阅读说明：本技术 一种包覆细胞的磁性微凝胶及其制备方法与应用 (Cell-coated magnetic microgel and preparation method and application thereof ) 是由 王秀瑜 楼琦 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种包覆细胞的磁性微凝胶及其制备方法与应用。它的制备方法,包括如下步骤：1)将细胞与氧化铁纳米颗粒子在细胞培养液中进行共培养,得到表面粘附磁性纳米颗粒的细胞；2)将所述表面粘附磁性纳米颗粒的细胞与藻酸盐水溶液混合,形成分散相；通过PDMS微流控设备,用连续相对所述分散相进行切割,形成微液滴；在所述PDMS微流控设备中,所述微液滴中释放Fe～(3+)和藻酸盐水溶液进行交联反应,即得到包覆细胞的磁性微凝胶。本发明制备方法可控,制得的产品具有超顺磁性,具有T1/T2双模态MRI医疗成像能力,成像更为清晰。(The invention discloses a cell-coated magnetic microgel and a preparation method and application thereof. The preparation method comprises the following steps: 1) co-culturing the cells and the iron oxide nanoparticles in a cell culture solution to obtain cells with magnetic nanoparticles adhered to the surfaces; 2) mixing the cells with the magnetic nanoparticles adhered on the surfaces with an alginate aqueous solution to form a dispersed phase; cutting the dispersed phase continuously through PDMS microfluidic equipment to form micro droplets; in the PDMS microfluidic device, Fe is released in the micro-droplets 3+ And carrying out crosslinking reaction with an alginate aqueous solution to obtain the cell-coated magnetic microgel. The preparation method is controllable, and the prepared product has superparamagnetism, T1/T2 bimodal MRI medical imaging capability and clearer imaging.)

1. A preparation method of a cell-coated magnetic microgel comprises the following steps: 1) co-culturing the cells and the iron oxide nanoparticles in a cell culture solution to obtain cells with magnetic nanoparticles adhered to the surfaces;

2) mixing the cells with the magnetic nanoparticles adhered to the surfaces with an alginate aqueous solution to obtain a dispersed phase; cutting the dispersed phase continuously through PDMS microfluidic equipment to form micro droplets; in the PDMS microfluidic device, Fe is released in the micro-droplets³⁺And carrying out crosslinking reaction with an alginate aqueous solution to obtain the cell-coated magnetic microgel.

2. The method of claim 1, wherein: the iron oxide nanoparticles are selected from FePt @ Fe₃O₄Core/shell magnetic nanoparticles;

the co-culture temperature is 4 ℃ and the co-culture time is 1-6 hours.

3. The production method according to claim 1 or 2, characterized in that: the feeding speed of the dispersed phase is 50-60 mu L/min;

the concentration of the iron oxide nanoparticles in the dispersed phase is 20-80 mug/mL;

said Fe³⁺The mass ratio of the alginate to the alginate is 1/250-1/1000;

the alginate is at least one selected from alginic acid, potassium alginate and sodium alginate.

4. The production method according to any one of claims 1 to 3, characterized in that: the feed flow rate ratio of the continuous phase to the dispersed phase is 1/3-1/4.

5. The production method according to any one of claims 1 to 4, characterized in that: the continuous phase is formed by adding a surfactant and acetic acid into fluorinated silicone oil; wherein the acetic acid accounts for 0.05-5% of the total volume of the continuous phase, the surfactant accounts for 0.5-1.5% of the total mass of the continuous phase, and the balance is the fluorinated silicone oil;

the surfactant is selected from at least one of PEG, Span80, Span60, Span20, Span40, Tween85 and PVA.

6. The production method according to any one of claims 1 to 5, characterized in that: the PDMS microfluidic device is formed by connecting a Y-shaped pipeline and a snake-shaped pipeline through a tapered pipeline;

the pipe diameter of the inlet end to the outlet end of the gradually-reduced pipeline is gradually reduced, the pipe diameter of the inlet end of the gradually-reduced pipeline is equal to that of the tail end of the Y-shaped pipeline, and the pipe diameter reduction ratio of the inlet end to the outlet end of the gradually-reduced pipeline is 3/4-3/5.

7. The method of claim 6, wherein: the snakelike pipeline is formed by a plurality of sections of U-shaped pipelines;

the number of the U-shaped pipelines is specifically 3-4 sections;

the middle part of the reducing pipeline is provided with a secondary annular magnetic field generator, and the rotation direction of the magnetic field of the secondary annular magnetic field generator is the same as that of the reducing pipeline.

8. The cell-coated magnetic microgel prepared by the preparation method of any one of claims 1 to 7.

9. Use of the cell-coated magnetic microgel of claim 8 in preparing a cell pretreatment product for cell therapy and diagnosis having any one of the following functions 1) to 3);

1) bimodal medical imaging;

2) controlled release of therapeutic cells;

3) under an external magnetic field, the microgel containing cells is magnetically navigated.

10. A PDMS microfluidic device as claimed in claim 6 or 7.

Technical Field

The invention relates to a cell-coated magnetic microgel and a preparation method and application thereof, belonging to the field of biomedical materials.

Background

At present, cell membrane damage caused by shearing force can be caused by cells in the injection process, or cell necrosis caused by high shearing force continuously applied to complex fluid flow in vivo or cell lysis and apoptosis caused by host immune cells, and at present, a mode of generating microgel on the cell surface through chemical reaction by micron-sized alginate particles is often adopted to encapsulate living cells so as to form a protective film to protect the cells. Ideally, the microgel particles should be composed of a uniform network structure so that the encapsulated cells can be stably encapsulated in a controlled microenvironment. Because the monodispersity, size, morphology and microstructure of the microgel have important influence on the properties and functions of cells, the size, shape and form of the alginate microgel can be accurately controlled by utilizing the strong production and control capability of micro-droplets of the droplet microfluidics technology. The currently known techniques for alginate microgel coating of cells include four methods:

firstly, the sodium alginate solution is emulsified into small sodium alginate water drops by the oil phase and is mixed with Ca in calcium chloride²⁺Ion crosslinking to form alginate microgel;

dispersing water-insoluble calcium carbonate nanoparticles in a sodium alginate solution, cutting the mixed solution of the calcium carbonate nanoparticles and sodium alginate to form micro-droplets, and dissolving calcium carbonate under an acidic condition to generate Ca²⁺Ions are crosslinked with the sodium alginate solution to form alginate microgel;

conveying calcium chlorate or acetic acid particles through an oil phase, uniformly mixing the calcium chlorate or acetic acid particles with a sodium alginate solution, and crosslinking to form alginate microgel;

fourthly, calcium ions are conveyed in the form of water-soluble ethylene diamine tetraacetic acid disodium calcium (EDTA-disodium calcium for short, Ca-EDTA), the emulsified alginate and the compound are uniformly mixed, and the mixture is cut by carbon fluoride oil (dissolved with acetic acid) to form liquid drops, and then the liquid drops are dissociated by Ca-EDTA under acidic conditions to release Ca²⁺And ions are crosslinked with the sodium alginate solution to form alginate microgel.

However, these alginate microgel coated techniques suffer from the following disadvantages:

1. in the first method, calcium ions can immediately contact with alginate chains in the ionic crosslinking process, so that the crosslinking reaction occurs before the calcium ions are uniformly distributed, and the microgel has non-uniform thickness; meanwhile, the rapid reaction of the calcium ions and the alginate chains generally causes uncontrolled gelation in the microfluidic device, and blocks the microfluidic pipeline;

2. in the second method, due to the water insolubility of calcium carbonate nano particles, calcium ions are unevenly distributed in the liquid drops after calcium carbonate is dissolved by acid, so that a uniform gel process is generated in the liquid drops; meanwhile, when the particles are gathered, the microfluid blocks a microfluidic channel, so that the available size range of the microgel is limited;

3. in the third method, calcium chlorate or acetic acid particles are conveyed by the oil phase, and crosslinking occurs before calcium ions are uniformly distributed, so that the uniformity of the generated microgel is reduced;

4. method four, while solving the problem of uniform distribution of calcium ions, Ca-alginate microgels in vivo deliver therapeutic cells primarily through mechanisms involving diffusion and migration, resulting in the trapping of most of the intravenously delivered cells in the lung and reticuloendothelial system, with up to 60% of the cells not even reaching the target site; the cell encapsulation rate of the Ca-alginate microgel is low, usually 20-30%, so that a large amount of acellular microgel is generated, and the treatment effect is seriously influenced; finally, none of the above methods is an effective in vivo imaging technique for real-time tracking of cells, resulting in non-invasive monitoring and long-term assessment of therapeutic efficacy.

Disclosure of Invention

The invention aims to provide a cell-coated magnetic microgel and a preparation method and application thereof.

The preparation method is controllable, and the prepared product has superparamagnetism, T1/T2 bimodal MRI medical imaging capability and clearer imaging.

The invention provides a preparation method of a cell-coated magnetic microgel, which comprises the following steps: 1) co-culturing the cells and the iron oxide nanoparticles in a cell culture solution to obtain cells with magnetic nanoparticles adhered to the surfaces;

2) mixing the cells with the magnetic nanoparticles adhered on the surfaces with an alginate aqueous solution to form a dispersed phase; cutting the dispersed phase continuously through PDMS microfluidic equipment to form micro droplets; in the PDMS microfluidic device, Fe is released in the micro-droplets³⁺And carrying out crosslinking reaction with an alginate aqueous solution to obtain the cell-coated magnetic microgel.

In the present invention, the cells are all conventional therapeutic cells in the art.

In the preparation method, the iron oxide nanoparticles are selected from FePt @ Fe₃O₄Core/shell magnetic nanoparticles.

In the invention, the FePt @ Fe₃O₄The specific preparation method of the core/shell magnetic nanoparticle comprises the following steps:

mixing acetylacetone platinum, 1, 2-hexadecanediol and 1-octadecene, and adding oleic acid, oleylamine and acetylacetone iron in the inert atmosphere to obtain a mixture 1; heating the prepared mixture to reflux, cooling the mixture to room temperature (25 ℃), centrifuging to obtain a precipitate, and dispersing the product into cyclohexane; mixing 3, 4-dihydroxycinnamic acid (DHCA, pH 3-12) and Tetrahydrofuran (THF), and heating to 50 ℃ to obtain a mixture 2; then, dropwise adding a BMNP (magnetic nanoparticle)/THF solution into the mixture 2 for reaction, and cooling to room temperature; adding NaOH solution for centrifugation, and taking black precipitate to obtain the FePt @ Fe₃O₄The core/shell magnetic nano-particles and the black precipitate are dissolved in deionized water and are placed in a refrigerator at 4 ℃ for standby.

In the preparation method, the co-culture temperature can be 4 ℃ and the time can be 1-6 hours.

In the invention, in the step 1), the usage amount of the iron oxide nanoparticles is the conventional usage amount in the field, namely, the usage amount is within the toxicity range; in a specific embodiment, the concentration of the iron oxide nanoparticles can be 20-40 μ g/mL, and the concentration is 250 μ g/mL, so that toxicity is generated.

In the above preparation method, the feeding speed of the dispersed phase may be 50 to 60 μ L/min, specifically 50 μ L/min or 50 to 55 μ L/min.

In the above preparation method, the concentration of the iron oxide nanoparticles in the dispersed phase may be 10 to 20 μ g/mL.

In the above production method, the Fe³⁺The mass ratio of the alginate to the alginate is 1/250-1/1000;

the alginate is at least one selected from alginic acid, potassium alginate and sodium alginate.

In the preparation method, the feed flow rate ratio of the continuous phase to the dispersed phase can be 1/3-1/4.

In the preparation method, the continuous phase is formed by adding a surfactant and acetic acid into fluorinated silicone oil; wherein the volume percentage of the acetic acid in the total amount of the continuous phase can be 0.05-5%, specifically 1%, 0.05-1%, 1-5% or 0.5-2.5%, the mass percentage of the surfactant in the total amount of the continuous phase can be 0.5-1.5%, specifically 1%, 1-1.5%, 0.5-1% or 0.75-1.25%, and the balance is fluorinated silicone oil;

the surfactant is selected from at least one of PEG, Span80, Span60, Span20, Span40, Tween85 and PVA.

In the invention, the PEG is commonly used in the field and has a molecular weight of 200-20000, and specifically can be PEG-200, PEG-400, PEG-600, PEG-800, PEG-1000, PEG-1500, PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG-10000 or PEG-20000.

In the preparation method, the PDMS microfluidic device is formed by connecting a Y-shaped pipeline and a snake-shaped pipeline through a tapered pipeline;

the pipe diameter of the inlet end to the outlet end of the gradually-reduced pipeline is gradually reduced, the pipe diameter of the inlet end of the gradually-reduced pipeline is equal to that of the tail end of the Y-shaped pipeline, and the pipe diameter reduction ratio of the inlet end to the outlet end of the gradually-reduced pipeline is 3/4-3/5.

In the preparation method, the serpentine pipeline is formed by a plurality of sections of U-shaped pipelines;

the number of the U-shaped pipelines is specifically 3-4 sections;

the middle part of the reducing pipeline is provided with a secondary annular magnetic field generator, and the rotation direction of the magnetic field of the secondary annular magnetic field generator is the same as that of the reducing pipeline.

The invention also provides the cell-coated magnetic microgel prepared by the preparation method.

The cell-coated magnetic microgel is applied to preparation of cell pretreatment products for cell therapy and diagnosis with any one of the following functions 1) to 3);

1) bimodal medical imaging (i.e., T1 and/or T2 imaging for MRI);

2) controlled release of therapeutic cells;

3) under an external magnetic field, the microgel containing cells is magnetically navigated.

The invention further provides the PDMS microfluidic device.

The PDMS microfluidic device is formed by connecting a Y-shaped pipeline and a snake-shaped pipeline through a tapered pipeline;

the pipe diameter of the inlet end to the outlet end of the gradually-reduced pipeline is gradually reduced, the pipe diameter of the inlet end of the gradually-reduced pipeline is equal to that of the tail end of the Y-shaped pipeline, and the pipe diameter reduction ratio of the inlet end to the outlet end of the gradually-reduced pipeline is 3/4-3/5.

Furthermore, the snakelike pipeline is formed by a plurality of sections of U-shaped pipelines;

the number of the U-shaped pipelines is specifically 3-4 sections;

the middle part of the reducing pipeline is provided with a secondary annular magnetic field generator, and the rotation direction of the magnetic field of the secondary annular magnetic field generator is the same as that of the reducing pipeline.

The invention has the following advantages:

(1) FePt @ Fe used₃O₄The core/shell magnetic nanoparticles are modified on the surface by PEG, have hydrophilicity and better biocompatibility with cells, can be uniformly distributed in an alginate aqueous solution, are favorable for forming a uniform mixture of alginate and the core/shell magnetic nanoparticles, and prevent accidental gel;

(2) by using FePt @ Fe₃O₄The core/shell magnetic nanoparticles are beneficial to increasing the probability of microgel carrying cells, and the probability is increased from 10-20% of the original calcium ions to 80-90%;

(3) when acetic acid releases H⁺FePt @ Fe when the pH value is made acidic₃O₄Decomposition of core/shell magnetic nanoparticles to Fe³⁺Uniformly separated out due to Fe³⁺The alginate cross-linking agent is generated on the surface of cells, and is beneficial to controlling the alginate cross-linking process from inside to outside;

(4) the micro-fluidic pipeline formed by multiple sections of U-shaped pipes is designed by adopting a snake-shaped structure, so that the produced Fe-alginate microgel shows a perfect spherical shape, the average diameter is 61 mu m, the microgel is easy to fuse with host tissues, and the transfer of cells from the microgel to the tissues is promoted.

(5)Fe³⁺Has a ligand number of 6 in comparison with Ca²⁺The number of the ligands (2) is more significant, and thus the gelation efficiency is more significant;

(6) the thickness of the hydrogel layer can be changed by changing sodium alginate/Fe³⁺The proportion of the microgel is easily adjusted, and the anti-shearing effect of the microgel can be effectively improved by controlling the thickness of gelation;

(7) residual FePt @ Fe on cells and microgels₃O₄The core/shell magnetic nanoparticles have superparamagnetism, so that the performance of no agglomeration of the microgel group in navigation in the blood vessel and/or tissue gap of the system is ensured, and meanwhile, the microgel group can be magnetically driven by an external magnetic field to guide the transmission of cells;

(8) superparamagnetic BMNPs (with high saturation magnetization) and Fe in the presence of Fe-alginate microgels³⁺Ionic paramagnetism has been engineered into application, with T1/T2 bimodal MRI medical imaging capability, imaging is clearer.

Drawings

FIG. 1 is a schematic diagram of the operation process and the device structure of the present invention.

Fig. 2 is a schematic structural diagram of a PDMS microfluidic device according to the present invention.

FIG. 3 is a schematic diagram of the gel reaction of alginate in the present invention.

1 cell culture fluid, 2 FePt @ Fe₃O₄Core/shell magnetic nanoparticles, 3 alginate, 4 dispersed phase, 5 continuous phase, 6PDMS microfluidic device, 601Y-channel, 602 serpentine channel, 603 tapered channel, 7 microdroplets, 701 alginate liquid film, 702H generated by acetic acid ionization⁺，703 Fe³⁺704 Fe-alginate crosslinked microgel, 8 magnetic microgel coating cells.

FIG. 4 is a graph of cell coverage; wherein FIG. 4(a) is CaCO₃Coating the nano particles; FIG. 4(b) shows Fe₃O₄And (4) coating the nano particles.

FIG. 5 is an image of T1/T2 in example 1 of the present invention; wherein fig. 5(a) is an imaging diagram of T1; fig. 5(b) is an imaging diagram of T2.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, sodium alginate was used as alginate.

In the following examples, FePt @ Fe₃O₄The preparation method of the core/shell magnetic nanoparticle specifically comprises the following steps: 1. platinum acetylacetonate (0.5mmol, alatin), 1, 2-hexadecanediol (1.5mmol) and 20mL 1-octadecene were combined in a three-necked flask and magnetically stirred under a nitrogen stream (1600rpm) to provide mixture 1; heating the mixture 1 to 80 ℃, and heating at 80 ℃ for 30 min; then oleic acid (0.5mmol), oleylamine (0.5mmol) and iron acetylacetonate (3mmol, alatin) were added to the flask under nitrogen protection; heating the prepared mixture to 300 ℃, refluxing for 30min, cooling the mixture to room temperature (25 ℃), washing for 3 times by using ethanol, centrifuging (6000rpm) for 10min, taking the centrifuged precipitate to obtain a product, and finally re-dispersing the product into cyclohexane for later use;

2. aqueous phase transfer of hydrophobic BMNPs: 50mg of 3, 4-dihydroxycinnamic acid (DHCA, Sigma-Aldrich) and 6ml of Tetrahydrofuran (THF) were magnetically stirred (1600rpm) and heated to 50 ℃ to give mixture 2; then magnetic Fe₃O₄Dropwise adding a nano particle (20mg)/THF (1mL) solution into the mixture 2, reacting for 3h after dropping, cooling to room temperature, adding into a 500 mu L centrifuge tube, adding 0.5mol NaOH solution, centrifuging at 3000rpm for 10min, and taking black precipitate to obtain FePt @ Fe₃O₄The core/shell magnetic nano-particles and the black precipitate are dissolved in deionized water and are placed in a refrigerator at 4 ℃ for standby.

As shown in fig. 1, a schematic diagram of a cell-coated magnetic microgel, a preparation method thereof, and an application apparatus thereof is shown.

Comprises cell culture solution 1, FePt @ Fe₃O₄Core/shell magnetic nanoparticles 2, alginate 3, dispersed phase 4, continuous phase 5 and PDMS microfluidic device 6.

Further, the continuous phase is formed by adding a surfactant and acetic acid which is easily dissolved in polar and non-polar solvents to fluorinated silicone oil, for FePt @ Fe₃O₄Fe of core/shell magnetic nanoparticles₃O₄Partial dissociation of the shell and release of the precise Fe³⁺Iron ions are essential for gelation of the micro-sized droplets.

Further, FePt @ Fe₃O₄The concentration of the core/shell magnetic nanoparticles is preferably 20-80 mu gmL^-1Selecting the concentration of 20 mu gmL^-1。

Further, Fe³⁺The mass ratio of the alginate to the alginate is preferably 1/250-1/1000, and the mass ratio of the alginate to the alginate is 1/250, so that the thickness of the microgel can be effectively controlled, and the magnetic microgel which is perfectly spherical and coats cells can be formed.

Further, the feeding speed of the dispersed phase is 50-60 mu L/min; the feed rate was selected to be 50. mu.L/min.

Further, the continuous phase is formed by adding a surfactant and acetic acid into fluorinated silicone oil; wherein the acetic acid accounts for 0.05-5% of the total volume, the surfactant accounts for 0.5-1.5% of the total mass, and further 1%;

the surfactant is at least one selected from PEG, Span80, Span60, Span20, Span40, Tween85 and PVA.

Furthermore, the feeding flow rate ratio of the continuous phase to the dispersed phase is preferably 1/3-1/4, and the feeding flow rate ratio is preferably 1/3, so that uniform alginate droplets are formed.

As shown in fig. 2, the structure of the PDMS microfluidic device is schematically illustrated.

The PDMS microfluidic device 6 consists of a Y-shaped pipe 601, a serpentine pipe 602, and a tapered pipe 603.

Further, the pipe diameter of the tapered pipeline 603 is gradually reduced, the pipe diameter of the inlet end of the tapered pipeline 603 is equal to that of the tail end of the Y-shaped pipeline 601, the pipe diameter reduction ratio from the pipe diameter of the inlet end to the pipe diameter of the outlet end of the tapered pipeline 603 is selected to be 3/4-3/5, and the pipe diameter reduction ratio 3/4 is selected, so that the blockage of the PDMS microfluidic device 6 channel due to the reduction of the volume of liquid drops is prevented.

Taking submicron-sized magnetic microgel as an example, the working process of the invention is as follows:

FePt @ Fe₃O₄Adding the core/shell magnetic nanoparticles into a cell culture solution to precoat cells, and fully dispersing FePt @ Fe by using an alginate aqueous solution₃O₄The cells adsorbed by the core/shell magnetic nano particles are fully stirred and mixed to form a dispersed phase, and the cells are covered with an alginate liquid film.

Murine bone Marrow Stromal Cells (MSC) were grown in Dulbecco's Modified Eagle Medium Medium (DMEM, Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, SAFC Biosciences, Lenexa, KS) and 1% (v/v) penicillin/streptomycin. Cells were split every 2 days under sterile conditions and at 37 ℃ and 5% CO₂And (4) incubating.

The flow rates of the continuous phase and the dispersed phase are adjusted through an injection pump, micro droplets with uniform size are generated in a Y-shaped pipeline 601 by cutting the dispersed phase through the continuous phase, and the micro droplets comprise FePt @ Fe adhered to the surface₃O₄Cells of core/shell magnetic nanoparticles, alginate, and aqueous solutions of cells.

H produced by ionization of acetic acid in serpentine 602 and tapered 603 channels⁺702 diffused into alginate coated cells, with FePt @ Fe₃O₄Fe of core/shell magnetic nanoparticle 2₃O₄Shell reaction, released Fe³⁺703 cross-linking with the alginate liquid film 701 to form Fe-alginate cross-linked microgel 704, and passing through the serpentine pipe 602 and the tapered pipe 603 to form magnetic microgel 8 for coating cells.

After gelation, 20% by volume of PFO (poly (9, 9-dioctylfluorene-2, 7-diyl) was added to the perfluorocarbon oil, the microgel was transferred to an aqueous medium and then centrifuged (1000rpm, 1 minute). The resulting oil phase is discarded and the resulting iron microgel is resuspended in cell culture medium.

After the preparation is completed, the magnetic microgel coated with cells is injected from the tail artery. By using an in vitro magnetic field, the magnetic microgel coated with cells is remotely controlled to navigate to a target area with high precision in complex biological fluids, and the minimal minimally invasive way to deliver the cells is realized.

In order to accomplish navigation in a complex biological environment through feedback control, the motion of the microgel needs to be tracked and monitored in real time in vivo, and noninvasive modality imaging is an effective means for this. Since FePt @ Fe₃O₄The core/shell magnetic nanoparticles have superparamagnetism and high saturation magnetization, and simultaneously Fe³⁺The magnetic microgel coated with the cells has paramagnetism, so that the magnetic microgel coated with the cells has the capacity of double T1/T2 MRI imaging, and stronger and clearer MRI images are finally obtained.

Example 1

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase is formed by adding acetic acid and PEG800 into fluorinated silicone oil, wherein the volume percentage of the acetic acid in the total amount of the continuous phase is 1% (1.575 mug/min), and the mass percentage of the PEG800 in the total amount of the continuous phase is 1% (0.153mg/min)

(3) The pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

20. mu.g/ml of FePt @ Fe₃O₄The core/shell magnetic nanoparticles are added to the cell culture solution, then 20 wt% of alginate aqueous solution is added to the cell culture solution 1, and the dispersed phase 4 is formed by fully stirring and mixing. The flow rates of the continuous and dispersed phases are adjusted by a syringe pump, and micro-droplets are generated in the Y-tubing 601 by cutting the dispersed phase through the continuous phase, the micro-droplets comprising an aqueous solution of alginate and cells. H produced by ionization of acetic acid in serpentine and tapered tubes 603⁺702 diffused into the alginate-coated cells,with FePt @ Fe₃O₄Fe of core/shell magnetic nanoparticle 2₃O₄Shell reaction, released Fe³⁺703 cross-linking with the alginate liquid film 701 to form Fe-alginate cross-linked microgel 704, and passing through the serpentine pipe 602 and the tapered pipe 603 to form magnetic microgel 8 for coating cells.

After the preparation is completed, the magnetic microgel coated with cells is injected from the tail artery. By using an in vitro magnetic field, the motion track of the magnetic microgel coated with the cells is remotely controlled, so that the magnetic microgel can be navigated to a target area in complex biological fluid with high precision, and the cells can be delivered in a minimally invasive manner.

After 10min, the PDMS microfluidic device is adjusted on site, and reaches a stable working state, the encapsulation rate of cells in the microgel is 90% when the device is stable, the size range of the magnetic microgel coating the cells is 50-70 μm, and the average diameter is 61 μm. The magnetic microgel coated with cells is injected from the old rat tail artery, so that the droplet movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

Example 2

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/1000 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The rest of the devices and conditions are the same as those in example 1, the cell-coated magnetic microgel is prepared in the manner of example 1, and after being debugged in the field, the PDMS microfluidic device reaches a stable working state after 10min, the encapsulation rate of cells in the microgel is 80% when being stable, the size range of the cell-coated magnetic microgel is 45-60 μm, and the average diameter is 52 μm. The cell-coated magnetic microgel is injected from the old rat tail artery, so that the microgel movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

Example 3

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/4; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The rest of the devices and conditions are the same as those in example 1, the cell-coated magnetic microgel is prepared in the manner of example 1, and after being debugged on site, the PDMS microfluidic device reaches a stable working state after 15min, the encapsulation rate of cells in the microgel is 88% when being stable, the size range of the cell-coated magnetic microgel is 48-65 μm, and the average diameter is 58 μm. The cell-coated magnetic microgel is injected from the old rat tail artery, so that the microgel movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

Example 4

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/5;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The rest of the devices and conditions were the same as those of example 1, the cell-coated magnetic microgel 8 was prepared in the same manner as in example 1, and after 10min from field commissioning, the PDMS microfluidic device reached a stable operating state, the encapsulation rate of cells in the microgel was 90% at the time of stabilization, the size range of the cell-coated magnetic microgel was 47 to 63 μm, and the average diameter was 55 μm. The cell-coated magnetic microgel is injected from the old rat tail artery, so that the microgel movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

Example 5

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 4U-shaped pipe sections.

The rest devices and conditions are the same as those of the example 1, the cell-coated magnetic microgel is prepared in the way of the example 1, and after being debugged on site, the PDMS microfluidic device reaches a stable working state after 10min, the encapsulation rate of cells in the microgel is 90% when being stable, the size range of the cell-coated magnetic microgel is 55-70 μm, and the average diameter is 62 μm. The cell-coated magnetic microgel is injected from the old rat tail artery, so that the microgel movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

Comparative example 1

The experimental conditions are as follows:

(1) ca in calcium disodium ethylenediamine tetraacetate²⁺The mass ratio to alginate 3 was 1/250;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

Adding 20 mu g/ml disodium calcium ethylene diamine tetraacetate into the cell culture solution 1, adding 20 wt% alginate water solution 3 into the cell culture solution 1, and fully stirring and mixing to form a dispersed phase 4. The flow rates of the continuous phase 5 and the disperse phase 4 are adjusted through the injection pump, the disperse phase 4 is cut through the continuous phase 5, and micro droplets are generated in the Y-shaped pipeline 601, and the micro droplets contain alginate and the aqueous solution of the cells 7. H produced by ionization of acetic acid in serpentine 602 and tapered 603 channels⁺702 diffusing into the micro-droplets, and dissociating the Ca released by the disodium calcium ethylene diamine tetraacetate²⁺Cross-linking with alginate 3 to finally produce the magnetic microgel coating cells.

After 10min of field debugging, the PDMS microfluidic device reaches a stable working state, the encapsulation rate of cells in the microgel is 30% when the device is stable, the size range of the magnetic microgel coating the cells is 78-90 μm, and the average diameter is 85 μm. Injection of cell-coated magnetic microgel from old rat tail artery failed to remotely control droplet transport and dual MRI imaging of T1/T2.

Comparative example 2

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/100 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The rest of the devices and conditions are the same as those in example 1, the cell-coated magnetic microgel is prepared in the manner of example 1, and after 10min of field debugging, the PDMS microfluidic device reaches a stable working state, the encapsulation rate of cells in the microgel is 91% during stabilization, but the overall size of the microgel becomes large, the size range of the cell-coated magnetic microgel is 70-90 μm, the average diameter is 83 μm, and the cell treatment is not facilitated. The magnetic gel pack for injecting micro-coated cells from the old rat tail artery can remotely control the motion of the gel pack and realize the dual MRI imaging of T1/T2.

Comparative example 3

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/5; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is gradually reduced compared with the pipe diameter before, and the pipe diameter of the U-shaped pipe is 120 mu m, and the reduction ratio is 3/4;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The remaining apparatus and conditions were the same as in example 1, and the fabrication of magnetic droplets of microgel coated cells was performed in the same manner as in example 1, and the PDMS microfluidic device could not be brought to a stable operating state by field commissioning.

Comparative example 4

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline 603 is the same as that before, and the pipe diameter of the U-shaped pipe is 120 mu m;

(4) the serpentine pipe 602 is designed using 3U-shaped pipe sections.

The rest of the devices and conditions are the same as those of the example 1, the magnetic droplet preparation of the microgel coated cells is carried out according to the method of the example 1, and after 10min, the PDMS microfluidic device can not reach a stable working state and the phenomenon of droplet overlapping can occur occasionally, so that the shape of part of the droplets is deformed from a sphere to an olive sphere. The encapsulation rate of the cells in the microgel is 87%, the size range of the magnetic microgel for coating the cells is 59-75 μm, and the average diameter is 65 μm. Injection of microgel coated cell magnetic droplets 8 from old rat tail arteries allowed remote control of droplet motion, but the T1/T2 dual MRI imaging was unclear.

Comparative example 5

The experimental conditions are as follows:

(1)[email protected]₃O₄fe in core/shell magnetic nanoparticles³⁺1/250 mass ratio to alginate;

(2) the flow rate of the dispersed phase was 50. mu.L/min, and the flow rate ratio of the continuous phase and the dispersed phase was 1/3; the continuous phase was the same as in inventive example 1;

(3) the pipe diameter of the tapered pipeline (603) is gradually reduced compared with the pipe diameter of the prior tapered pipeline, the pipe diameter of the U-shaped pipe is 120 mu m, and the pipe diameter reduction proportion is 3/4;

(4) the serpentine pipe 602 is designed using 6U-shaped pipe sections.

The rest of the devices and conditions are the same as those in example 1, the magnetic droplet of the microgel coated cells is manufactured in the manner of example 1, and after 10min from field debugging, the PDMS microfluidic device reaches a stable working state, the encapsulation rate of the cells in the microgel is 91% when being stable, the size range of the magnetic droplet of the microgel coated cells is 55-72 μm, and the average diameter is 65 μm. The magnetic liquid drop of the microgel coated cells is injected from the old rat tail artery, the liquid drop movement can be remotely controlled, and the T1/T2 dual MRI imaging can be realized.

The above experimental results show that the increase of the number of U-shaped tubes in comparative example 5 relative to example 1 of the present invention has little effect on cell coating, because the number of U-shaped tubes reaches 3 to 4, and the cell encapsulation rate reaches 90%. However, the number of the U-shaped tubes is more, the encapsulation efficiency is only improved by 1%, but the movement distance of the cells is doubled, so that the encapsulation time is increased, and therefore, in order to improve the cell encapsulation efficiency of the microfluidic device, the number of the U-shaped tubes is preferably 3-4.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.