CN112980786A - T cell and nanoparticle connection method based on click chemistry - Google Patents
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Abstract
The invention relates to the field of nano medicine, in particular to a T cell and nanoparticle connection method based on click chemistry. The nanoparticles are covalently bound to azide groups on proteins on the surface of cell membranes by a click chemistry approach of diphenylcyclooctyne group-azide linkage to form efficient linkage of T cells to the nanoparticles. The existing connection method is unstable, low in efficiency and poor in specificity, the T cell and nanoparticle connection method based on click chemistry overcomes the defects, is simple and easy to implement, mild in reaction condition and stable in connection, and can realize nano-engineering transformation of T cells.
Description
Technical Field
The invention relates to the field of nano medicine, in particular to a T cell and nanoparticle connection method based on click chemistry.
Background
Click chemistry (click chemistry) was a modular synthetic concept first proposed in 2001 by the nobel prize chemist k. The method is a new combinatorial chemistry method which selects easily available raw materials, realizes carbon heteroatom connection through modularized, reliable, high-efficiency and high-selectivity chemical transformation, and quickly synthesizes various new compounds at low cost.
The most widely used click chemistry is currently the copper ion catalyzed terminal alkyne and azide Huisgen dipolar cycloaddition reaction. However, since copper ions are introduced as a catalyst during the click reaction, the physiological toxicity of the remaining copper ions may cause degradation of DNA and denaturation of proteins. And copper ions cannot be introduced by the novel cycloalkynyl-azide linkage-based copper-free novel click chemistry method, so that a good foundation is laid for biomedical application.
T cell therapy as a viable cell therapy regimen is one direction for future drugs. On 19/10/2017, the U.S. government approved a second therapy based on modification of the patient's autoimmune cells (yescarta therapy) to treat patients with specific lymphoid cancers. yescarta therapy belongs to chimeric antigen receptor T cell (CAR-T) therapy. However, genetic methods for engineering modification of T cell surfaces are large in engineering volume, have high technical threshold, and are limited by inefficient primary cell modification.
Prior art methods such as the method of adsorption of positively charged nanoparticles onto the surface of T cells [ Anselmo AC et al, stabilizing nanoparticles to cavities in suspension and spoke through adsorption on red blood cells, 2013.7 (12): 11129-37.] and conjugation of maleimide groups to thiols on T cells to achieve selective modification of T cells [ Huang B et al, Active targeting of chemotherapy to dispersed tumors using nanoparticle-carrying T cells, 2015.7 (291): 291ra94 ] all suffer from various degrees of shortcomings. In the method for adsorbing the positive charge nano particles on the surface of the T cell, the positive charge and the negative charge are adsorbed into non-covalent connection and are easy to fall off in a complex solution environment. The newly generated C-S bond in the process of conjugation of a maleimide group to a thiol group on a T cell to achieve selective modification of the T cell is not stable enough under physiological conditions, undergoes reverse michael addition, and is less stable in the case of competitive thiol compounds (e.g., cysteine).
T cells and nanoparticles are connected by click chemistry to realize nano-engineering transformation of the T cells, and related functions of the T cell nanoparticles are endowed, such as carrying fluorescent nanoparticle tracing, carrying chemotherapy nanoparticles to enhance anti-tumor effect, and carrying photosensitive nanoparticles to realize combined treatment of immunotherapy and photothermal therapy. In order to further expand the application of nano-engineering modification of T cells and increase the possibility of clinical application thereof, it would be a feasible solution to apply copper-free catalyzed click chemistry as a tool for chemical assembly. The copper-free catalytic click reaction is generated between a diphenyl cyclooctyne group and an azide group, so that the key for solving the problems is to realize the nano-engineering modification of the T cell by a novel, simple, specific and stable click chemistry-based T cell and nanoparticle connection method.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of instability, low efficiency and poor specificity of a connection method in the prior art, the invention provides a T cell and nanoparticle connection method based on click chemistry.
Technical scheme
The invention discloses a T cell and nanoparticle connection method based on click chemistry, and aims to provide a method which is mild in reaction condition, stable in connection and capable of realizing T cell nano-engineering modification. Incubating tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) of the azido sugar molecule with T cells to make the surface of the T cells metabolically labeled with azido groups (-N)3) Optionally with a compound carrying a diphenylcyclooctyne group (a)-DBCO) nanoparticles (inp-DBCO) recognize each other and react, thus achieving efficient covalent attachment of T cells to the nanoparticles.
A T cell and nanoparticle connection method based on click chemistry is characterized by comprising the following steps:
step 1: preparation of T cells
(1) T cell extraction and expansion: peripheral blood of healthy volunteers is collected and human Peripheral Blood Mononuclear Cells (PBMCs) are isolated. Using AIM-V culture medium containing 2% fetal calf serum, culturing and amplifying PBMCs in a 6-well plate for 10 days at 37 ℃ in an environment of 5% carbon dioxide/95% air, then adding anti-CD 3/CD28 magnetic beads and interleukin 2 to stimulate T cells to differentiate, and culturing for 3-4 days to obtain the amplified T cells.
(2) T cell metabolism labeling of azide groups: after 100. mu.M of the sugar molecule tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) was added to the above (1) and incubated for 48 hours, the azido group was labeled on the surface of the T cell membrane by metabolism.
Step 2: preparation of indocyanine green polymer nanoparticles
(1) Polyglycolide Lactide (PLGA) was dissolved in a 1mg/mL acetonitrile solution.
(2) Mu.g of soy lecithin, 120. mu.g of distearoylphosphatidylethanolamine-polyethylene glycol-diphenylcyclooctyne (DSPE-PEG-DBCO) and 1mg of indocyanine green were dissolved in 3mL of 4% ethanol.
(3) And (3) carrying out ultrasonic treatment on the solution (2) by using an ultrasonic cell disruptor at the frequency of 20kHz and the power of 35W, and simultaneously dropwise adding 1mL of polyglycolide-lactide (PLGA) acetonitrile solution (1mg/mL) into the solution for 5min, and carrying out ultrafiltration for 2-3 times by using a 10kDa ultrafiltration tube to obtain the indocyanine green polymer nanoparticles (INPs).
And step 3: ligation of T cells to nanoparticles
T cells were first washed 1 time with PBS buffer, cells were harvested by centrifugation, fresh AIM-V medium without calf serum was changed, indocyanine green polymer nanoparticles (INPs, 50 μ g/mL) were then added, reaction was carried out for 1 hour, and then unlinked nanoparticles were washed with PBS buffer.
Compared with the prior art, the method has the beneficial effects that
The biological system does not contain cycloalkynyl and azido, so that the whole reaction system only can ensure that cells containing cycloalkynyl can react with the nanoparticles containing azido, and the labeling method has obvious specificity.
In relatively complex biological systems such as blood, human tissues and the like, the labeling method can greatly avoid pseudo signals caused by the conventional nonspecific labeling based on the amino and carboxyl reactions. Meanwhile, the labeled product is more stable compared with a positive and negative charge adsorption method due to chemical bonding.
Because the T cell and nanoparticle connection is based on the metabolic labeling of the T cell, the method can be used for carrying out nanoparticle engineering connection modification on organisms such as cells, stem cells, viruses and bacteria.
The novel cell and nanoparticle connection method based on non-copper ion catalysis click chemistry has the advantages of mild reaction conditions, quick and simple marking and good reproducibility, and the marked product has very good stability due to the formation of covalent bonds.
The preparation method is simple and easy to implement, and convenient to operate and popularize.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic representation of T cell attachment to nanoparticles.
FIG. 2 is a flow cytometer for detecting metabolic expression of azide groups on the surface of T cell membranes.
Fig. 3 shows the particle size of indocyanine green polymer nanoparticles (inp).
FIG. 4 shows the scanning electron microscope results of T cell attachment to nanoparticles.
Fig. 5 shows the results of confocal laser microscopy on T cell ligation with indocyanine green polymer nanoparticles (inp).
Detailed Description
Various embodiments of the present disclosure will be described more fully hereinafter. The present disclosure is capable of various embodiments and of modifications and variations therein.
Example 1
The working principle of the invention for carrying out fluorescence quantum dot marking on cells by using click chemistry is shown in figure 1. By means of diphenylcyclooctyne groups (-DBCO) -azide (-N)3) Click chemistry of ligation, nanoparticles (INPs-DBCO) are ligated with azide (-N) on cell membrane surface proteins3) Covalently bound to form a highly efficient linkage of the T cells to the nanoparticles.
The method comprises the following specific steps:
step 1: preparation of T cells
(1) T cell extraction and expansion: peripheral blood from healthy volunteers was collected and human Peripheral Blood Mononuclear Cells (PBMCs) were isolated. The specific operation is as follows: collecting blood by an anticoagulation tube, and adding 1: 1, adding a separation solution with the same volume as the physiological saline into the other tube, adding a physiological saline and blood mixed solution into the separation solution along the tube wall, centrifuging at 800g for 30min, then layering, generally dividing into 4 layers, namely a serum layer, a T cell layer, a separation solution and red blood cells, and sucking the T cell layer. And culturing and amplifying the sucked T cell layer, namely PBMCs, in a 6-well plate for 10 days by using an AIM-V culture medium containing 2% fetal calf serum under the environment of 5% carbon dioxide/95% air at 37 ℃, adding anti-CD 3/CD28 magnetic beads and interleukin 2 into the culture medium to stimulate cell differentiation, and culturing for 3-4 days to obtain the amplified T cells.
(2) T cell metabolism labeling of azide groups: after 100. mu.M of the sugar molecule tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) was added to the medium of the above (1) and incubated for 48 hours, the T cell membrane surface was labeled with an azido group by metabolism.
Step 2: preparation of indocyanine green polymer nanoparticles
(1) The PLGA polymer was first dissolved in a 1mg/mL acetonitrile solution.
(2) Mu.g of soya lecithin, 120. mu.g of DSPE-PEG-DBCO and 1mg of indocyanine green were dissolved in 3mL of 4% ethanol.
(3) And (3) carrying out ultrasonic treatment on the solution (2) by using an ultrasonic cell disruptor at the frequency of 20kHz and the power of 35W, and simultaneously dropwise adding a PLGA acetonitrile solution (1mg/mL) into the solution for 5min, and carrying out ultrafiltration for 2-3 times by using a 10kDa ultrafiltration tube to obtain the indocyanine green polymer nanoparticles (INPs).
And step 3: ligation of T cells to nanoparticles
T cells were first washed 1 time with PBS buffer, cells were harvested by centrifugation, fresh AIM-V medium without calf serum was changed, indocyanine green polymer nanoparticles (INPs, 50 μ g/mL) were then added, reaction was carried out for 1 hour, and then unlinked nanoparticles were washed with PBS buffer.
Example 2
Method for detecting metabolic expression of azide group on surface of T cell membrane by flow cytometry
As shown in the flow cytometry detection data of FIG. 2, azide groups (-N) on the surface of T cells3) Labeling by using FITC dye, and drawing a remarkable difference between the flow peak of a T cell group for incubating the sugar molecule tetraacetyl-N-azidoacetylmannosamine (Ac4ManAz) and the flow peak of a Control group (Control) without incubating the sugar molecule, which indicates that the T cell group for incubating the sugar molecule is successfully labeled by the FITC dye and the surface of a T cell membrane successfully expresses an azido group.
Example 3
Particle size detection of indocyanine green polymer nanoparticles (INPs)
As shown in FIG. 3, the particle size of the nanoparticles of INPs prepared by ultrasonic hydration method was measured by Malvern particle sizer, and the average particle size of the nanoparticles of INPs was about 160 nm.
Example 4
Scanning electron microscope for observing connection of T cells and nanoparticles
The connected T cells are treated by gold spraying and observed under a scanning electron microscope, and as shown in fig. 4, indocyanine green polymer nanoparticles (INPs, about 160nm in size) are successfully connected to the surfaces of the T cells.
Example 5
Laser confocal microscopy of T cell junctions with indocyanine green polymer nanoparticles (INPs)
T cells seeded in 8-well plates: (
Nunc, USA), 200. mu.L of AIM-V medium was added to each well, and the number of cells was 2X 104. After 24 hours, the medium was replaced with AIM-V medium of indocyanine green polymer nanoparticles. After 1 hour incubation, washed twice with PBS buffer, nuclear stained with Hoeches 33258 dye, Hoeches 33258 stainable T cell nuclei, indocyanine green polymer nanoparticles themselves fluorescent, and 8-well plates were observed under a laser scanning confocal microscope (Leica TCS SP5, germany) using two channel excitation wavelengths of 405nm and 633nm, respectively.
As shown in fig. 5, the DAPI channel shows T cell nuclei stained with fluorescent dye, the ICG channel shows indocyanine green polymer nanoparticles (inp), and the superimposed results indicate successful attachment of inp to the T cell surface.
Those skilled in the art will appreciate that the figures are merely schematic representations of one preferred implementation scenario and that the blocks or flow diagrams in the figures are not necessarily required to practice the present invention.
Those skilled in the art will appreciate that the modules in the devices in the implementation scenario may be distributed in the devices in the implementation scenario according to the description of the implementation scenario, or may be located in one or more devices different from the present implementation scenario with corresponding changes. The modules of the implementation scenario may be combined into one module, or may be further split into a plurality of sub-modules.
The above-mentioned invention numbers are merely for description and do not represent the merits of the implementation scenarios.
The above disclosure is only a few specific implementation scenarios of the present invention, however, the present invention is not limited thereto, and any variations that can be made by those skilled in the art are intended to fall within the scope of the present invention.
Claims (7)
1. A T cell and nanoparticle connection method based on click chemistry is characterized by comprising the following steps:
(1) extracting and expanding T cells;
(2) labeling a azido group on the surface of a T cell membrane;
(3) preparing indocyanine green polymer nanoparticles;
(4) t cells are linked to nanoparticles.
2. The ligation method according to claim 1, wherein the T cells in step (1) are derived from isolated human peripheral blood mononuclear cells, which are cultured in a 6-well plate using AIM-V medium containing 2% fetal bovine serum at 37 ℃ in an atmosphere of 5% carbon dioxide/95% air for 10 days, and then anti-CD 3/CD28 magnetic beads and interleukin 2 are added to the medium to stimulate the differentiation of the T cells, and the cultured cells are cultured for 3-4 days to obtain expanded T cells.
3. The ligation method according to claim 1, wherein 100 μ M of the sugar molecule tetraacetyl-N-azidoacetylmannosamine is added to the medium of step (1) in step (2) and incubated for 48 hours.
4. The connection method according to claim 1, wherein the indocyanine green polymer nanoparticles are prepared in step (3) by an ultrasonic hydration method, and the indocyanine green polymer nanoparticles are prepared by firstly dissolving polyglycolide lactide in 1mg/mL acetonitrile, then dissolving 180 μ g of soybean lecithin, 120 μ g of distearoyl phosphatidyl ethanolamine-polyethylene glycol-diphenylcyclooctyne and 1mg of indocyanine green in 3mL of 4% ethanol, ultrasonically treating the mixed solution containing the indocyanine green with an ultrasonic cell disruptor at a frequency of 20kHz and a power of 35W while dropwise adding 1mL of the polyglycolide lactide acetonitrile solution into the mixed solution, wherein the ultrasonic time is 5min, and ultrafiltering the mixed solution with a 10kDa ultrafilter tube for 2-3 times.
5. The ligation method according to claim 1, wherein in step (4), the T cells are washed 1 time with PBS buffer, the cells are collected by centrifugation, fresh AIM-V medium without calf serum is replaced, then 50 μ g/mL indocyanine green polymer nanoparticle solution is added, the reaction is carried out for 1 hour, and then unligated nanoparticles are washed away with PBS buffer.
6. A method according to claim 1 or 5, wherein nanoparticles are used to which diphenylcyclooctyne groups are attached.
7. A nanoengineered T-cell prepared by the ligation method according to any one of claims 1 to 6.
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