Method for in-situ polymerization of macromolecules on surface of living cell by selecting loci

Abstract

The invention relates to a method for in-situ polymerization of macromolecules on the surface of a living cell by selecting loci, which comprises the following steps: introducing bio-orthogonal click reaction groups to specific biological loci (such as proteins, lipids and glycans) of the living cell by using a metabolism labeling technology, and covalently linking a chain transfer agent (CTA) to the selected biological loci on a cell membrane through a click chemical reaction. The method comprises the following steps: mixing a living cell anchored with CTA with a solution containing a monomer, free CTA and hydrogen peroxide, adding ammonium ferrous sulfate to start a Fenton-RAFT polymerization reaction, and growing a polymer at a selected site of the living cell. The method has huge application potential in multiple fields such as drug delivery, biosensing and bioimaging.

Description

1. Technical Field

[0001] The present invention relates to a method for in-situ polymerizing macromolecules at selected sites on the surface of living cells. 2. Background Technology

[0002] Living cell surface engineering is to use chemistry, biology, materials science and other methods to engineer cells while maintaining cell activity, so as to achieve artificial manipulation of living cell surface groups, properties and functions. Artificial polymers are not only the easiest material form to simulate the structure and function of natural macromolecules, but can also achieve functions that are difficult to achieve with natural macromolecules by adjusting monomers. Using artificial macromolecules to engineer living cells can provide a rich model for understanding the structure and physiological functions of complex biological systems, promote the engineering research of human cells and tissues, and create new disease treatment methods based on living cells.

[0003] On the surface of living cells, the traditional polymer engineering method is to bind the synthesized polymer to the cell surface through lipid insertion, electrostatic adsorption, covalent bonding, etc. The problem with these methods is that the steric hindrance between the polymer and the cell surface molecules will lead to low connection efficiency and fail to achieve efficient and accurate grafting of polymers at the selected sites of the cell. This will affect the synthetic polymers from playing the preset regulatory functions on the cell surface.

[0004] To solve this problem, the present invention has developed a method for in situ initiation of redox-type controlled free radical polymerization at selected sites on the surface of living cells. Bioorthogonal reaction groups are introduced into selected sites such as proteins, lipids or polysaccharides on the surface of living cells through metabolic labeling technology to mediate the anchoring of chain transfer agents at designated sites, further initiating ultrafast Fenton-reversible addition fragmentation chain transfer (Fenton-RAFT) polymerization, and growing polymers in situ at different biological sites of living cells. This method is of great significance for the development of cell and tissue engineering technology, as well as cell-based disease therapy. III. Summary of the invention

[0005] The purpose of the present invention is to develop a method for in situ polymerizing polymers at selected sites on the surface of living cells.

[0006] In order to accurately control the growth site of the polymer, the present invention utilizes a metabolic labeling reagent modified with a bioorthogonal click reaction group to participate in the properties of a specific synthetic pathway in the cell, thereby realizing covalent labeling of specific biological sites of the cell. The label can be connected to a chain transfer agent (CTA) modified with a complementary group through a click chemistry reaction, thereby realizing the anchoring of CTA at a selected biological site on the cell membrane. After the living cells anchored with CTA are mixed with a solution containing monomers, free CTA and hydrogen peroxide, a small amount of ammonium ferrous sulfate is added to start the Fenton-RAFT reaction. After 2 minutes of polymerization, the polymerization reaction is terminated by exposing the reaction system to the air and diluting the solution. After polymerization, the cell activity is maintained at more than 95%. By adding olefin monomers and functional monomers to the polymerization system at the same time, in situ copolymerization can be achieved at selected sites on the surface of living cells.

[0007] The present invention is achieved through the following technical solutions:

[0008] 1) If Figure 1 As shown, an azide-modified metabolic labeling reagent is added to the cell culture medium, and after culturing the cells for a certain period of time, an azide modification can be introduced at the selected site of the cell. 1,3,4,6-tetra-O-acetyl-N-azidoacetylaminomannose (Ac4ManNAz), 1,3,4,6-tetra-O-acetyl-2-[(azidoacetyl)amino]-2-deoxy-β-D-pyranose galactose (Ac4GalNAz), L-azidohomoalanine hydrochloride (AHA) and 1-azidoethylcholine (AECho) are used as metabolic labeling reagents to introduce N3 groups on sialic acid (glycans), N-acetylgalactosamine (glycans), methionine (proteins) and choline (lipids) on the cell surface, respectively.

[0009] 2) DBCO-modified chain transfer agent (DBCO-BTPA) was synthesized by reacting the amino group of dibenzocyclooctyne (DBCO) with the carboxyl group of chain transfer agent 2-{[(butylsulfonyl)carbonylsulfonyl]sulfonyl}propionic acid (BTPA). Cells modified with azide at selected sites were incubated with DBCO-BTPA for 1 hour, and DBCO-BTPA was covalently linked to the N3 group at the selected site of the cell using copper-free click chemistry reaction, thereby achieving anchoring of the chain transfer agent at the selected site on the surface of living cells.

[0010] 3) The living cells anchored with BTPA were mixed with a solution containing monomers, free BTPA and hydrogen peroxide, and then ferrous ammonium sulfate (Fe 2+ ) solution to initiate Fenton-RAFT polymerization. The ratio of each component in the polymerization system is [monomer]∶[BTPA]∶[H2O2]∶[Fe 2+ After 2 minutes of reaction, the reaction system was exposed to air and the solution was diluted to terminate the polymerization reaction.

[0011] Compared with the prior art, the present invention has the following characteristics:

[0012] A method for in situ polymerizing polymers at selected sites on the surface of living cells is proposed. Compared with existing polymer-based living cell surface engineering methods:

[0013] (1) The present invention realizes the controllable polymer polymerization of growth sites on the surface of living cells for the first time. By growing polymers at different sites, the recognition characteristics of engineered cells can be precisely controlled.

[0014] (2) A strategy for covalently anchoring chain transfer agents on the surface of living cells using metabolic labeling technology was proposed, which reduced unnecessary interference to the cells and improved the stability of polymer modification.

[0015] (3) A controlled free radical polymerization method initiated in situ by cells has been developed, which is extremely fast and can be completed in just two minutes. Compared with the method of grafting synthesized polymers onto the cell surface, this invention breaks through the steric hindrance of macromolecules on the cell surface and artificial high molecular weight polymers, effectively improving the grafting efficiency.

[0016] (4) After polymerization, the activity, proliferation ability, cytoskeleton, intracellular reactive oxygen species, protein and DNA expression levels, metabolic capacity, and cell functions are all well maintained.

[0017] (5) The present invention is applicable to various olefin monomers and can flexibly customize polymers with different functions on the surface of living cells by copolymerizing different types of monomers. IV. Description of the drawings

[0018] Figure 1 Schematic diagram of the method for in situ polymerizing polymers at selected sites on the surface of living cells Figure 2 Schematic diagram of the reaction principle of in situ polymerization of polymers at selected sites on the surface of living cells V. Specific implementation methods

[0019] Example 1: Combination Figure 1 , polymerizing polymers in situ at selected sites on the surface of living cells.

[0020] The polymerization method is divided into three steps: (1) metabolic labeling of cells; (2) anchoring of chain transfer agents; and (3) in situ polymerization.

[0021] (1) Metabolic labeling of cells

[0022] The metabolic labeling method using Jurkat T cells as an example is as follows.

[0023] Glycan labeling: Jurkat T cells were suspended in RPMI-1640 complete medium (RPMI-1640 incomplete medium supplemented with 10% fetal bovine serum, 2mM GlutaMAX supplement and 1mM sodium pyruvate) containing 40μM Ac4ManNAz or Ac4GalNAz and cultured in a humidified incubator at 37°C and 5% CO2 for 48 hours to achieve azide labeling of cell surface sialic acid (Sia) or mucin-type O-acetylgalactosamine (GalNAc).

[0024] Protein labeling: Jurkat T cells were suspended in serum-free methionine-free RPMI-1640 medium and cultured in a humidified incubator at 37°C and 5% CO2 for 1 hour to deplete residual methionine (Met). The cells were then collected by centrifugation and dispersed in methionine-free RPMI-1640 complete medium containing 0.1 mM AHA and cultured in an incubator for 4 hours to install AHA to the Met site of cell surface proteins.

[0025] Lipid labeling: Jurkat T cells were suspended in RPMI-1640 complete medium containing 1 mM AECho and cultured in a humidified incubator at 37°C and 5% CO2 for 24 hours to install azide at the choline (Cho) site on the cell surface.

[0026] (2) Anchoring of chain transfer agents

[0027] The metabolically labeled cells were collected by centrifugation and washed three times with RPMI-1640 incomplete medium without phenol red. The cells were then evenly dispersed in a PBS solution containing 40 μM DBCO-BTPA. After reacting at 4°C for 1 hour, the cells were collected by centrifugation and washed three times with RPMI-1640 incomplete medium without phenol red to obtain cells modified with a chain transfer agent at the selected site.

[0028] (3) In situ polymerization

[0029] The concentration ratio of each component in the polymerization system is [monomer]∶[CTA]∶[H2O2]∶[Fe 2+]=80∶1∶0.3∶0.1. The polymerization medium is 4900μL of phenol red-free RPMI-1640 incomplete medium with 100μL of B-27 antioxidant-free additive. The polymerization process is as follows: 120μmol monomer, 1.5μrnol BTPA and 0.45μrnol H2O2 are dissolved in 190μL of polymerization medium, added to a glass bottle and sealed. The mixture is bubbled with nitrogen for 30 minutes. The cells modified with chain transfer agent obtained in step (2) are dispersed in 100μL of pre-deoxygenated polymerization medium and then quickly added to the glass bottle. After sealing the glass bottle, 10μL of ammonium ferrous sulfate solution (15mM) is added through a degassed syringe to start the polymerization reaction. The reaction is carried out in a shaker at 200-250rpm for 2 minutes, and then the reaction solution is exposed to air, and 700μL of RPMI-1640 complete medium is immediately added to dilute the reaction solution to terminate the polymerization. The polymerized cells were collected by centrifugation at 4°C, washed three times with phenol red-free RPMI-1640 incomplete medium, and resuspended in phenol red-free RPMI-1640 incomplete medium to obtain engineered cells that grow polymers at selected sites. This method is suitable for homopolymerization of olefin monomers or copolymerization with functional monomers on the surface of living cells. The proportions of each component in different polymerization systems are shown in the following table:

[0030]

[0031] Note: HPMA: 2-hydroxypropyl methacrylamide; AA-Bio: acrylamide-polyethylene glycol (PEG) 4-biotin, AA-N3: acrylamide-PEG4-azide; MA-Man: methacrylamide-mannose, MA-Gal: methacrylamide-galactose.

Claims

1. A method for in situ polymerizing polymers at selected sites on the surface of living cells, characterized in that the azide groups are covalently modified at selected biological sites (proteins, lipids, polysaccharides) on the cell surface using metabolic labeling technology, and then a chain transfer agent is connected to the azide group through a click chemistry reaction, and then a Fenton-reversible addition fragmentation chain transfer polymerization reaction is initiated to generate polymers at selected sites on the surface of living cells.

2. The method for in situ polymerizing polymers at selected sites on the surface of living cells according to claim 1, characterized in that polymers can be generated in situ at different biomolecular sites of cells, including amino acids, choline, and sugars, by selecting different metabolic labeling reagents modified with azide.

3. The method for in situ polymerizing a polymer at a selected site on the surface of a living cell according to claim 1, characterized in that: The azide groups introduced at selected sites of cells through metabolic labeling technology can undergo click chemistry reactions with dibenzocyclooctyne-modified chain transfer agents in physiological buffer to achieve covalent anchoring of the chain transfer agents at selected sites on the surface of living cells.

4. The method for in situ polymerizing a polymer at a selected site on the surface of a living cell according to claim 1, characterized in that: Under anaerobic conditions, living cells with chain transfer agents anchored at selected sites are mixed with a solution containing monomers, free chain transfer agents and hydrogen peroxide, and then ammonium ferrous sulfate is added to initiate the Fenton-reversible addition-fragmentation chain transfer polymerization reaction to grow polymers at selected sites of the living cells.

5. The method for in situ polymerizing a polymer at a selected site on the surface of a living cell according to claim 1, characterized in that: The method can be used to homopolymerize olefin monomers on the surface of living cells or copolymerize with functional monomers to generate homopolymers or functional copolymers on the surface of living cells.

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