CN113493545B - Composition and method for producing linear polymer by light-operated RAFT polymerization in living cells - Google Patents
Composition and method for producing linear polymer by light-operated RAFT polymerization in living cells Download PDFInfo
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- C08F283/00—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G
- C08F283/06—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals
- C08F283/065—Macromolecular compounds obtained by polymerising monomers on to polymers provided for in subclass C08G on to polyethers, polyoxymethylenes or polyacetals on to unsaturated polyethers, polyoxymethylenes or polyacetals
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Abstract
The invention discloses a composition and a method for producing linear polymer by light-controlled RAFT polymerization in living cells. The uptake of the composition by the cells is achieved by incubating the monomers, chain transfer agent and photoinitiator with living cells, and then in situ synthesis of the polymer in the cells is achieved by illumination. Further, the polymer synthesized in the cells is extracted, characterized, tested and compared with the polymer obtained by in vitro simulation, and the polymer synthesized in the cells is found to have higher molecular weight and narrower molecular weight distribution, and belongs to a typical controllable polymerized high polymer product. Therefore, the invention can realize the purpose of introducing the structure-controllable non-natural polymer into living cells, and can further realize the purpose of artificially regulating and controlling the composition and structure in the cells, and even can realize the purpose of controlling the cell behaviors and cell functions.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a composition and a method for generating a linear polymer by light-operated RAFT polymerization in living cells.
Background
Because of the high complexity of the intracellular chemistry and the sensitivity of living cells to external stimuli, many organic synthetic reactions are difficult to perform within cells without damaging the cells, and thus the number of artificial chemical reactions that can be performed within living cells is currently known to be limited. However, the synthesis of the non-natural compound in living cells by an artificial method has great development potential in the field of biological medicine, and is an important research field in biological research all the time, and the purposes of synthesis of artificial organelles, regulation of cell structures (such as viscosity and cytoskeletal structures) and even artificial regulation of cell behaviors are expected to be realized by artificially synthesizing the non-natural compound in cells.
With the rapid development of bio-orthogonal chemistry, an increasing number of unnatural chemical reactions have been found to be applicable in living cells as well as biological tissues, and have been widely used in research in the biomedical field. Common bio-orthogonal chemical reactions mainly include cycloaddition reactions between azide-cyclic alkynes, electron-withdrawing Diels Alder reactions between tetrazine-olefins, nucleophilic reactions between aldehydes/ketones-hydroxylamine/hydrazines, diels Alder reactions between conjugated dienes and olefins, and the like. The reactants that are generally reacted in cells are often modified with natural polymers and small organic molecules, and typical polymer synthesis achieved in cells is rarely performed.
It is found that intracellular polymerization can significantly change cell properties such as cell cycle, cytoskeletal structure and cell motility, etc., and that fluorescent polymers synthesized in situ in cells can be used for long-term tracking of specific cells and can be applied to disease diagnosis and treatment due to the characteristic of slow metabolism of polymers, meanwhile, the invention realizes in-situ construction of polymer nanoparticles in cells through hydrophilic and hydrophobic changes generated during polymerization, and artificially introduces polymer nanostructures in living cells.
However, due to the high reactivity of the radical reaction itself, the produced polymer mostly has a relatively wide molecular weight distribution (the reaction is not controllable), and due to the complex chemical environment in the cell, the high-reactivity radical intermediate is easy to react with active groups such as amino groups, mercapto groups and the like to undergo chain transfer, so that the molecular weight of the obtained polymer is relatively small. Compared with the traditional free radical polymerization, the novel light-operated reversible addition fragmentation chain transfer (RAFT) polymerization has stronger time and space controllability and more stable active intermediate due to the unique polymerization reaction mechanism, so that the synthesis of the polymer with higher molecular weight can be realized in a relatively complex reaction system in a controllable manner (the molecular weight distribution is narrower). Meanwhile, the initiation efficiency of RAFT polymerization is obviously higher than that of the traditional free radical polymerization, so that the use amount of monomers and an initiator can be greatly reduced while the molecular weight of the generated polymer is ensured, and the toxicity of a polymerization system on cells and organisms is reduced.
Disclosure of Invention
The invention provides a composition and a method for generating a linear polymer by light-controlled RAFT polymerization in living cells, wherein the composition is taken up by the cells through incubating a monomer, a chain transfer agent and a photoinitiator with the living cells, and the in-situ synthesis of the polymer in the cells is realized through illumination. The synthesized polymers have a relatively high molecular weight (19-32 kDa) and a relatively narrow molecular weight distribution (< 1.1), which are typically polymeric polymer products of controlled polymerization.
A composition comprising a monomer, a RAFT chain transfer agent, and a photoinitiator,
the monomer comprises a first monomer and a second monomer, wherein the first monomer is N, N-dimethylacrylamide, and the second monomer is biotin modified acrylate monomer, and the structure is as follows:
the RAFT chain transfer agent is 2- (butylthiocarbonylthio) thio) propionic acid;
the photoinitiator is eosin Y;
the composition is capable of producing linear polymers in living cells by light controlled RAFT polymerization.
Further, in order to ensure polymerization efficiency and the amount of polymer produced, the amounts of the first monomer and the second monomer are controlled to be 2 to 5mM and 1mM, respectively, at the maximum allowable doses for toxicity.
The RAFT chain transfer agent 2- (butylthiocarbonylthio) propanoic acid and the photoinitiator eosin Y were added at a 10:1 molar ratio, with the concentration of 2- (butylthiocarbonylthio) propanoic acid being 0.01-0.1mM to ensure formation of a typical linear polymer.
A linear polymer produced by light-controlled RAFT polymerisation in living cells, the linear polymer being prepared by:
(1) Incubating a composition with cells to allow the cells to ingest the composition, the composition comprising a first monomer, a second monomer, 2- (butylthiocarbonylthiothiothio) propionic acid, eosin Y; the first monomer is N, N-dimethylacrylamide, the second monomer is biotin modified acrylate monomer, and the structure is as follows:
(2) Light is used as a stimulation signal, so that the time/space controllability is higher, and the intracellular polymerization reaction is realized through illumination.
Further, the living cells in the step (1) are inoculated in a cell culture plate for incubation, the first monomer, the second monomer, the 2- (butylthiocarbonylthio-thio) propionic acid and eosin Y are dissolved in the cell culture medium in proportion, and the washed living cells are added for incubation; and (2) adding a cell culture medium into the cells obtained in the step (1) to realize polymerization reaction by illumination.
Further, in order to ensure polymerization efficiency and polymer production, the amounts of the first monomer and the second monomer are controlled to be 2-5mM and 1mM, respectively, at the maximum allowable doses for toxicity.
RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propanoic acid and photoinitiator eosin Y at 10:1 molar ratio, 2- (butylthiocarbonylthio) thio) acrylic acid concentration was 0.01-0.1mM to ensure formation of a typical linear polymer.
Further, the maximum excitation wavelength of the light source in the step (2) is 470nm, so that the damage to cells is small.
The linear polymer generated by light-controlled RAFT polymerization in living cells has the following structure:
the linear polymer, the living cells in the step (1) are at a ratio of 2x10 5 Inoculating the culture medium into a cell culture plate, and incubating for 18 hours at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; the first monomer, the second monomer, 2- (butylthiocarbonylthio) thio) propionic acid and eosin Y are dissolved in the cell culture medium in proportion, and cells washed with PBS are added in an amount of 2mL per well, and incubated at a constant temperature of 37 ℃ for 4 hours. In the step (2), the cells obtained in the step (1) are washed by PBS, 1mL of fresh cell culture medium is added to each well, and the polymerization reaction is realized by irradiating the culture plate with a light source for 10min vertically from the lower side of the culture plate.
A method of producing a linear polymer by optically controlled RAFT polymerization in living cells comprising the steps of:
(1) Incubating a composition with cells to allow the cells to ingest the composition, the composition comprising a first monomer, a second monomer, 2- (butylthiocarbonylthiothiothio) propionic acid, eosin Y; the first monomer is N, N-dimethylacrylamide, the second monomer is biotin modified acrylate monomer, and the structure is as follows:
(2) Light is used as a stimulation signal, so that the time/space controllability is higher, and the intracellular polymerization reaction is realized through illumination.
The method for producing the linear polymer comprises the steps that in the step (1), living cells are inoculated in a cell culture plate for incubation, a first monomer, a second monomer, 2- (butylthiocarbonylthio-thio) propionic acid and eosin Y are dissolved in the cell culture medium in proportion, and the washed living cells are added for incubation; and (2) adding a cell culture medium into the cells obtained in the step (1) to realize polymerization reaction by illumination.
Further, in order to ensure polymerization efficiency and polymer production, the amounts of the first monomer and the second monomer are controlled to be 2-5mM and 1mM, respectively, at the maximum allowable doses for toxicity.
RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propanoic acid and photoinitiator eosin Y at 10:1 molar ratio, 2- (butylthiocarbonylthio) thio) acrylic acid concentration was 0.01-0.1mM to ensure formation of a typical linear polymer.
In the method for producing the linear polymer, the maximum excitation wavelength of the light source in the step (2) is 470nm, so that the cell damage is small.
The method for producing a linear polymer, wherein in the step (1), living cells are used in an amount of 2X10 5 Inoculating the culture medium into a cell culture plate, and incubating for 18 hours at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; the first monomer, the second monomer, 2- (butylthiocarbonylthio) thio) propionic acid and eosin Y are dissolved in the cell culture medium in proportion, and cells washed with PBS are added in an amount of 2mL per well, and incubated at a constant temperature of 37 ℃ for 4 hours. In the step (2), the cells obtained in the step (1) are washed by PBS, 1mL of fresh cell culture medium is added to each well, and the polymerization reaction is realized by irradiating the culture plate with a light source for 10min vertically from the lower side of the culture plate.
The composition is applied to the aspect of regulating the cell composition and the structure.
The composition is applied to the synthesis of artificial organelles.
The composition is applied to controlling cell behaviors and cell functions.
The linear polymer is applied to the aspect of regulating and controlling the cell composition and structure.
The linear polymer is applied to the synthesis of artificial organelles.
The linear polymer is applied to control the cell behavior and the cell function.
In conclusion, compared with the prior art, the invention achieves the following technical effects:
1. the construction of controllable polymers in living cells is realized for the first time.
2. The visible light control polymerization reaction is realized, and the polymerization reaction can be controlled in time and space.
3. The used polymeric raw materials have low cytotoxicity and can not obviously influence the physiological activity of cells.
4. The polymer synthesized in the cell meets the standard of controllable polymerization, and has higher molecular weight and lower molecular weight distribution.
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 an intracellular polymerization reaction;
FIG. 2 is a synthetic route to RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propionic acid (RAFTCTA);
FIG. 3 is a schematic illustration of RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propanoic acid (RAFTCTA) in deuterated DMSO 1 HNMR profile;
FIG. 4 is a synthetic route to biotin modified acrylate monomers (BiotinMA);
FIG. 5 is a biotin modified acrylate monomer (BiotinMA) in deuterated DMSO 1 HNMR profile;
FIG. 6 is a graph showing the effect of 470nm illumination time on HeLa cell activity (test results obtained by CCK-8 assay, n=6);
FIG. 7 is a graph showing the effect of varying concentrations of N, N-Dimethylacrylamide (DMA) on HeLa cell activity (test results obtained by CCK-8 assay, n=6);
FIG. 8 is the effect of varying concentrations of biotin-modified acrylate monomer (BiotinMA) on HeLa cell activity (test results obtained by CCK-8 assay, n=6);
FIG. 9 is a graph showing the effect of varying concentrations of RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propionic acid (RAFTCTA) on HeLa cell activity (test results obtained by CCK-8 test, n=6);
fig. 10 is the effect of varying concentrations of eosin Y on HeLa cell activity (test results obtained by CCK-8 test, n=6);
FIG. 11 is a measurement of the intracellular synthesized polymer of example 5 after extraction and re-dissolution in deuterated water 1 HNMR profile;
FIG. 12 is a GPC chart of the intracellular synthesized polymer in example 5 after extraction and re-dissolution in DMF;
FIG. 13 is a graph showing the in vitro polymerization of the composition of example 5 in deuterated PBS buffer based on intracellular concentration, with real-time monitoring of monomer conversion (by comparison of 1 Integral calculation of monomer signals in HNMR map;
FIG. 14 is a measurement of the polymer synthesized in the in vitro PBS buffer according to the intracellular concentration of the composition of example 5 after dissolution in deuterated water 1 HNMR profile;
FIG. 15 is a GPC chart of the composition according to example 5 as measured by dissolving a polymer synthesized in PBS buffer in vitro according to intracellular concentration in DMF.
FIG. 16 is a measurement of the re-dissolution in deuterated water of the intracellular synthesized polymer of example 6 after extraction 1 HNMR profile;
FIG. 17 is a GPC chart showing the result of the polymer synthesized in the cells in example 6 after extraction and redissolution in DMF;
FIG. 18 is a measurement of the intracellular synthesized polymer of example 7 after extraction and re-dissolution in deuterated water 1 HNMR profile;
FIG. 19 is a GPC chart showing the result of the extraction of the intracellular synthesized polymer in example 7 and redissolution in DMF;
FIG. 20 is a measurement of the re-dissolution in deuterated water of the intracellular synthesized polymer extracted in example 8 1 HNMR profile;
FIG. 21 is a GPC chart showing the result of the extraction of the intracellular synthesized polymer in example 8 and redissolution in DMF;
FIG. 22 is a graph showing the results of the extraction of the intracellular synthesized polymer of example 9Measured after redissolving in deuterated water 1 HNMR profile;
FIG. 23 is a GPC chart showing the result of the extraction of the intracellular synthesized polymer in example 9 and redissolution in DMF.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
A method for producing linear polymer by light-controlled RAFT polymerization in living cells comprises the following steps:
(1) Incubating a composition with cells to allow the cells to ingest the composition, the composition comprising N, N-Dimethylacrylamide (DMA), biotin modified acrylate monomer (BiotinMA), RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propionic acid (RAFTCTA), photoinitiator eosin Y;
living cells at 2x10 5 Inoculating the cells to a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide to ensure cell adhesion; DMA, biotinMA, RAFT chain transfer agent RAFTCTA and photo initiator eosin Y are dissolved in a high-sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) in proportion, and cells washed with PBS (3 times) are added according to the dosage of 2mL per hole, and the cells are incubated for 4 hours at the constant temperature of 37 ℃ to realize the uptake of the composition by the cells;
in order to ensure polymerization efficiency and polymer production, the amounts of DMA and BiotinMA were controlled to be 5mM and 1mM, respectively, at the maximum allowable doses for toxicity. Rafcta and photoinitiator eosin Y were prepared at 10:1 molar ratio, RAFTCTA concentration of 0.01-0.1mM, to ensure formation of a typical linear polymer.
(2) Light is used as a stimulation signal, so that the time/space controllability is higher, and the intracellular polymerization reaction is realized through illumination.
Cells that had ingested the composition were washed 3 times with PBS to remove surface-adherent reactants and 1mL fresh DMEM medium was added to each well, with a 470nm LED blue light source (power: 260 mW/cm) 2 ) The polymerization reaction is realized by vertically irradiating for 10min from the lower part of the culture plate, the damage to cells by the light source of the wave band is small, and experiments prove that the polymerization reaction of the cells cannot be initiated by the light source of 405nm and the light source of 530 nm.
Example 1: synthesis step of RAFT chain transfer agent 2- (butylthiocarbonylthio thio) propanoic acid
As shown in FIG. 2, 50mL of a THF solution containing 16.0g of n-butanol was added dropwise to a 150mL THF suspension containing 9.0g of KOH over 30min, stirred at room temperature for 30min, and then 50mL of a THF solution containing 17.0g of CS was added dropwise 2 Stirring at room temperature for 24h, concentrating under reduced pressure to 50mL, dropwise adding 50mL of THF solution containing 22.4g of n-propyl ammonium bromide into the concentrated solution under stirring, stirring at room temperature for 24h, removing the solvent under reduced pressure to obtain a target crude product, and purifying by silica gel column chromatography to obtain a bright yellow crystalline target product.
Example 2: synthesis step of biotin modified acrylate monomer BiotinMA
As shown in FIG. 4, 1.0g of biotin, 1.5g of poly (ethylene glycol) methacrylate (Mn=500 Da), 0.78. 0.78g N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride, 0.05g of 4- (dimethylamino) pyridine were dissolved together in 50mL of DMF, stirred at room temperature for 24 hours, the solvent was removed under reduced pressure, and the resulting solid was redissolved in dichloromethane with 5% NaHCO respectively 3 Washing 1% hydrochloric acid and saturated saline for 3 times respectively, drying with anhydrous sodium sulfate, removing dichloromethane under reduced pressure to obtain a target crude product, and purifying by silica gel column chromatography to obtain a white viscous liquid target product.
Example 3: characterization of cell Activity by CCK-8 assay
In the CCK-8 test, heLa cells were first treated at 1X10 4 Is inoculated in 96-hole cell culture plate and incubated for 18h at constant temperature of 37 ℃ and 4% carbon dioxide to ensure cell adhesionA wall; then the cells are subjected to different concentrations of drug administration or irradiation (5-20 min,260 mW/cm) 2 ) Waiting for treatment, and continuing constant-temperature incubation for 24 hours; after 24h, the original culture medium is sucked out, the cells are washed 3 times by PBS, 100 mu L of diluted CCK-8 solution (the ratio of the CCK-8 solution to the culture medium is 1:10) is added into each hole, after incubation for 4h at constant temperature, the ultraviolet absorption at 450nm position in each hole is detected by an enzyme-labeling instrument, and the absorbance of the treated cells is compared with that of untreated cells to obtain the cell activity value.
In the above method, it was confirmed by CCK-8 test that 10min light and composition hardly caused toxicity to living cells in the working concentration range, as shown in FIGS. 6-10.
Example 4: in vitro simulation of polymerization reactions occurring within cells
The concentration of each component of the composition in the cell is detected by ultraviolet-visible spectrum, high performance liquid chromatography and other methods, the optimal administration concentration is obtained by in vitro simulation optimization, and the polymer extracted from the cell is compared with the polymer obtained by in vitro simulation, so that the structural property of the polymer obtained in the cell is verified. The polymer synthesized by intracellular polymerization has higher molecular weight and narrower molecular weight distribution range, and the controlled polymerization in the cell is successfully realized.
The absorption spectrum of the cell lysate was quantitatively analyzed by uv-vis absorption spectrum and compared with the standard of the corresponding compound according to the above method to obtain the concentrations of the respective components of the composition taken into the cells, the concentrations of which are shown in table 1.
The polymerized cells are lysed by RIPA cell lysate, and the generated polymer containing biotin unit structure is extracted by using the selective interaction of biotin and streptavidine, the extraction and separation of the generated polymer in cells are realized, the molecular weight and molecular weight distribution are further characterized by GPC, and the composition structure is analyzed by NMR.
Polymerization reactions occurring in cells can be simulated in vitro by polymerizing the measured concentrations of the components of the intracellular composition in vitro with PBS as a solvent, and by comparing with the extracted polymer, reaction parameters such as polymer conversion can be simulated, and further the composition, relative molecular mass, and molecular weight distribution of the intracellular polymer produced can be confirmed. As shown in Table 2, the polymer extracted from the cell has similar structure property to the polymer generated by in vitro simulation, has higher molecular weight and narrower molecular weight distribution, and belongs to a high molecular product generated by typical controllable polymerization.
TABLE 1
TABLE 2
Example 5: light controlled RAFT polymerization in HeLa cells to linear Polymer (5 mM DMA, 1mM Biotinma, 0.1mM RAFTCTA, 0.01mM eosin Y)
HeLa cells at 2X10 5 Inoculating the culture medium into a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; 5mM DMA, 1mM BiotinMA, 0.1mM RAFTCTA and 0.01mM eosin Y were dissolved in high sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) and HeLa cells washed with PBS (3 times) were added in an amount of 2mL per well, incubated at 37℃for 4 hours, heLa cells were washed 3 times with PBS to remove surface-adherent reactants, and 1mL fresh DMEM medium was added per well, and polymerization was effected by irradiation with 470nm LED blue light source from below the culture plate for 10 min.
By passing through 1 HNMR testing shows that the obtained polymer is DMA-BiotinMA copolymer, and from nuclear magnetic resonance spectrum, the polymer structure contains two monomers (as shown in fig. 11); it can be seen from GPC that the resulting polymer has a relatively large molecular weight (Mn=19.6 kDa) and a relatively narrow molecular weight distribution
(as shown in fig. 12). The reaction conversion was monitored by in vitro simulation in PBS at the intracellular reactant concentration (10 min conversion was 27.5% as shown in fig. 13), and the polymer obtained by in vitro polymerization was found to be similar in molecular structure, molecular weight and molecular weight distribution to the polymer obtained by in vitro simulation (as shown in fig. 14, 15 and table 2), indicating that the polymer obtained by in vitro polymerization was the desired controlled RAFT polymer.
Example 6: light controlled RAFT polymerization in HeLa cells to linear Polymer (5 mM DMA, 1mM Biotinma, 0.05mM RAFTCTA, 0.005mM eosin Y)
HeLa cells at 2X10 5 Inoculating the culture medium into a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; 5mM DMA, 1mM BiotinMA, 0.05mM RAFTCTA and 0.005mM eosin Y were dissolved in high sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) and HeLa cells washed with PBS (3 times) were added in an amount of 2mL per well, incubated at 37℃for 4 hours, heLa cells were washed 3 times with PBS to remove surface-adherent reactants, and 1mL fresh DMEM medium was added per well, and polymerization was effected by irradiation with 470nm LED blue light source from below the culture plate for 10 min.
By passing through 1 HNMR testing shows that the obtained polymer is DMA-BiotinMA copolymer, and from nuclear magnetic resonance spectrum, the polymer structure contains two monomers (as shown in fig. 16); the structure and monomer composition ratio are similar to those of the embodiment 1, which shows that the polymerization method is applicable to polymerization reactions under systems with different concentrations; it can be seen from GPC that the resulting polymer has a relatively large molecular weight (Mn=23.2 kDa) and a relatively narrow molecular weight distribution
As shown in FIG. 17, the polymer obtained in example 6 has a significantly higher molecular weight than that of example 5, mainly because the monomer concentration in example 6 is the same as that in example 5, but RAFTCTA is used in an amount of only half thereof, so that the number of monomers polymerized per polymer chain is larger and the molecular weight is larger; and the molecular weight distribution of the polymer obtained in example 6Similar to example 5, it is demonstrated that the polymerization reaction still belongs to controlled polymerization.
Example 7: light controlled RAFT polymerization in HeLa cells to linear Polymer (5 mM DMA, 1mM Biotinma, 0.01mM RAFTCTA, 0.001mM eosin Y)
HeLa cells at 2X10 5 Inoculating the culture medium into a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; 5mM DMA, 1mM BiotinMA, 0.01mM RAFTCTA and 0.001mM eosin Y were dissolved in high sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) and HeLa cells washed with PBS (3 times) were added in an amount of 2mL per well, incubated at 37℃for 4 hours, heLa cells were washed 3 times with PBS to remove surface-adherent reactants, and 1mL fresh DMEM medium was added per well, and polymerization was effected by irradiation with 470nm LED blue light source from below the culture plate for 10 min.
By passing through 1 HNMR testing shows that the obtained polymer is DMA-BiotinMA copolymer, and from nuclear magnetic resonance spectrum, the polymer structure contains two monomers (as shown in fig. 18); the structure and monomer composition ratio are similar to those of the embodiment 5, which shows that the polymerization method is applicable to polymerization reactions under systems with different concentrations; it can be seen from GPC that the resulting polymer has a relatively large molecular weight (Mn=31.7 kDa) and a relatively narrow molecular weight distribution
(as shown in FIG. 19), the polymer obtained in example 7 has a higher molecular weight than those obtained in examples 5 and 6; however, the molecular weight distribution of the resulting polymer was similar to examples 5 and 6, indicating that the polymerization still belongs to controlled polymerization.
Example 8: light-operated RAFT polymerization in 1205Lu cells to produce linear polymers
1205Lu cells at 2X10 5 Inoculating the culture medium into a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; 5mM DMA, 1mM BiotinMA, 0.05mM RAFTCTA, and 0.005mM eosin Y were dissolved in high sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) in proportion and per wellHeLa cells washed with PBS (3 times) were added in an amount of 2mL, incubated at 37℃for 4 hours, heLa cells were washed with PBS 3 times to remove surface-adhesive reactants, and 1mL of fresh DMEM medium was added to each well, and polymerization was effected by irradiating vertically from below the plate with 470nm LED blue light source for 10 min.
By passing through 1 HNMR testing shows that the obtained polymer is DMA-BiotinMA copolymer, and from nuclear magnetic resonance spectrum, the polymer structure contains two monomers (as shown in fig. 20); the structure and monomer composition ratio are similar to those of the embodiment 5, which shows that the polymerization method is applicable to polymerization reactions under different cell environments; the molecular weight (mn=19.5 kDa) and molecular weight distribution (d=1.02) of the resulting polymer were close to those of example 5 (as shown in fig. 21) by GPC test, indicating that the cell type did not affect the occurrence of polymerization and the properties of the resulting polymer.
Example 9: light-operated RAFT polymerization in A375 cell to generate linear polymer
A375 cells at 2x10 5 Inoculating the culture medium into a 6-hole cell culture plate, and incubating for 18h at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; 5mM DMA, 1mM BiotinMA, 0.05mM RAFTCTA and 0.005mM eosin Y were dissolved in high sugar DMEM cell culture medium (containing 10% calf serum, 100uni/mL penicillin and streptomycin) and HeLa cells washed with PBS (3 times) were added in an amount of 2mL per well, incubated at 37℃for 4 hours, heLa cells were washed 3 times with PBS to remove surface-adherent reactants, and 1mL fresh DMEM medium was added per well, and polymerization was effected by irradiation with 470nm LED blue light source from below the culture plate for 10 min.
By passing through 1 HNMR testing shows that the obtained polymer is DMA-BiotinMA copolymer, and from nuclear magnetic resonance spectrum, the polymer structure contains two monomers (as shown in fig. 22); the structure and monomer composition ratio are similar to those of the embodiment 5, which shows that the polymerization method is applicable to polymerization reactions under different cell environments; as can be seen from GPC measurement, the molecular weight (Mn=19.3 kDa) and molecular weight distribution (D=1.02) of the obtained polymer were close to those of example 5 (as shown in FIG. 23), indicating that the cell type did not affect the occurrence and progress of polymerizationPolymer properties are produced.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (5)
1. A method for producing linear polymers by light controlled RAFT polymerization in living cells for the purpose of non-disease diagnosis and treatment methods, comprising the steps of:
(1) Incubating a composition with cells to allow the cells to ingest the composition, the composition comprising a first monomer, a second monomer, 2- (butylthiocarbonylthiothiothio) propionic acid, eosin Y; the first monomer is N, N-dimethylacrylamide, the second monomer is biotin modified acrylate monomer, and the structure is as follows:
(2) Light is used as a stimulation signal, and intracellular polymerization reaction is realized through illumination.
2. The method of producing linear polymer according to claim 1, wherein the living cells of step (1) are inoculated in a cell culture plate for incubation, the first monomer, the second monomer, 2- (butylthiocarbonylthio-thio) propionic acid, eosin Y are dissolved in the cell culture medium in proportion, and the washed living cells are added for incubation; and (2) adding a cell culture medium into the cells obtained in the step (1) to realize polymerization reaction by illumination.
3. The method of claim 1, wherein the first monomer and the second monomer are present in step (1) at a concentration of 2 to 5mM and 1mM, respectively, the molar ratio of 2- (butylthiocarbonylthio) propanoic acid to eosin Y is 10:1 and the concentration of 2- (butylthiocarbonylthio) propanoic acid is 0.01 to 0.1mM.
4. The method of producing a linear polymer according to claim 1, wherein the maximum excitation wavelength of the light source in step (2) is 470nm.
5. The method of producing a linear polymer according to claim 1, wherein the living cells in step (1) are present in a ratio of 2x10 5 Inoculating the culture medium into a cell culture plate, and incubating for 18 hours at a constant temperature of 37 ℃ under the condition of 4% carbon dioxide; the first monomer, the second monomer, 2- (butylthiocarbonylthio) thio) propionic acid and eosin Y are dissolved in a cell culture medium in proportion, and cells washed by PBS are added according to the dosage of 2mL per hole, and the cells are incubated for 4 hours at the constant temperature of 37 ℃; in the step (2), the cells obtained in the step (1) are washed by PBS, 1mL of fresh cell culture medium is added to each well, and the polymerization reaction is realized by irradiating the culture plate with a light source for 10min vertically from the lower side of the culture plate.
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