polar organic solvent as an external phase, and a crosslinking agent solution as an additional phase;

(2) injecting the inner phase at the first flow rate, and injecting the outer phase into the inner phase fluid microchannel and the outer phase fluid microchannel of the microfluidic chip device at the second flow rate, the inner phase and the outer phase are merged and then in the output channel Forming a concentric axis fluid with an outer phase surrounding the inner phase, promoting rapid nucleation of gelatin molecules by rapid material diffusion between the two phases, and gradually growing gelatin nanoparticles; the time required for the formation of the inner and outer phases from confluence to gelatin nanoparticles In 0.01-10 seconds;

(3) injecting the additional phase to the additional phase fluid microchannel located downstream of the microfluidic chip device at a third flow rate, the crosslinker solution being mixed with the mixed solution of the gelatin nanoparticle-containing inner and outer phases formed in step (2) , the gelatin particles are cross-linked to form a gelatin nanoparticle suspension, and the chip is taken out from the outlet of the output channel and collected in the container;

(4) repeatedly collecting the collected gelatin nanoparticle suspension and resuspending in deionized water to finally obtain gelatin nanoparticles;

Wherein, the time required for the inner phase and the outer phase to merge to mix with the crosslinker solution is <10 seconds.

Further, in the above aspect, the gelatin concentration in the gelatin aqueous solution described in the step (1) is 0.1 to 12 w/v%, preferably 1 to 5%, more preferably 2.5 to 3%.

Further, in the above technical solution, the temperature of each fluid in the microfluidic chip device is maintained at 30 to 60 °C.

Further, in the above aspect, the gelatin aqueous solution has a pH of 1 to 5, preferably 2 to 4 or 9 to 12, preferably 10 to 11.

Further, in the above technical solution, the polar organic solvent is a polar organic solvent which is insoluble or slightly soluble in gelatin, preferably in methanol, ethanol, isopropanol, butanol, acetone, acetonitrile or tetrahydrofuran. One or a combination of several.

Further, in the above technical solution, the flow rate of the first, second and third flow rate were 0.05-10mL hr ^-1, 0.1-50mL hr ^-1 and 0.05-500μL hr ^-1.

Further, in the above technical solution, the ratio of the second flow rate to the first flow rate is 1.0 to 9.0, preferably 2.0 to 3.5; and the ratio of the third flow rate to the first flow rate is 0.0067 to 0.067, preferably 0.0067 to 0.013.

Further, in the above technical solution, the crosslinking agent is glutaraldehyde, glyceraldehyde, formaldehyde, carbodiimide, dihaloalkane, isocyanate, diisocyanate, glutamine transaminase, and genipin One or several.

Further, in the above technical solution, the molar ratio of the crosslinking agent to the gelatin amino group is from 0.25 to 10.0, preferably from 0.5 to 1.0.

The present invention also provides a microfluidic chip device for preparing gelatin nanoparticles, the device comprising an internal phase fluid microchannel, at least one external phase fluid microchannel, an additional phase fluid microchannel, and an output channel, the internal phase fluid microchannel The inner diameter is smaller than the inner diameter of the outer phase fluid microchannel; the inner phase fluid microchannel is for flowing into the inner phase, the outer phase fluid microchannel is for flowing into the outer phase, and the inner and outer phases respectively flow through the inner phase fluid microchannel and The outer phase fluid microchannels merge directly into the output channel, and form a concentric fluid surrounding the inner phase in the output channel; the additional phase fluid microchannel is intersected with the output channel, and the additional phase flow flows through the additional phase fluid microchannel The output channel is in fluid communication with the concentric shaft in the output channel. Preferably, the inner phase fluid microchannel, the outer phase fluid microchannel, the additional phase fluid microchannel, and the output channel are on the same horizontal plane.

In a preferred technical solution, the microfluidic chip device includes an inner phase fluid microchannel, an outer phase fluid microchannel, an additional phase fluid microchannel, and an output channel, and one end of the inner phase fluid microchannel is unsealed Inserted at one end of the outer phase fluid microchannel, one end of the output channel is sealingly inserted at the other end of the outer phase fluid microchannel, and the port of the inner phase fluid microchannel inserted in the outer phase fluid microchannel is non-end-to-end connected, the additional The phase inflow channel is in communication with an output channel that is not inserted into the outer phase fluid microchannel. Wherein, preferably, in the outer phase fluid microchannel, the distance between the port of the inner phase fluid microchannel and the output channel port is 50-500 [mu]m. Preferably, the port of the inner phase fluid microchannel port and the output channel inserted into the outer phase fluid microchannel is tapered. The microfluidic chip device may be a microfluidic chip fabricated using a capillary. Preferably, each channel is on the same level.

In still another preferred embodiment, the microfluidic chip device includes an internal phase fluid microchannel, two external phase fluid microchannels, an additional phase fluid microchannel, and an output channel, and the two external phase fluid microchannels and outputs respectively The channels are connected to each other to form a Y-shaped channel. The center position of the intersecting junction is intersected with the internal phase fluid microchannel. The center line of the internal phase fluid microchannel coincides with the center line of the output channel, and the two external phase fluid microchannels are completely symmetrical. The ground position is on both sides of the inner phase fluid microchannel, and the angle between the outer phase fluid microchannel and the inner phase fluid microchannel is 30 to 90, and the angle is preferably 45-60, more preferably 60. The angle between the inner and outer phases at the intersection of the Y-shaped channels can form a concentric fluid surrounding the inner phase in the output channel. It has a very important adjustment effect. If the angle is too small or too large , can not form a concentric shaft fluid well. The microfluidic chip device may be a polymer chip prepared by soft etching. Preferably, each channel is on the same level. Preferably, in the Y-shaped channel formed by the intersection of the inner phase fluid microchannel and the outer phase fluid microchannel and the output channel, the cross-sectional area of the intersection is 3×10 ^-4 to 8×10 ^-1 mm ² , It is preferably 3 × 10 ^-4 to 1.2 × 10 ^-1 mm ² , more preferably 2 × 10 ^-3 to 3 × 10 ^-2 mm ² .

In the above technical solution, the inner phase fluid microchannel has an inner diameter of 10 to 500 μm, preferably 10 to 200 μm, more preferably 25 to 100 μm; and the outer phase fluid microchannel has an inner diameter of 20 to 1000 μm, preferably 10 to 500 μm. More preferably, it is 50-100 micrometer.

The invention utilizes a fluid-focused microfluidic channel design to form a gelatin aqueous solution (internal phase) and a polar organic solvent (external phase) to form a concentric fluid, thereby significantly increasing the contact area of the two-phase fluid and accelerating the diffusion of the two phases. Increase the rate of nanoparticle formation. In the present invention, the design of the microfluidic chip and the flow rates of the inner and outer phases are important factors influencing the ability of the outer phase to surround the concentric fluid of the inner phase in the continuous preparation of gelatin.

It is worth mentioning that micron-scale fluid passages are not simply a reduction of millimeter-scale fluid passages. From the macroscale to the microscale, many physical properties, including specific surface area, diffusion-based material exchange, etc., do not linearly decrease with size reduction. In contrast, in a microfluidic chip, the fluid follows the laminar flow under the action of viscous forces; that is, the parallel flow of different fluids in the micron-sized channel without turbulence and perpendicular to the flow direction of the fluid. Therefore, in micron-scale channels, material exchange relies mainly on passive molecular diffusion rather than convection or turbulence.

Material diffusion is a nonlinear process. Assuming the time t required for a substance to diffuse over a square area of diameter X, we can calculate the diffusion of the substance through a simple one-dimensional diffusion process: X ² = 2D × t. The diffusion time of gelatin molecules in a 10 μm square microfluidic channel was calculated to be about 500 ms, based on the diffusion coefficient of gelatin in water of 1×10 ^-1 μm ² /ms. Therefore, the microchannel provides a higher reaction efficiency for the preparation method of synthesizing gelatin nanoparticles by means of two-phase diffusion.

The microfluidic chip device for preparing gelatin nanoparticles realizes the mixing of the two-phase fluid by utilizing the high diffusion rate of the two-phase fluid in the micrometer-scale space to promote the nucleation growth of the nanoparticles. At the same time, the device of the present invention utilizes a fluid-focused microfluidic channel design to form a concentric axis of the two-phase fluid, thereby increasing the contact area between the two phases and increasing the diffusion efficiency of the two phases. In addition, the two-phase fluid of the present invention does not need to design a complicated output channel after the confluence, but uses a horizontal microchannel to reduce the complexity of the chip design and save cost; and at the same time, the rapid diffusion of the two phases is used to promote the nucleation and growth of the nanoparticles. The reaction time from the confluence of the two-phase fluid to the formation of the nanoparticles is significantly shorter than previously reported, and the reaction can be completed in at least 100 milliseconds.

The present invention also provides a colloidal gel obtained by blending a lyophilized powder of gelatin nanoparticles prepared by the method of the present invention described above and an aqueous solution. Wherein, in the dispersion formed by blending the gelatin nanoparticles with the aqueous solution, the volume percentage of the composite nanoparticles is 5% to 150%. The colloidal gel prepared has an elastic modulus of 10 Pa to 100 kPa, preferably 10 Pa to 50 kPa. The colloidal gel has an injectable and repairable function, and the recovery value after storage (elastic) modulus after shear failure exceeds 60% of the initial storage (elastic) modulus value within 30 minutes. The colloidal gel can also be obtained by directly blending a lyophilized powder of gelatin nanoparticles prepared by the method described above with an aqueous solution in which cells are suspended or an aqueous solution in which bioactive molecules are dissolved. Wherein, the cell is selected from the group consisting of a primary cultured cell, a subcultured cell, a cell culture cell, and a hybrid; the bioactive molecule is one of a drug, a protein, and a signal factor. The colloidal gel can be applied to the preparation of implantable filler materials for tissue repair and treatment.

The beneficial effects of the invention:

1. The preparation method of the present invention adopts a micro-scale microfluidic chip capable of forming a concentric fluid in a gelatin aqueous solution and a polar organic solvent, and the specific surface area of the fluid in the microfluidic chip is increased compared with the conventional macroscopic preparation method. Material exchange and heat diffusion are more uniform and faster, thus facilitating increased nanoparticle yield, controlling particle size distribution and reducing unnecessary additional products. The traditional macro stirring preparation method requires a method of adding a polar organic solvent to a gelatin aqueous solution to promote the nucleation of gelatin into nanoparticles, and the reaction time is calculated in minutes or even hours, and the reaction time is long and the production efficiency is low; The microparticles are prepared by using a micro-scale microfluidic chip, and the gelatin particles are generated in the range of 0.01 to 10 seconds, so that the reaction efficiency is higher.

2. Conventional macro-laboratory preparation method When blending a gelatin aqueous solution with a polar organic solvent of a gelatin poor solvent, physical agitation is required to rapidly mix the two phases, but the preparation is slow due to the slow exchange rate of substances and heat on a macro scale. The gelatin nanoparticles have a wide scale distribution; and the microfluidic chip used in the invention ensures fast and efficient blending of the two-phase fluid on a microscopic scale, and the parameter control is more precise, so the prepared gelatin nanoparticle size and the same preparation parameters are conventional. The nanoparticles prepared by the method have smaller size and narrower size distribution.

3. The traditional method needs to be prepared in batches. The nanoparticles are very sensitive to the preparation parameters during the preparation process, which makes it difficult to maintain the stability of the nanoparticle product parameters obtained between different preparation batches. However, the microfluidic control preparation method in the present invention can be The continuous sample preparation is realized, the reaction conditions are more stable and more controllable, and the obtained product parameters are more stable and controllable.

4. The method of superimposing multiple microfluidic channels can realize the enlargement of production, improve the yield and the yield, and is beneficial to industrial production.

DRAWINGS

1 is a schematic view showing the structure of a microchannel of a capillary microfluidic chip device described in Embodiment 1.

2 is a schematic view showing the microchannel structure of the micro-fluidic chip device of the soft etching process described in Embodiment 2.

3 is a transmission electron micrograph of gelatin nanoparticles prepared according to Example 3.

4 is a scanning electron micrograph of gelatin nanoparticles prepared according to Example 3.

5 is a particle size distribution of gelatin nanoparticles prepared according to the microfluidic chip method described in Example 3 and the conventional stirring method described in Example 4.

Figure 6 is a schematic representation of the preparation of alkaline phosphatase (active protein) gelatin nanoparticles according to the method described in Example 9.

Figure 7 shows that alkaline phosphatase (active protein) gelatin nanoparticles prepared by the method described in Example 9 induced mineralization in a calcium glycinate phosphate solution.

Figure 8 is a graph showing the rheological test results of the self-healing behavior of a GelA+B colloidal gel having a mass fraction of 10% by weight prepared by the method described in Example 10.

Symbol Description: 1, internal phase fluid microchannel, 2, external phase fluid microchannel, 3, additional phase fluid microchannel, 4, output channel, 5, exhaust port, 6, abutment, 7, internal phase fluid sample end 8, the external phase fluid sample end, 9, the output channel output.

Detailed ways

The following non-limiting examples are provided to enable a person of ordinary skill in the art to understand the invention, but not to limit the invention in any way. In the following examples, unless otherwise stated, the experimental methods used are all conventional methods, and the materials, reagents and the like used can be purchased from a biological or chemical company.

Example 1

As shown in FIG. 1, a capillary microfluidic chip device having a fluid focusing function includes an inner phase fluid microchannel, an outer phase fluid microchannel, an additional phase fluid microchannel, an output channel, and a collection container, the inner phase One end of the fluid microchannel is non-sealedly inserted into one end of the outer phase fluid microchannel, one end of the output channel is sealingly inserted at the other end of the outer phase fluid microchannel, and the port of the inner phase fluid microchannel inserted in the outer phase fluid microchannel is not An end-to-end connection, the additional phase fluid microchannel is in communication with an output channel not inserted into the outer phase fluid microchannel, and the other end of the output channel is connected to the collection container; the device can be fixed to the base for ease of use, Each channel is on the same level, and the inner wall surface of each microchannel is subjected to hydrophilic treatment.

The port of the inner phase fluid microchannel inserted in the outer phase fluid microchannel and the output channel port inserted in the outer phase fluid microchannel are tapered; the inner phase fluid microchannel, the outer phase fluid microchannel and the additional phase fluid micro The channels are respectively connected to a micro-peristaltic pump or micro-injector to achieve automatic injection; in the outer-phase fluid microchannel, the distance between the port of the internal phase fluid microchannel and the port of the output channel is 200 μm. A portion of the output passage that is not inserted into the outer phase fluid passage is provided with an exhaust port for discharging the gas in the chip when the fluid is first injected into the chip.

In the microfluidic chip device, the outer phase fluid microchannel is a square AIT glass capillary having a uniform inner diameter (inner diameter of 1.05 μm). The inner phase fluid microchannel is a cylindrical AIT glass capillary having a uniform inner diameter (inner diameter of 560 μm), and the port inserted in the outer phase fluid passage is a tapered port having a port inner diameter of 30 μm. The output channel is a cylindrical AIT glass capillary with a uniform inner diameter (inner diameter of 560 μm), and the port inserted in the outer phase fluid microchannel is a tapered port with a port inner diameter of 60 μm.

Figure 1B is a cross-sectional view taken along line a-a' of Figure 1A.

Example 2

The microfluidic chip device of the present invention can be processed by soft etching, as shown in FIG. The inner phase fluid microchannel, the outer phase fluid microchannel, the additional phase fluid microchannel, the output channel and the collection container, the inner phase fluid, the outer phase fluid, and the additional phase fluid are respectively input into the corresponding microchannel through the corresponding sample delivery end, a, b, and c represent different regions within the microchannel, respectively. Figure 2B shows the formation of gelatin particles in three different regions a, b, c. The fluid in the a region acts as an internal phase gelatin aqueous solution, and merges with the external phase (organic solvent) to form a concentric fluid surrounding the inner phase in the output channel, and the material diffuses faster in the micron-sized channel between the two-phase microfluids. The gelatin molecules dissolved in water are rapidly supersaturated and nucleated, and gradually grow to form gelatin nanoparticles (b region), further gelatin nanoparticle solution is mixed with its downstream crosslinking agent, cross-linking reaction, and gradually form gelatin nanoparticle micro The ball suspension is discharged from the outlet of the output channel and collected in a container.

As a preferred technical solution, the microfluidic chip device of the present invention can be provided with two external phase fluid microchannels, and the two outer phase fluid microchannels are respectively connected with the output channels to form a Y-shaped channel. The center position of the intersecting junction is intersected with the internal phase fluid microchannel, the center line of the inner phase fluid microchannel coincides with the center line of the output channel, and the two outer phase fluid microchannels are completely symmetrically positioned in the inner phase fluid microchannel On both sides, the angle between the outer phase fluid microchannel and the outer phase fluid outer channel is 60°. The inner phase and the outer phase respectively flow through the corresponding microchannels, merge at the intersection of the Y-shaped channels, flow into the output channel, form a concentric fluid surrounding the inner phase in the output channel, and promote gelatin by rapid material diffusion between the two phases. The molecules rapidly grow in nucleation and gradually grow to form gelatin nanoparticles. 2C is a three-dimensional three-dimensional structural view of the microchannel at the inner and outer two-phase fluids in the microfluidic chip device of the present invention having two symmetric outer phase fluid channels. Figure 2D shows the structure of the microchannel cross section of the inner and outer phase fluid at the intersection of the output channels.

In the above microfluidic chip device, the inner phase fluid microchannel, the outer phase fluid microchannel and the additional phase fluid microchannel are respectively connected to a microperistaltic pump or a microsyringe to realize automatic injection.

The microfluidic chip device is prepared by soft etching, and each channel is on the same horizontal surface, and the inner wall surface of each microchannel is subjected to hydrophilic treatment.

In the microfluidic chip device, the inner phase fluid microchannel is a uniform pipeline structure having an inner diameter of 50 μm, the outer phase fluid microchannel is a uniform pipeline structure having an inner diameter of 100 μm, and the crosslinker microchannel is an inner diameter of 50 μm. Uniform pipe structure, the output channel is a uniform pipe structure with an inner diameter of 200μm.

Example 3

The gelatin nanospheres are continuously prepared by using the capillary microfluidic chip device shown in FIG. 1, and the specific steps include:

(1) Preparation of gelatin aqueous solution: gelatin is mixed with deionized water at 40 ° C, and a gelatin solution with a gelatin concentration of 5 w/v% is disposed; the gelatin is completely dissolved to obtain a transparent clear solution, and the pH of the gelatin aqueous solution is adjusted. 3, the gelatin aqueous solution was filtered and injected into a syringe, and the gelatin aqueous solution was heated using a heating mantle, and the temperature was maintained at 40 ° C;

(2) The gelatin aqueous solution was used as the internal phase to be injected into the internal phase fluid microchannel at a flow rate of 3 mL/h, and pure acetone was used as the external phase to be injected into the external phase fluid microchannel at a flow rate of 9 mL/h; the microfluidic chip was continued using a heating jacket. Heating at 40 ° C, the temperature remains stable, the inner phase and the outer phase merge to form a concentric fluid surrounding the inner phase in the output channel, and the diffusion of the material between the two-phase microfluids in the micro-scale channel is faster, prompting dissolution The gelatin molecules in the water are rapidly supersaturated and nucleated, and gradually grow to form nanoparticles; according to different flow rates, the time for the nanoparticles to form in the microfluidic chip is 0.1 to 0.5 seconds;

(3) Injecting a cross-linking agent solution (25% by weight aqueous solution of glutaraldehyde) into the additional phase fluid microchannel at a flow rate of 19.8 μL/h, and then the cross-linking agent solution flows into the output channel and is inside and outside the gelatin-containing particles. The mixed solution is mixed to crosslink the gelatin particles to form a gelatin nanoparticle suspension, and the chip is taken out from the outlet of the output channel and collected in the collecting container;

(4) The gelatin nanoparticle suspension in the collection container was continuously stirred at 600 rpm overnight at room temperature, and the gelatin nanoparticle dispersion was repeatedly centrifuged at room temperature and sufficiently dispersed three times to obtain a gelatin colloidal particle dispersion.

Among them, due to the size of the microchannel, the Reynold constant of the fluid is small, and after the inner phase and the outer phase meet, the inner phase and the outer phase form a stable laminar fluid in the output channel.

The prepared gelatin nanoparticles were dispersed in deionized water, dropped on a copper mesh, and naturally dried to obtain a product for transmission electron microscopy to examine the morphology and size of the product. As a result, as shown in FIG. 3, the prepared gelatin nanoparticles were spherical, and the particle diameter was distributed in the range of 200 to 400 nm.

The above gelatin nanoparticles were dispersed in water and then freeze-dried to obtain powder of gelatin particles and subjected to scanning electron microscopic analysis. As a result, as shown in FIG. 4, the gelatin nanoparticles after lyophilization were spherical, and the size distribution was narrow, and the average particle diameter was about 200 nm.

Example 4

Gelatin nanospheres prepared by traditional physical stirring method: 3.75 g of dried gelatin is dissolved in 75 mL of deionized water at 40 ° C, stirring is continued for 30 min to obtain a colorless clear gelatin aqueous solution, and the pH value of the gelatin aqueous solution is adjusted with hydrochloric acid. 3, the gelatin aqueous solution was continuously stirred using a magnetic stirrer at a speed of 1200 rpm and a temperature of 40 °C. A 225 mL of acetone was added using a syringe pump to finally obtain a suspension of gelatin colloidal microspheres. Then, gelatin microspheres were chemically crosslinked by adding 495 μL of an aqueous solution of glutaraldehyde (25 wt%), and stirred at room temperature for 60 hours at 600 rpm. Centrifugal washing gives a dispersion of gelatin colloidal particles.

The particle size of the nanoparticles in the water was analyzed by a laser particle size analyzer using the above-described conventional method and the dispersion of gelatin nanocolloid particles obtained in the microfluidic chip device preparation method of Example 3 in deionized water. The results are shown in Fig. 5. In the case where the preparation parameters (including temperature, gelatin aqueous solution/acetone two-phase mixed volume ratio, cross-linking degree) are the same, the gelatin nanoparticles prepared by the conventional method are compared with the average particle prepared by the microfluidic chip. The particle size is larger and the size distribution is wider. It was confirmed that the gelatin nanoparticles prepared by the method of the present invention are superior in performance.

Example 5

The gelatin nanospheres are continuously prepared by using the capillary microfluidic chip device shown in FIG. 1, and the specific steps include:

(1) Preparation of gelatin aqueous solution: Gelatin was blended with deionized water at 37 ° C, 50 ° C or 60 ° C, respectively, and a gelatin aqueous solution having a gelatin concentration of 5 w/v% was placed; the pH of the gelatin aqueous solution was adjusted to 3. Then add gelatin aqueous solution to the syringe, and use a heating jacket to heat the gelatin solution therein, the temperature is maintained at 37 ° C, 50 ° C or 60 ° C;

(2) The gelatin aqueous solution was used as the internal phase to be injected into the internal phase fluid microchannel at a flow rate of 3 mL/h, and pure acetone was used as the external phase to be injected into the external phase fluid microchannel at a flow rate of 9 mL/h; Continuously heating 37 ° C, 50 ° C or 60 ° C, the temperature remains stable, the inner phase and the outer phase meet to form a concentric fluid surrounding the inner phase in the output channel;

(3) The cross-linking agent solution was flowed into the additional phase fluid microchannel of the microfluidic chip of different temperature at a flow rate of 19.8 μL/h, and the gelatin nanoparticle suspension was obtained by cross-linking the gelatin microparticles, which were exported and collected in a collecting container. After stirring the crosslinking reaction, centrifugation and redispersion treatment, a gelatin nanoparticle dispersion prepared at three different temperatures is finally obtained.

The preparation method of the above gelatin nanoparticle dispersion liquid is the same as that of the third embodiment except that the preparation temperature is different.

The particle size of the gelatin nanoparticles in the dispersion prepared at different temperatures was analyzed by a laser particle size analyzer. As shown in Table 1, the particle size of the gelatin increased as the preparation temperature increased.

Table 1. Particle size distribution of gelatin nanoparticles prepared at different preparation temperatures

Example 6

The microfluidic chip device prepared by the soft etching technique shown in FIG. 2 is used to continuously prepare gelatin nano microspheres, and the specific steps include:

(1) Preparation of gelatin aqueous solution: the gelatin is blended with deionized water at 40 ° C, and a gelatin solution having a gelatin concentration of 5 w/v% is disposed; the pH of the gelatin solution is adjusted to 11, and the gelatin solution is added to the syringe. And using a heating jacket to heat the gelatin solution therein, the temperature is maintained at 40 ° C;

(2) Injecting the gelatin aqueous solution as an internal phase into the internal phase fluid microchannel, and using ethanol as the external phase to inject into the external phase fluid microchannel, wherein the input flow rate of the gelatin aqueous solution is 3 mL/h (the first flow rate) remains unchanged. The input flow rate of ethanol (second flow rate) is changed according to the second/first flow rate ratio (as shown in Table 2), and the microfluidic chip is continuously heated by 40 ° C using a heating jacket, and the temperature is kept stable;

(3) The cross-linking agent solution was flowed into the additional phase fluid microchannel of the microfluidic chip of different temperature at a flow rate of 19.8 μL/h, and the gelatin nanoparticle suspension was obtained by cross-linking the gelatin microparticles, which were exported and collected in a collecting container. After stirring the crosslinking reaction, centrifugation and redispersion treatment, a gelatin nanoparticle dispersion prepared at different flow rates of the internal phase and the external phase is finally obtained.

The gelatin nanoparticles prepared under different conditions were subjected to particle size analysis using a laser particle size analyzer. The results are shown in Table 2.

Table 2. Effect of internal and external phase flow rates on the particle size of prepared gelatin colloidal particles

Example 7

The microfluidic chip device prepared by the soft etching technique shown in FIG. 2 is used to continuously prepare gelatin nano microspheres, and the specific steps include:

(1) Preparation of gelatin aqueous solution: the gelatin is blended with deionized water at 40 ° C, and a gelatin solution having a gelatin concentration of 5 w/v% is disposed; the pH of the gelatin solution is adjusted to 11, and the gelatin solution is added to the syringe. And using a heating jacket to heat the gelatin solution therein, the temperature is maintained at 40 ° C;

(2) Injecting a gelatin aqueous solution as an internal phase into the internal phase fluid microchannel, and using ethanol as an external phase to inject into the external phase fluid microchannel, wherein the microfluidic chip is continuously heated by 40 ° C using a heating sleeve, and the temperature is kept stable; The input flow rate (first flow rate) of the aqueous solution of the gelatin, the input flow rate of the external phase ethanol (second flow rate), and the flow rate of the cross-linking agent solution are different (as shown in Table 3), specifically: the second flow rate/first flow rate ratio is maintained at 3.0. Under varying conditions, changing the input flow rate of the internal phase and the external phase; changing the flow rate of the cross-linking agent solution, but the change is such that the ratio of the cross-linking agent solution to the first flow rate remains unchanged at 0.0066, as shown in Table 3;

(3) The cross-linking agent glutaraldehyde solution was flowed into the additional phase fluid microchannel of the microfluidic chip of different temperature at a flow rate of 19.8 μL/h, and the gelatin nanoparticle suspension was obtained by cross-linking the gelatin particles to change the cross-linking agent solution. The flow rate, but the change is to maintain the ratio of the cross-linking agent solution to the first flow rate remains unchanged at 0.0066, as shown in Table 3; the cross-linked gelatin particles are exported and collected in a collection container, stirred and cross-linked, centrifuged and redispersed. After the treatment, a gelatin nanoparticle dispersion prepared under flow rate parameters was finally obtained.

The particle size analysis of the gelatin nanoparticles prepared under different conditions was carried out using a laser particle size analyzer, and the results are shown in Table 3.

Table 3. Effect of flow rate on size of gelatin nanoparticles produced

Example 8

The gelatin nanospheres are continuously prepared by using the capillary microfluidic chip device shown in FIG. 1, and the specific steps include:

(1) Preparation of gelatin aqueous solution: the gelatin is blended with deionized water at 40 ° C, and a gelatin solution having a gelatin concentration of 5 w/v% is disposed; the pH of the gelatin solution is adjusted to 11, and the gelatin solution is added to the syringe. And using a heating jacket to heat the gelatin solution therein, the temperature is maintained at 40 ° C;

(2) Injecting the gelatin aqueous solution as an internal phase into the internal phase fluid microchannel, and using ethanol as the external phase to inject into the external phase fluid microchannel, wherein the input flow rate of the gelatin aqueous solution is 3 mL/h (the first flow rate) remains unchanged. Under the condition that the input flow rate (second flow rate) of ethanol is kept unchanged at 9 mL/h, the micro-flow chip is continuously heated at 40 ° C using a heating sleeve, and the temperature is kept stable;

(3) Injecting the cross-linking agent glutaraldehyde solution into the additional phase fluid microchannel of the microfluidic chip at different flow rates (as shown in Table 4), cross-linking the gelatin particles to obtain a gelatin nanoparticle suspension; and crosslinking the gelatin particles after derivatization And collected in a collection container, after stirring cross-linking reaction, centrifugation and redispersion treatment, finally obtained gelatin nanoparticle dispersion prepared under flow rate parameters.

The gelatin nanoparticles prepared under different conditions were subjected to particle size analysis using a laser particle size analyzer, and the results are shown in Table 4.

Table 4. Effect of flow rate of crosslinker solution on size of gelatin nanoparticles

Example 9

In the present embodiment, alkaline phosphatase (ALP) is used as a model drug, and a microfluidic chip device prepared by the soft etching technique shown in FIG. 2 is used to prepare gelatin nanoparticles in which a macromolecular drug is embedded, and the specific preparation method is as follows:

(1) Dissolve gelatin and ALP in deionized water at 40 ° C, the final concentration of gelatin is 5 w / v%, the concentration of ALP is 0.2 w / v%; the pH of the aqueous gelatin solution is adjusted to 3 using hydrochloric acid, added to the syringe , using a heating jacket to heat the gelatin solution therein, the temperature is maintained at 40 ° C;

(2) The gelatin aqueous solution obtained in the step (1) was injected as an internal phase into the internal phase inflow microchannel at a flow rate of 3 mL/h, and pure acetone was used as an external phase to be injected into the external phase into the microchannel at a flow rate of 9 mL/h, using heating. The micro-flow chip is continuously heated at 40 ° C, and the temperature is kept stable. After the inner phase and the outer phase meet, a concentric fluid surrounding the inner phase is formed in the output channel, and the material diffusion between the two-phase microfluids in the micro-scale channel is further Fast, the gelatin molecules dissolved in water are rapidly supersaturated and nucleated, and gradually grow to form nanoparticles; the time for the nanoparticles to form in the microfluidic chip is 0.1 to 0.5 seconds;

(3) Injecting a cross-linking agent solution (25% by weight aqueous solution of glutaraldehyde) into the microchannel at a flow rate of 19.8 μL/h, the cross-linking agent solution flows into the output channel and is inside and outside the gelatin-containing particles. The mixed solution is mixed to crosslink the gelatin particles to form a gelatin nanoparticle suspension, and the chip is taken out from the outlet of the output channel and collected in the container;

(4) The gelatin nanoparticle suspension collected in the container was continuously stirred at 600 rpm overnight at room temperature, and the same volume of 100 mM aqueous solution of guanidine hydrochloride (or lysine) was added to neutralize the unreacted aldehyde group. Gelatin nanoparticles;

(5) After stirring for 1 hour, the gelatin nanoparticle dispersion was filtered, resuspended in deionized water, and repeatedly centrifuged and redispersed 5 times at room temperature to obtain a gelatin nanoparticle dispersion in which ALP was embedded.

In order to confirm that ALP was successfully embedded in gelatin nanospheres and still maintain its activity, ALP gelatin nanospheres were resuspended in 10 mM aqueous calcium glycinate solution, and calcium glycerophosphate could diffuse into gelatin microspheres, ALP. The macromolecule decomposes calcium glycerophosphate into phosphate PO4 ^3- and Ca ²⁺ calcium ions, thereby forming calcium phosphate crystals grown in gelatin microspheres, as shown in FIG. It can be seen in Fig. 7 that platelet-shaped calcium phosphate crystals are clearly formed in the gelatin particles, indicating that ALP in the ALP-containing gelatin nanoparticles prepared by the above method retains its activity.

A schematic diagram of the above preparation method is shown in FIG.

Example 10

The gelatin nanospheres are continuously prepared by using the capillary microfluidic chip device shown in FIG. 1, and the specific steps include:

(1) Blending A or B gelatin with deionized water at 40 ° C, dissolving a gelatin solution with a gelatin concentration of 5 w/v%; adjusting the pH of the gelatin solution to 3, and then adding the gelatin solution to the syringe. And using a heating jacket to heat the gelatin solution therein, the temperature is maintained at 40 ° C;

(2) Injecting a gelatin aqueous solution as an internal phase into the internal phase fluid microchannel, and using acetone as an external phase to inject into the external phase fluid microchannel, wherein the input flow rate of the gelatin aqueous solution is 3 mL/h (the first flow rate) remains unchanged. Under the condition that the input flow rate (second flow rate) of acetone is kept unchanged at 9 mL/h, the micro-flow chip is continuously heated at 40 ° C using a heating sleeve, and the temperature is kept stable;

(3) The cross-linking agent glutaraldehyde solution was injected into the additional phase fluid microchannel of the microfluidic chip at a flow rate of 19.8 μL/h, and the gelatin microparticles were crosslinked to obtain a gelatin nanoparticle suspension; the crosslinked gelatin particles were exported and collected. In the collection container, the A-type or B-type gelatin nanoparticle dispersion liquid is obtained by stirring cross-linking reaction, centrifugation and redispersion treatment, respectively. The particle size and zeta potential of the type A and type B gelatin nanoparticles prepared by the above method were tested using a laser particle size analyzer, and the results are shown in Table 5.

Table 5. Performance parameters of different types of gelatin particles

The above gelatin nanoparticle dispersion was freeze-dried to obtain a lyophilized powder of type A gelatin particles (labeled as GelA) or a lyophilized powder of type B gelatin particles (labeled as GelB). The lyophilized powder of the above GelA or GelB gelatin colloidal particles was blended with an appropriate amount of 1 mM NaCl solution, and rapidly stirred and mixed to obtain an injectable colloidal gel.

Dispersing type A gelatin and type B gelatin microgel particles in a 20 mM aqueous solution of NaOH to obtain positively charged type A gelatin microgel particles and negatively charged type B gelatin microgel particles. The dispersion is thoroughly mixed and stirred to obtain a dispersion in which two different microgel particles are dispersed, wherein the ratio of the particles of type A gelatin to type B gelatin is 1:1; and 100 mM is added to the dispersion. The hydrochloric acid was adjusted to pH 7.0, stirred and mixed, and lyophilized to give a lyophilized powder containing two different gelatin colloidal particles, designated as GelA+B. The above GelA+B mixture lyophilized powder was blended with an appropriate amount of 1 mM NaCl solution, and rapidly stirred and mixed to obtain an injectable self-healing colloidal gel. The resulting colloidal gels of the different components were prepared, and the viscoelastic properties of the resulting colloidal gels of different components were evaluated by a rheometer. The results are shown in Table 6. As the colloidal volume fraction increases, the elastic modulus of the gel increases. When the volume fraction is the same, the gel elastic modulus of the oppositely charged colloidal particles is significantly stronger than that of the single component colloidal gel. The colloidal gel elastic modulus of the GelA+B component was >40 kPa at a microgel colloidal particle mass fraction of 25 vol%.

The self-repairing behavior of the colloidal gel is characterized by a rheometer, and the specific test method is as follows. Continuous rheological testing of the colloidal gel: firstly, the oscillating time scan is performed, and an external force of 1 Hz and a strain of 0.5% is applied to the sample, and the storage modulus (or elastic modulus, G') and the loss mode of the test sample are tested. The amount (or viscous modulus, G"), at which point the gel exhibits a rigid behavior of the solid under low shear conditions, so the storage modulus G' is greater than the loss modulus G" and remains stable. The G' value at this stage is the initial elastic modulus of the sample. Then gradually increase the applied strain from 0.1% to 1000%. During this process, the sample is destroyed by applying an external force, and the elastic modulus G' is gradually decreased, and finally lower than G", that is, the colloidal system changes from a rigid solid to a viscous fluid, and the structure It was destroyed. Immediately after the external force was removed, the recovery of the elastic modulus of the sample was examined. After the external force was released, the percentage of the stored energy (elastic) modulus of the sample and its initial storage elastic modulus (%) was quantitatively investigated. Repair efficiency. The self-healing efficiency of the gel is shown in Table 7. The gel elastic modulus composed of the oppositely charged colloidal particles is significantly stronger than that of the single component colloidal gel. Among them, the mass fraction is 10 wt% of GelA+B. The self-repairing process of the colloidal gel is shown in Fig. 8. The elastic modulus of the gel recovers instantaneously after shear failure, and the self-repairing elastic modulus recovers to more than 85% of the initial modulus within 5 minutes. Repeatedly: during the shear failure of applying multiple cycles to the sample, the elastic modulus of the gel is quickly restored and restored to 80% of the initial elastic modulus each time the external force is removed. On.

Table 6. Rheological storage (elastic) modulus G' of different colloidal gel materials

Table 7. Self-healing efficiency of different colloidal gel materials

*Note: The self-repair efficiency is 1000% strain. After you cut the gel material for 60s, the percentage of elastic modulus recovery (%) within 5 minutes after stress release is detected.

Claims (15)

1. A continuous preparation method for preparing gelatin nanoparticles based on a microfluidic chip device, comprising the following steps:

   (1) Dissolving gelatin in deionized water to obtain an aqueous solution of gelatin as an internal phase, a polar organic solvent as an external phase, and a crosslinking agent solution as an additional phase;

   (2) injecting the inner phase at the first flow rate, and injecting the outer phase into the inner phase fluid microchannel and the outer phase fluid microchannel of the microfluidic chip device at the second flow rate, the inner phase and the outer phase are merged and then in the output channel Forming a concentric axis fluid with an outer phase surrounding the inner phase, promoting rapid nucleation of gelatin molecules by rapid material diffusion between the two phases, and gradually growing gelatin nanoparticles; the time required for the formation of the inner and outer phases from confluence to gelatin nanoparticles In 0.01 to 10 seconds;

   (3) injecting the additional phase to the additional phase fluid microchannel located downstream of the microfluidic chip device at a third flow rate, the crosslinker solution being mixed with the mixed solution of the gelatin nanoparticle-containing inner and outer phases formed in step (2) , the gelatin particles are cross-linked to form a gelatin nanoparticle suspension, and the chip is taken out from the outlet of the output channel and collected in the container;

   (4) repeatedly collecting the collected gelatin nanoparticle suspension and resuspending in deionized water to finally obtain gelatin nanoparticles;

   Wherein, the time required for the inner phase and the outer phase to merge to mix with the crosslinker solution is <10 seconds.
2. The method according to claim 1, wherein the microfluidic chip device comprises an internal phase fluid microchannel, an additional phase fluid microchannel, an output channel, and at least one external phase fluid microchannel, the internal phase fluid micro The inner diameter of the passage is smaller than the inner diameter of the outer phase fluid microchannel; the inner phase fluid microchannel is for flowing into the inner phase, the outer phase fluid microchannel is for flowing into the outer phase, and the inner and outer phases respectively flow through the inner phase fluid The channel and the outer phase fluid microchannel merge directly into the output channel, and form a concentric fluid surrounding the inner phase in the output channel; the additional phase fluid microchannel is intersected with the output channel, and the additional phase flows through the additional phase fluid The channel flows into the output channel and merges with the concentric shaft fluid in the output channel.
3. The preparation method according to claim 2, wherein the inner phase fluid microchannel has an inner diameter of 10 to 500 μm, and the outer phase fluid microchannel has an inner diameter of 20 to 1000 μm.
4. The method according to claim 2, wherein one end of the inner phase fluid microchannel is non-sealedly inserted into one end of the outer phase fluid microchannel, and one end of the output channel is sealingly inserted into the outer phase fluid microchannel The other end is non-end-to-end connected to the port of the internal phase fluid microchannel inserted in the outer phase fluid microchannel, the additional phase inflow channel being in communication with the output channel not interposed in the outer phase fluid microchannel.
5. The preparation method according to claim 4, wherein in the outer phase fluid microchannel, the distance between the port of the inner phase fluid microchannel and the output channel port is 50 to 500 μm.
6. The preparation method according to claim 2, wherein the microfluidic chip device comprises two external phase fluid microchannels, and the two outer phase fluid microchannels are respectively connected to the output channel to form a Y-shaped channel, intersecting The center of the connection is intersected with an internal phase fluid microchannel, the centerline of the internal phase fluid microchannel coincides with the centerline of the output channel, and the two external phase fluid microchannels are completely symmetrically positioned in the inner phase fluid microchannel. The angle between the side, outer phase fluid microchannel and the inner phase fluid microchannel is 30 to 90°.
7. The preparation method according to claim 1, wherein the gelatin aqueous solution in the step (1) has a gelatin concentration of 0.1 to 12 w/v%, and the gelatin aqueous solution has a pH of 1 to 5 or 9. ~12.
8. The method according to claim 1, wherein the temperature of each fluid in the microfluidic chip device is maintained at 30 to 60 °C.
9. The preparation method according to claim 1, wherein the polar organic solvent is one or a combination of methanol, ethanol, isopropanol, butanol, acetone, acetonitrile or tetrahydrofuran. The crosslinking agent is one or more of glutaraldehyde, glyceraldehyde, formaldehyde, carbodiimide, dihaloalkane, isocyanate, diisocyanate, glutamine transaminase, and genipin.
10. The preparation method according to claim 1, wherein the first flow rate, the second flow rate, and the third flow rate are 0.05 to 10 mL hr ^-1 , 0.1 to 50 mL hr ^{-1 ,} and 0.05 to 500 μL hr ^{-1 , respectively.} .
11. The preparation method according to claim 1, wherein the ratio of the second flow rate to the first flow rate is 1.0 to 9.0; and the ratio of the third flow rate to the first flow rate is 0.0067 to 0.067.
12. The method according to claim 1, wherein the molar ratio of the crosslinking agent to the gelatin amino group is 0.25 to 10.0.
13. An injectable, self-healing colloidal gel, characterized in that the colloidal gel is a lyophilized powder of a gelatin nanoparticle prepared by the method according to any one of claims 1 to 12 and an aqueous solution directly It is obtained by blending; in the prepared colloidal gel, the volume percentage of the gelatin nanoparticles is 30% to 150%.
14. The colloidal gel according to claim 13, wherein the aqueous solution is an aqueous solution in which cells are suspended or an aqueous solution in which bioactive molecules are dissolved.
15. Use of the colloidal gel of claim 13 in the preparation of an implantable filler material for tissue repair and treatment.

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