CN112980786B - T cell and nanoparticle connection method based on click chemistry - Google Patents
T cell and nanoparticle connection method based on click chemistry Download PDFInfo
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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 nanoparticle is covalently bound with an azide group on a cell membrane surface protein by a click chemistry method of diphenyl cyclooctynyl group-azide connection to form an efficient connection of a T cell with the nanoparticle. The existing connection method is unstable, low in efficiency and poor in specificity, and the connection method of T cells and nano particles based on click chemistry overcomes the defects, is simple, convenient and feasible, has mild reaction conditions and stable connection, and can realize the nano engineering transformation of the 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) is a modular synthetic concept first proposed in 2001 by the nobel chemical prize acquirer k.barry shared. The method is a novel combinatorial chemistry method for quickly synthesizing various novel compounds at low cost by selecting easily available raw materials and realizing carbon heteroatom connection through modularized, reliable, high-efficiency and high-selectivity chemical transformation.
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 residual copper ions may lead to degradation of DNA and denaturation of proteins. The novel copper-free novel click chemistry method based on cycloalkynyl-azide connection does not introduce copper ions, which lays a good foundation for biomedical application.
T cell therapy is one direction of future drugs as a viable cell therapy regimen. The united states government approved a second therapy based on engineering patient's autoimmune cells (yescarta therapy) for the treatment of specific lymphoma patients on day 10 and day 19 2017. yescarta therapy belongs to chimeric antigen receptor T cell (CAR-T) therapy. However, genetic methods for engineering modification of T cell surfaces are engineering intensive, have high technical thresholds, and are limited by the inefficiency of primary cell modification.
Prior art methods such as adsorption of positively charged nanoparticles to T cell surfaces [ Anselmo AC et al Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells,2013.7 (12): 11129-37 ] and maleimide groups and thiol groups on T cells to achieve selective modification of T cells [ Huang B et al Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells,2015.7 (291): 291ra94 ] all suffer from various degrees of disadvantage. In the method of adsorbing positive charge nano particles on the surface of T cells, positive and negative charges are adsorbed to be non-covalent connection, and are easy to fall off in a complex solution environment. The lack of stability of the newly formed C-S bond under physiological conditions in the method of coupling maleimide groups to thiols on T cells to effect selective modification of T cells can result in reverse michael addition, which is less stable in competing thiol compounds (e.g., cysteine).
The T cells and the nanoparticles are connected by click chemistry to realize the nano engineering transformation of the T cells, so that the T cells and the nanoparticles are endowed with related functions, such as tracing by carrying fluorescent nanoparticles, enhancing anti-tumor effect by carrying chemotherapy nanoparticles, and carrying photosensitive nanoparticles to realize the combined treatment of immunotherapy and photothermal therapy. In order to further expand the application of the nano engineering of the T cells, the possibility of clinical application of the T cells is increased, and the application of a click chemical reaction without copper catalysis as a tool for chemical assembly is a feasible solution. The click reaction without copper catalysis occurs between the diphenyl cyclooctyne group and the azido group, so that the novel simple, specific and stable method for connecting T cells and nano particles based on click chemistry to realize the nano engineering transformation of the T cells is a key for solving the problems.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of unstable connection method, low efficiency and poor specificity in the prior art, the invention provides a T cell and nanoparticle connection method based on click chemistry.
Technical proposal
The invention discloses a method for connecting T cells and nano particles 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 transformation. Incubating the azided sugar molecule tetraacetyl-N-azidoacetamannonamine (Ac 4 ManAz) with T cells to make the metabolic mark on the surface of the T cells to be azido (-N) 3 ) Can mutually identify and react with nano particles (INPs-DBCO) carrying diphenyl cyclooctyne groups (-DBCO), thereby realizing the efficient covalent connection of T cells and the nano particles.
A method for connecting T cells and nano particles 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 was collected from healthy volunteers and human Peripheral Blood Mononuclear Cells (PBMCs) were isolated. PBMCs were cultured and expanded in 6-well plates at 37℃in a 5% carbon dioxide/95% air environment using AIM-V medium containing 2% fetal bovine serum, followed by stimulation of T cell differentiation by addition of anti-CD 3/CD28 magnetic beads and interleukin 2 for 3-4 days to obtain expanded T cells.
(2) T cell metabolism marks the azide group: 100. Mu.M sugar molecule tetraacetyl-N-azidoacetamannonamine (Ac 4 ManAz) was added to the above (1) and incubated for 48 hours, and the surface of the T cell membrane was labeled with an azide group by metabolism.
Step 2: indocyanine green polymer nanoparticle preparation
(1) Polyglycolide Lactide (PLGA) was dissolved in 1mg/mL acetonitrile solution.
(2) 180 μg of soybean lecithin, 120 μg of distearoyl phosphatidylethanolamine-polyethylene glycol-diphenylcyclooctyne (DSPE-PEG-DBCO) and 1mg of indocyanine green were dissolved in 3mL of 4% ethanol.
(3) And (3) ultrasonically treating the solution (2) by using an ultrasonic cell disruption instrument at the frequency of 20kHz and the power of 35W, and dropwise adding 1mL of Polyglycolide Lactide (PLGA) acetonitrile solution (1 mg/mL) into the solution, wherein the ultrasonic treatment time is 5min, and the ultrafiltration is carried out for 2-3 times by using a 10kDa ultrafiltration tube, thus obtaining indocyanine green polymer nano-particles (INPs).
Step 3: attachment of T cells to nanoparticles
T cells were first washed 1 time with PBS buffer, cells were collected by centrifugation, fresh AIM-V medium without calf serum was replaced, indocyanine green polymer nanoparticles (inp, 50 μg/mL) were then added, reacted for 1 hour, and then unbound nanoparticles were washed off 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 only cells containing cycloalkynyl can react with nanoparticles with azido in the whole reaction system, and the labeling method has obvious specificity.
For relatively complex biological systems such as blood, human tissue and the like, the use of this labeling method can greatly avoid the artifacts associated with conventional nonspecific labeling based on amino and carboxyl reactions. Meanwhile, due to chemical bonding, compared with a positive and negative charge adsorption method, the marked product is more stable.
Because the connection of the T cells and the nano particles is based on metabolic markers performed on the T cells, the method can perform nano particle engineering connection modification on organisms such as cells, stem cells, viruses, bacteria and the like.
The novel cell and nanoparticle connection method based on non-copper ion catalysis has the advantages of mild reaction conditions, rapid and simple marking and good reproducibility, and the marked product has very good stability due to covalent bond generation.
The preparation method is simple and easy to implement, and is convenient to operate and popularize.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the connection of T cells to nanoparticles.
FIG. 2 shows the metabolic expression of azide groups on the surface of T cell membrane detected by a flow cytometer.
FIG. 3 is a graph of particle size of indocyanine green polymer nanoparticles (INPs).
FIG. 4 shows the result of scanning electron microscopy of T-cell attachment to nanoparticles.
FIG. 5 is a laser confocal microscope observation of T-cell ligation to indocyanine green polymer nanoparticles (INPs).
Detailed Description
Hereinafter, various embodiments of the present disclosure will be more fully described. 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 fluorescent quantum dot labeling on cells by utilizing click chemistry is shown in figure 1. Through diphenyl cyclooctynyl group (-DBCO) -azide (-N) 3 ) Click chemistry for ligation of nanoparticles (INPs-DBCO) to azides (-N) on cell membrane surface proteins 3 ) Covalent binding to form an efficient attachment of T cells to the nanoparticle.
The specific steps of the invention are as follows:
step 1: preparation of T cells
(1) T cell extraction and expansion: peripheral blood was collected from healthy volunteers and human Peripheral Blood Mononuclear Cells (PBMCs) were isolated. The specific operation is as follows: anticoagulation tube to collect blood, add 1:1, adding a separation liquid with the same volume as the physiological saline into the other tube, adding a mixture of the physiological saline and blood into the separation liquid along the tube wall, centrifuging at 800g for 30min, layering, namely, separating into 4 layers, namely, serum, a T cell layer, the separation liquid and erythrocytes, and sucking the T cell layer. The sucked T cell layer, namely PBMCs, is cultured and amplified in a 6-hole plate for 10 days at 37 ℃ under the environment of 5% carbon dioxide/95% air by using AIM-V culture medium containing 2% fetal bovine serum, and anti-CD 3/CD28 magnetic beads and interleukin 2 are added into the culture medium to stimulate cell differentiation, and the culture is carried out for 3-4 days, so that the amplified T cells are obtained.
(2) T cell metabolism marks the azide group: 100. Mu.M sugar molecule tetraacetyl-N-azidoacetamannonamine (Ac 4 ManAz) was added to the medium of (1) above and incubated for 48 hours, and the surface of the T cell membrane was labeled with an azide group by metabolism.
Step 2: indocyanine green polymer nanoparticle preparation
(1) The PLGA polymer was first dissolved in 1mg/mL acetonitrile solution.
(2) 180 μg of soybean lecithin, 120 μg of DSPE-PEG-DBCO and 1mg of indocyanine green were dissolved in 3mL of 4% ethanol.
(3) And (3) ultrasonically treating the solution (2) by using an ultrasonic cell disruption instrument at the frequency of 20kHz and the power of 35W, dropwise adding PLGA acetonitrile solution (1 mg/mL) into the solution, and ultrafiltering the solution for 2 to 3 times by using a 10kDa ultrafiltration tube for 5min to obtain indocyanine green polymer nano particles (INPs).
Step 3: attachment of T cells to nanoparticles
T cells were first washed 1 time with PBS buffer, cells were collected by centrifugation, fresh AIM-V medium without calf serum was replaced, indocyanine green polymer nanoparticles (inp, 50 μg/mL) were then added, reacted for 1 hour, and then unbound nanoparticles were washed off with PBS buffer.
Example 2
Flow cytometry for detecting metabolic expression of azide group on surface of T cell membrane
As shown in the flow cytometer detection data of FIG. 2, the T cell surfaceAzido groups (-N) 3 ) Labeling with FITC dye, the flow peak of the T cell group incubated with the sugar molecule tetraacetyl-N-azidoacetamannonamine (Ac 4 ManAz) was significantly separated from the flow peak of the Control group (Control) without incubating the sugar molecule, indicating that the T cell group incubated with the sugar molecule was successfully labeled with FITC dye and the surface of the T cell membrane successfully expressed the azide group.
Example 3
Particle size detection of indocyanine green polymer nanoparticles (INPs)
As shown in fig. 3, the particle size of the nanoparticles was measured using an infs prepared by ultrasonic hydration method using a malvern particle sizer, and the average particle size of the infs nanoparticles was about 160nm.
Example 4
Scanning electron microscope observation of T cell and nanoparticle connection
The connected T cells are subjected to metal spraying treatment and observed under a scanning electron microscope, and indocyanine green polymer nanoparticles (INPs, about 160nm in size) are successfully connected on the surfaces of the T cells as shown in FIG. 4.
Example 5
Laser confocal microscope observation of T cell and indocyanine green polymer nanoparticle (INPs) connection
T cells are inoculated in an 8-hole culture plate
Nunc, usa), 200 μl of AIM-V medium was added to each well, and the number of cells was 2×10 4 . After 24 hours, AIM-V medium was changed to indocyanine green polymer nanoparticles. After 1 hour incubation, washing twice with PBS buffer, nuclear staining with Hoeches 33258 dye, hoeches 33258 staining T cell nuclei, indocyanine green polymer nanoparticles themselves having fluorescence, and observing the 8-well plate under a laser scanning confocal microscope (Leica TCS SP5, germany), the two channels used excitation light wavelengths of 405nm and 633nm, respectively.
As shown in fig. 5, DAPI channel shows T cell nuclei stained with fluorescent dye, ICG channel shows indocyanine green polymer nanoparticles (inp), and the superimposed results indicate that the inp is successfully attached to the T cell surface.
Those skilled in the art will appreciate that the drawing is merely a schematic illustration of a preferred implementation scenario and that the modules or flows in the drawing are not necessarily required to practice the invention.
Those skilled in the art will appreciate that modules in an apparatus in an implementation scenario may be distributed in an apparatus in an implementation scenario according to an implementation scenario description, or that corresponding changes may be located in one or more apparatuses different from the implementation scenario. 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 inventive sequence numbers are merely for description and do not represent advantages or disadvantages of the implementation scenario.
The foregoing disclosure is merely illustrative of some embodiments of the invention, and the invention is not limited thereto, as modifications may be made by those skilled in the art without departing from the scope of the invention.
Claims (4)
1. A method for connecting T cells and nanoparticles based on click chemistry, comprising the steps of:
(1) Extracting and expanding T cells;
(2) Labeling an azide group on the surface of a T cell membrane;
(3) Preparing indocyanine green polymer nano particles;
(4) T cells are linked to the nanoparticle;
preparing indocyanine green polymer nano particles by adopting an ultrasonic hydration method in the step (3), firstly dissolving polyglycolide lactide in 1mg/mL acetonitrile, then dissolving 180 mug of soybean lecithin, 120 mug of distearoyl phosphatidyl ethanolamine-polyethylene glycol-diphenyl cyclooctyne and 1mg of indocyanine green in 3mL of 4% ethanol, ultrasonically treating the mixed solution containing indocyanine green by using an ultrasonic cell breaker at the frequency of 20kHz and the power of 35W, dropwise adding 1mL of polyglycolide lactide acetonitrile solution into the mixed solution while the ultrasonic time is 5min, and ultrafiltering for 2-3 times by using a 10kDa ultrafilter tube to obtain indocyanine green polymer nano particles;
in the step (4), firstly, washing T cells for 1 time by using PBS buffer solution, centrifugally collecting the cells, changing fresh AIM-V culture medium without calf serum, then adding 50 mug/mL indocyanine green polymer nanoparticle solution, reacting for 1 hour, and then washing the unconnected nanoparticles by using PBS buffer solution;
the nanoparticles used are connected with diphenyl cyclooctyne groups.
2. The method of claim 1, wherein the T cells in step (1) are derived from isolated human peripheral blood mononuclear cells, which are cultured in 6-well plates at 37 ℃ in a 5% carbon dioxide/95% air environment using AIM-V medium containing 2% fetal bovine serum, for 10 days, and then anti-CD 3/CD28 magnetic beads and interleukin 2 are added to the medium to stimulate T cell differentiation, and cultured for 3 to 4 days, to obtain expanded T cells.
3. The method of claim 1, wherein 100 μm of tetraacetyl-N-azidoacetaminonamide is added to the medium of step (1) and incubated for 48 hours in step (2).
4. A nanoengineered T cell made by the ligation method of any of claims 1 to 3.
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