CN112472685A

CN112472685A - Preparation method of hybrid neutrophil granulocytes robot

Info

Publication number: CN112472685A
Application number: CN202011434838.3A
Authority: CN
Inventors: 吴志光; 张红玥; 贺强; 李泽生
Original assignee: Harbin Institute of Technology
Current assignee: Harbin Institute of Technology
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-03-12
Anticipated expiration: 2040-12-10
Also published as: CN112472685B

Abstract

A preparation method of a hybrid neutrophil robot relates to the technical field of micro-nano robots. The invention aims to construct a hybrid neutrophil robot which has both biomass activity and a function of a synthetic material. The method comprises the following steps: obtaining neutrophils; preparing medicament-carrying nano gel particles doped with ferroferric oxide magnetic nano particles with outer membranes disguised of escherichia coli by adopting an emulsification evaporation and nano extrusion method; co-culturing the neutrophil obtained in the first step and the nano gel particles obtained in the second step, and inducing the neutrophil to phagocytose the nano gel particles to obtain the hybrid neutrophil robot. The hybrid neutrophil robot is still a living cell, can perform directional chemotactic movement under the stimulation of substances such as cytokines, inflammatory factors, chemotactic peptides and the like, has good biocompatibility, can cross blood brain barriers, can avoid immune clearance, can survive well in vivo, has good biomedical application prospect, and is applied to the field of medicine.

Description

Preparation method of hybrid neutrophil granulocytes robot

Technical Field

The invention relates to the technical field of micro-nano robots, in particular to a preparation method of a hybrid neutrophil granulocyte robot.

Background

Neutrophils, which are the most abundant leukocytes in the body, are part of the innate immunity and are first recruited in the innate immune response process to play a role in eliminating pathogens, phagocytosing foreign bodies, and the like. The neutrophilic granulocyte derived from organisms is combined with the synthetic material nano particles to construct a biological hybrid robot which has good biocompatibility and is not easy to be eliminated by immunity, and the biological hybrid robot can be abundantly applied to in-vivo treatment scenes such as in-vivo targeted drug delivery, crossing of special biological barriers and the like. However, at present, a nano robot is mostly prepared by using nano magnetic particles, and drug sites or markers and the like are attached to the surface of the nano robot, or DNA fragments and the like are combined with the magnetic particles to prepare the nano robot, and no report of combination of neutrophils and the nano magnetic particles is adopted.

Disclosure of Invention

The invention aims to construct a hybrid neutrophil robot which has both biomass activity and a function of a synthetic material. And provides a method for preparing the hybrid neutrophil robot.

The invention relates to a preparation method of a hybrid neutrophil robot, which is carried out according to the following steps:

step one, obtaining neutrophils;

step two, preparing the drug-loaded nano gel particles doped with the ferroferric oxide magnetic nano particles with outer membranes disguised of escherichia coli by adopting an emulsification evaporation and nano extrusion method;

and step three, co-culturing the neutrophils obtained in the step one and the nano gel particles obtained in the step two, inducing the neutrophils to phagocytose the nano gel particles, and preparing the hybrid neutrophil robot.

Further, the obtaining of neutrophils in the step one is separated from freshly isolated mouse bone marrow.

Further, the neutrophils are obtained by the following method:

1) cutting off two ends of a hind femur, a tibia or an forelimb humerus of a mouse, flushing a bone inner cavity for multiple times by using an RPMI1640 culture medium until the bone is white and semitransparent, collecting flushing liquid of the RPMI1640 culture medium, centrifuging, collecting a solid phase substance, and suspending the solid phase substance in a phosphate buffer solution to obtain a cell suspension;

2) adding the Percoll separating medium with the volume percentage of 71 percent and the Percoll separating medium with the volume percentage of 61 percent into a centrifugal tube in sequence to form an interface; the Percoll separating medium with the volume percentage of 71 percent and the Percoll separating medium with the volume percentage of 61 percent are prepared by the Percoll separating medium with the volume percentage of 100 percent and normal saline;

3) adding the cell suspension obtained in the step 1) to the surface of the Percoll separating medium with the volume percentage of 61% obtained in the step 2), centrifuging, and sucking interfacial liquid between the Percoll separating medium with the volume percentage of 71% and the Percoll separating medium with the volume percentage of 61% to obtain the neutrophils.

Further, the centrifugation described in step 1) was performed at 1600rpm for 5 min.

Further, the centrifugation described in step 3) was performed at 3000rpm for 30 min.

Furthermore, the Percoll separation solution with the volume percentage of 100 percent is prepared by mixing Percoll stock solution and NaCl solution with the concentration of 1.5M according to the volume ratio of 9: 1.

Further, the specific preparation method of the drug-loaded nano gel particles in the step two is as follows:

1) 1-1.5 g by mass volume: mixing gelatin and water in a ratio of 20mL, adding a 0.1mol/L NaOH solution, stirring at 70 ℃ for 30min, adding hexanoic anhydride, and reacting at 70 ℃ for 5 h; the volume-mass ratio of the NaOH solution to the gelatin is 2mL: 1-1.5 g; the volume-mass ratio of the hexanoic anhydride to the gelatin is 4mL: 1-1.5 g;

2) cooling to room temperature after the reaction in the step 1), adjusting the pH value to 7.4, dialyzing by using ethanol with the volume percentage of 25% for 24 hours, and drying the dialyzate in a drying oven at the temperature of 60 ℃ to obtain amphiphilic gelatin;

3) dispersing paclitaxel and ferroferric oxide nano particles with the diameter of 6nm by using chloroform to obtain an organic phase; dissolving the amphiphilic gelatin of step 2) with water to obtain a water phase; adding the organic phase into the water phase, ultrasonically cleaning for 20min, stirring at 50 deg.C to remove organic solvent to obtain nanometer gel particles, centrifuging and cleaning for 3 times, and standing at 4 deg.C; wherein the mass-volume ratio of chloroform to ferroferric oxide nanoparticles to paclitaxel is 0.2-0.5 mL: 2-3 mg: 1 mg;

4) centrifuging the cultured Escherichia coli at 5000rpm for 10min to remove thallus, filtering with 0.45 micrometer filter membrane, concentrating the supernatant with ultrafiltration centrifuge tube, and centrifuging at 150000rpm at 4 deg.C for 2h to obtain Escherichia coli outer membrane vesicle;

5) extruding the outer membrane vesicles of the escherichia coli for 11 times through a 200nm filter membrane by using a nano extrusion method to obtain bacterial membrane vesicles;

6) mixing the bacterial membrane vesicles obtained in the step 5) with the nano gel particles obtained in the step 3), extruding the mixture for 21 times through a 200nm filter membrane, and centrifugally cleaning the mixture for 3 times to obtain the drug-loaded nano gel particles.

Further, the centrifugal cleaning rotating speed is 10000 rpm.

Further, the molecular weight cut-off of the ultrafiltration centrifuge tube is 100 kDa.

Further, the co-culturing of the neutrophils obtained in the step one and the nanogel particles obtained in the step two in the step three is performed by the following steps:

diluting the neutrophils obtained in the step one by using PBS, and then centrifuging to remove Percoll separating medium used for separating the neutrophils; and dispersing the nanogel particles obtained in the step two into the neutrophilic granulocyte suspension without the Percoll separating medium, performing co-culture for 30min under the condition of 37 ℃ water bath, and performing centrifugal cleaning to complete the co-culture.

The motion principle of the hybrid neutrophil robot in the invention is as follows: the hybrid neutrophilic granulocyte robot carries ferroferric oxide magnetic nanoparticles inside, so that the hybrid neutrophilic granulocyte robot can rotate under the action of an alternating magnetic field; and secondly, the neutrophil is responsive to the concentration gradient of the inflammatory factor, so that the concentration gradient of the inflammatory factor can be constructed, and the hybrid neutrophil robot is induced to perform chemotactic movement.

The bacteria membrane camouflage gel particle has the functions of loading drugs, loading fluorescent probes and the like, and can perform the functions of drug release, fluorescence imaging and the like in application. The surface molecules of the bacterial membrane can act with receptors on the surfaces of the neutrophils, the phagocytosis speed of the neutrophils on gel particles is improved through the bacterial membrane camouflage, the hybrid neutrophil robot can be quickly prepared, meanwhile, the drug release can be slowed down through the bacterial membrane camouflage, and the leakage of the loaded drugs in the hybrid neutrophil robot is reduced.

The invention has the following beneficial effects:

the hybrid neutrophil robot prepared by the invention can be prepared by a magnetic field (A) and (B) because the interior of the hybrid neutrophil robot is loaded with magnetic gel particles>6mT), and the hybrid neutrophil robot is still a living cell and can perform directional chemotactic movement under the stimulation of cytokines, inflammatory factors, chemotactic peptides and other substances. The invention fully utilizes the phagocytic capacity and the movement capacity of the neutrophil to prepare the double-response hybrid neutrophil robot with cell activity and magnetic response, the preparation process is safe, efficient and quick, and the hybrid neutrophil motor can reach the aim of the hybrid neutrophil motor under the rotating magnetic field with the strength of 18mT>The movement speed of 14 mu m/s can reach about 0.2 mu m s under the chemotactic condition^-1The compound has good biological compatibility, can cross blood brain barrier, can avoid immune clearance, can survive in vivo well, and has good biomedical application prospect. The hybrid neutrophil robot has the functions of loading drugs and loading fluorescent probes, has the effect of being not easy to be eliminated by an immune system, and can be abundantly applied to in-vivo treatment scenes such as in-vivo targeted drug delivery, crossing of special biological barriers and the like.

Drawings

FIG. 1 is an optical microscope photograph of a hybrid neutrophil robot with a scale of 20 μm;

FIG. 2 is an optical microscope photograph of the hybrid neutrophil robot moving in an alternating magnetic field with a scale of 10 μm;

FIG. 3 is an optical microscope photograph of the movement of the hybridized neutrophil robot in a chemokine concentration gradient with a scale of 10 μm.

FIG. 4 is a fluorescent microscopic photograph of calcein acetoxymethyl ester-propidium iodide co-staining of a hybrid neutrophil robot with a scale of 200 μm;

FIG. 5 shows fluorescence photographs taken by a microscope of a hybridized neutrophil robot, wherein (A) is a DiI fluorescence channel, (B) is a coumarin-6 fluorescence channel, and (C) is a superposition channel with a scale of 20 μm.

Detailed Description

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples for carrying out the invention, and that various changes in form and details may be made therein without departing from the spirit and scope of the invention in practice.

To make the objects, aspects and advantages of the embodiments of the present invention more apparent, the following detailed description clearly illustrates the spirit of the disclosure, and any person skilled in the art, after understanding the embodiments of the disclosure, may make changes and modifications to the technology taught by the disclosure without departing from the spirit and scope of the disclosure.

The exemplary embodiments of the present invention and the description thereof are provided to explain the present invention and not to limit the present invention.

Example 1

The methods for obtaining neutrophils are described in detail in this example:

1. the test material used was male Kunming Mouse (KM) (purchased from Experimental animals center of second Hospital, affiliated to Harbin medical university) weighing 30g or more.

2. Mice were sacrificed by cervical dislocation, dissected to remove hind femoral, tibial, forelimb humerus, and bones were soaked in RPMI1640 medium (purchased from Hyclone Laboratories, usa) for use.

3. The bone was cut into two ends and the bone lumen was flushed with RPMI1640 medium (purchased from Hyclone Laboratories, usa) multiple times using a 1mL syringe until the bone was white translucent at which time the cells were present in the RPMI1640 medium flush.

4. The wash solution was centrifuged at 1600rpm for 5min to obtain a cell pellet and resuspended in 2mL phosphate buffered saline (PBS, pH 7.4).

5. Preparation for gradient centrifugation: a100% Percoll separation medium was prepared by mixing 45mL of Percoll stock solution with 5mL of 1.5M NaCl solution using a commercially available Percoll separation medium (purchased from Shanghai assist, san Yang Sheng Biotech Co., Ltd.), mixing the 100% Percoll separation medium with physiological saline in respective proportions to give 61% and 71% Percoll separation medium, and stacking 3mL of the 71% Percoll separation medium and 3mL of the 61% Percoll separation medium in 10mL centrifuge tubes in this order to form an interface between the 71% and 61% Percoll separation medium.

6. The 2mL cell suspension of step 4 was overlaid on the surface of the 61% Percoll fraction of step 5 and centrifuged at 3000rpm for 30 min.

7. After the centrifugation in step 6 is finished, sucking the cells on the interfaces of 71% and 61% Percoll separating medium, namely the needed neutrophils.

Example 2

The preparation method of the escherichia coli outer membrane-camouflaged drug-loaded nano-gel particles doped with ferroferric oxide magnetic nanoparticles is described in detail in the embodiment:

1.1.25 g gelatin was dissolved in 20mL water, 2mL of 0.1mol/L NaOH solution was added, and the mixture was stirred at 70 ℃ for 30 minutes. 4mL of hexanoic anhydride was added and the reaction was carried out at 70 ℃ for 5 hours.

2. And cooling to room temperature, adjusting the pH to 7.4 by using a dilute sodium hydroxide solution, dialyzing for 24 hours by using a 25% ethanol solution, and drying the solution in an oven at the temperature of 60 ℃ to obtain the amphiphilic gelatin.

3. Dispersing 2.5mg of ferroferric oxide nano particles with the diameter of 6nm and 1mg of paclitaxel by using 0.25mL of chloroform to obtain an organic phase, dissolving 50mg of amphiphilic gelatin in 1mL of water to obtain a water phase, adding the organic phase into the water phase, carrying out ultrasonic treatment for 20 minutes by an ultrasonic cleaning machine, and stirring at 50 ℃ to remove the organic solvent. The obtained nanogel particles were washed 3 times by centrifugation at 10000rpm and stored at 4 ℃.

4. Culturing Escherichia coli (Escherichia coli was cultured in LB broth (purchased from Qingdao Gaokuan Haibo Biotechnology Co., Ltd.) under conditions of 37 ℃ and 200rpm using a shaker, removing the cells by centrifugation at 5000rpm for 10min, and concentrating the supernatant using an ultrafiltration centrifugal tube (cut-off molecular weight of 100kDa) by filtration through a 0.45-micron filter membrane, and ultracentrifuging at 4 ℃ and 150000g for 2h to obtain Escherichia coli outer membrane vesicles.

5. The outer membrane vesicles of Escherichia coli were extruded 11 times through a 200nm filter membrane using a nano-extrusion method to obtain bacterial membrane vesicles. And mixing the bacterial membrane vesicles with the prepared nanogel, extruding the mixture for 21 times through a 200nm filter membrane, and centrifugally cleaning the mixture for 3 times to obtain the bacterial membrane camouflage nanogel particles.

Example 3

The method for preparing a hybrid neutrophil robot by co-culture is described in detail in this example:

1. the neutrophil-containing Percoll fraction obtained in example 1 was diluted with PBS and then centrifuged at 260 × G for 5min to remove the Percoll fraction.

2. Dispersing the nanogel particles prepared in the embodiment 2 into the neutral granulocyte suspension containing 1 separated Percoll, co-culturing in a water bath at 37 ℃ for 30min, and centrifuging and cleaning to obtain the hybrid neutral granulocyte robot.

This example is according to 0.5-3X 10⁶Freshly isolated neutrophils and 0.1-10 mg/mL^-1The bacterial membrane camouflage gel particles are mixed to obtain the hybrid neutrophilic granulocyte robot.

The performance of the hybrid neutrophil robot prepared in the above example was verified:

firstly, morphology:

evaluation method and results of the morphology of the hybrid neutrophil robot prepared in examples 1 to 3 (as shown in fig. 1).

The morphology of the hybrid neutrophil robot is characterized by using an upright microscope (Olympus BX-53, Japan), and the prepared hybrid neutrophil robot is observed to retain the cell appearance.

Secondly, alternating magnetic field movement:

the movement of the hybrid neutrophil robot prepared in examples 1 to 3 was verified in the form of alternating magnetic field driving. The alternating magnetic field source is a self-made triaxial Helmholtz coil system, and is matched with a virtual instrument signal source for use, the applied magnetic field frequency is 2Hz, and the applied magnetic field intensity is 12 mT. The movement of the hybrid neutrophil robot (as shown in fig. 2) was observed and recorded using an optical microscope system (Olympus IX-71, Japan) (the white line in the figure is the movement trajectory of the neutrophil robot). The applied magnetic field frequency is 25Hz, the moving speed of the hybrid neutrophil granulocytic robot under the condition that the applied magnetic field intensity is 18mT is shown in Table 1, the Table 1 records the moving speed of the hybrid neutrophil granulocytic robot in an alternating magnetic field, and respectively records the moving speed of 5 hybrid neutrophil granulocytic robots; the movement speed of the hybrid neutrophil robot can reach 14.3 mu m/s.

The result shows the feasibility of the hybrid neutrophil robot moving in the alternating magnetic field.

TABLE 1

Thirdly, chemokine concentration gradient movement:

the movement of the hybrid neutrophil robots prepared in examples 1 to 3 was verified in the form of constructing a concentration gradient of chemokines. Live bacteria are added into experimental conditions, and the live bacteria generate chemotactic factor concentration gradient to attract the hybrid neutrophil robot to perform deformed chemotactic movement. The movement of the hybridized neutrophil robot was observed and recorded using an optical microscope system (Olympus IX-71, Japan). The recorded data show that the hybrid neutrophil robot stretches out of the false foot to perform deformation movement, and the speed can reach 0.2 mu m/s (as shown in figure 3) (the white line in the figure is the movement track of the neutrophil robot).

The results show the feasibility of the hybrid neutrophil robot movement in a chemokine concentration gradient.

Example 4

The biological activity of the hybrid neutrophil robots prepared in examples 1 to 3 was verified in this example:

the hybrid neutrophil robot prepared in example 3 was simultaneously labeled with two fluorescent dyes of Calcein acetoxymethyl ester (Calcein-AM) and propidium iodide (purchased from beijing solibao technologies ltd.), and the staining of the hybrid neutrophil robot was observed using a fluorescence microscope (Olympus BX-53, Japan), and the results are shown in fig. 4. Calcein acetoxy methyl ester is a cell staining reagent capable of carrying out fluorescent labeling on living cells, penetrates through cell membranes, enters the cells, and is sheared by esterase in the cells to form calcein, so that the calcein is retained in the cells and emits strong green fluorescence. Propidium iodide is a nuclear staining reagent that stains DNA, releases red fluorescence upon intercalation into double stranded DNA, and does not pass through a living cell membrane, but can stain nuclei through a damaged cell membrane. Calcein acetoxymethyl ester in combination with propidium iodide stains both live (green) and dead (red) cells simultaneously. As can be seen from the observation of FIG. 4, more than 90% of the hybridized neutrophil robots are green fluorescent, which indicates that most of the hybridized neutrophil robots retain the biological activity of the cells during the preparation process.

Example 5

The drug loading capacity of the hybrid neutrophil robots prepared in examples 1 to 3 was verified in this example:

coumarin-6 (purchased from beijing solibao technologies ltd.) is added in the process of synthesizing nanogel particles in step 3 of example 2, and coumarin-6 is a fat-soluble laser dye with high laser conversion rate and relatively stable performance, the emission wavelength of the fat-soluble laser dye is about 504nm, and the fat-soluble laser dye is green fluorescence, and coumarin-6 is used as a visual drug model for drug loading effect of a hybrid neutrophil robot. The cell membrane of neutrophils was labeled with a fluorescent orange-red dye DiI (all known as 1,1' -dioctadecyl-3,3,3',3' -tetramethylenecarbocyanine perchlorate, purchased from beijing solibao technologies ltd.) and then a hybrid neutrophil robot was prepared as described in example 3. The prepared hybrid neutrophil robot was observed by using an upright fluorescence microscope (Olympus BX-53, Japan), and the result is shown in fig. 5, and the fluorescence photograph of the microscope shows that the green fluorescence of the nanogel particles is distributed in the red fluorescence of the cell membrane, thereby verifying that the hybrid neutrophil robot internally carries the simulated drug molecule coumarin-6.

The above description is only a preferred embodiment of the present invention, and these embodiments are based on different implementations of the present invention, and the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The preparation method of the hybrid neutrophil granulocyte robot is characterized by comprising the following steps of:

step one, obtaining neutrophils;

2. The method for preparing a hybrid neutrophil robot according to claim 1, wherein the obtaining of neutrophils in the first step is separated from freshly isolated mouse bone marrow.

3. The method for preparing a hybrid neutrophil robot according to claim 1 or 2, wherein the neutrophil is obtained by:

4. The method for preparing a hybrid neutrophil robot according to claim 3, characterized in that the centrifugation in step 1) is at 1600rpm for 5 min.

5. The method for preparing a hybrid neutrophil robot according to claim 3, wherein the centrifugation in step 3) is performed at 3000rpm for 30 min.

6. The method for preparing the hybrid neutrophil granulocytes robot according to claim 3, wherein the Percoll separation solution with the volume percentage of 100% is prepared by mixing a Percoll stock solution and a NaCl solution with the concentration of 1.5M according to the volume ratio of 9: 1.

7. The preparation method of the hybrid neutrophil granulocyte robot as claimed in claim 1, wherein the specific preparation method of the drug-loaded nanogel particles in the step two is as follows:

8. The method for preparing a hybrid neutrophil robot according to claim 7, wherein the centrifugal cleaning rotation speed is 10000 rpm.

9. The method for preparing the hybrid neutrophil granulocytes robot according to claim 7, wherein the molecular weight cut-off of the ultrafiltration centrifugal tube is 100 kDa.

10. The method for preparing hybridized neutrophil robot according to claim 1, wherein the co-culturing the neutrophil obtained from the first step and the nanogel particle obtained from the second step in the third step is performed by the following steps: