CN108841775B

CN108841775B - Method for dynamic functionalization of cell membranes

Info

Publication number: CN108841775B
Application number: CN201810553009.3A
Authority: CN
Inventors: 李永勇; 董海青; 何良华; 李艳
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2018-05-31
Filing date: 2018-05-31
Publication date: 2021-09-03
Anticipated expiration: 2038-05-31
Also published as: CN108841775A

Abstract

The invention relates to a method for dynamically functionalizing a cell membrane, which is characterized in that a disulfide substance with high reaction activity performs exchange reaction with a sulfhydryl on the surface of the cell membrane in a physiological environment to dynamically functionalize the cell membrane to obtain a cell with a cell membrane engineered by a material. Compared with the prior art, the invention is suitable for different cell types in the process of functionalizing the cell membrane, the whole process has high cell compatibility, and the used disulfide substance with high reaction activity can be automatically degraded after a period of time, thereby effectively avoiding the long-term influence on the cell function. The technology can temporarily regulate and control the functions of cells such as proliferation, period and endocytosis efficiency, and has wide application prospect in cell fate and function regulation and control.

Description

Method for dynamic functionalization of cell membranes

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a method for dynamic functionalization of cell membranes.

Background

The cell membrane is used as a boundary on a cell structure, is composed of a phospholipid bilayer and mosaic proteins, not only maintains a natural barrier of a relatively independent environment between a cell and the surrounding environment, but also is an intelligent portal for ensuring the complex substance, energy and information exchange between the cell and the surrounding environment. The cell membrane itself is a complex, precise, dynamic, ordered and disordered molecular engineering that is difficult to compare favorably. The dynamic and biological functional properties of cell membrane structures present a common key technical problem for cell membrane macromolecule engineering. Because the cell structure is always in dynamic change, the cell is not sufficiently constructed and is easy to be directly endocytosed by the cell, and the cell is easy to be accidentally damaged by cell functions or even to be apoptotic after being excessively constructed. Nevertheless, efforts by researchers continue to overcome difficulties, developing a range of strategies.

Recently, Langer predicted "live Biomaterials" to be the future development direction in a prospective review. In addition, cell membrane macromolecular engineering is expected to answer technical bottlenecks associated with many cellular applications. Therefore, the relation between nature and artificial engineering highlights important scientific and technical values from the viewpoint of both foundation and application research. At present, cell membrane engineering makes great progress in the aspects of regulating and controlling the immunogenicity, the mobility and the adhesiveness of cells, improving the capability of the cells in resisting severe environment, controlling cell metabolism, assembling artificial receptors, assembling cell groups, assisting in stimulating the cell functions, removing environmental heavy metals, inducing stem cell differentiation, delivering functional molecules and the like. The main idea of cell membrane engineering is to modify the structure of cell membrane by using the physicochemical properties of cell membrane, mainly including electronegativity of cell membrane and rich carboxyl, amino and sulfhydryl of protein, in order to achieve the purpose of regulation and control (expbiol Med.2016; 241: 1098-106). The method for coating based on the electrostatic interaction of the cationic polymer and the negatively charged cell membrane is more and relatively mature. For example, based on electrostatic adsorption, a polylysine and ascorbic acid polymer layer, and a gelatin and sodium alginate layer are deposited on the cell surface (Chemnanomat.2016; 2: 376-84.). The strategy has high efficiency, can accurately control the number of polymer layers, but is limited by the toxicity of the cationic polymer, and is difficult to exert larger influence. Chemical modification by addition reaction of thiol group with maleimide group based on carboxyl amino condensation reaction is also widely used. For example, the reaction of amino groups on the surface of islet cells and active ester is utilized to modify the surface of the islet cells with a PEG layer, which can help to inhibit the immune response of the islet cells and is beneficial to the success of islet transplantation.

In response to some extreme environments (heat, drying, radiation, chemical, etc.), bacillus temporarily forms a strong protective layer of spores, mainly composed of peptidoglycan. The buds break down later and the bacteria return to normal physiological conditions. (Accounts Chem Res.2016; 49:792-800) cell membrane macromolecule engineering may also play a similar protective role, however, less research has been directed towards designing how to disrupt. This relationship is a "good after" problem after completion of the engineering mission-avoiding the persistent presence of artificial structures on the cell membrane. This is of crucial importance in many situations, since most of the time the regulation is temporary. For example, modulation of stem cell differentiation by stem cell surface engineering does not require that the engineered introduced material be carried after differentiation. In targeted delivery, it is also desirable that the functional material carried on the surface is shed and infiltrated into the tissue to function after the cells chemotaxis the targeted tissue.

Disclosure of Invention

The invention provides a method for dynamic functionalization of cell membranes by aiming at the targeted design of the disruption, and the method is based on covalent bonding of disulfide bonds and the cell membrane surface to dynamically functionalize the cell membranes.

The purpose of the invention can be realized by the following technical scheme:

a method for dynamically functionalizing cell membrane features that the disulfide with high reaction activity and the mercapto group on the surface of cell membrane are effectively exchanged to dynamically functionalize the cell membrane to obtain the cell engineered by the material.

The disulfide with high reaction activity is selected from one or more of the following substances:

(1) substances only containing free sulfydryl react with substances with activated sulfydryl to form disulfide substances with high reaction activity;

(2) the disulfide bond is broken by the substance capable of breaking the disulfide bond to obtain the free sulfhydryl, and then the free sulfhydryl is activated and reacted with the substance with activated sulfhydryl to form the disulfide substance with high reaction activity;

(3) the substance containing free sulfydryl and disulfide bond reacts with the substance with activated sulfydryl to form disulfide substance with high reaction activity.

The substance containing free sulfydryl comprises a macromolecular compound or a small molecular compound;

the substance which does not contain free sulfydryl and contains disulfide bonds comprises a macromolecular compound or a small molecular compound;

the substance containing both free sulfydryl and disulfide bonds comprises a macromolecular compound or a small molecular compound;

macromolecular compounds include proteins and the like. The macromolecular compounds or the small molecular compounds may be compounds that contain functionality.

The substance containing only free mercapto groups refers to a substance containing 2 or more free mercapto groups.

The substances that can break the disulfide bond include, but are not limited to, Dithiothreitol (DTT), glutathione, or the like.

The substance having an activated mercapto group includes 5,5' -dithiobis (2-nitrobenzoic acid) and the like.

The cell membrane is derived from human or animal tumor cells, stem cells, immune cells or lymphocytes, etc. with rich sulfhydryl on the surface.

The exchange reaction can be completed within 2 hours, and no toxic and harmful substances are used, so that the method is rapid and efficient.

After the exchange reaction, the process of removing the disulfide substance with high reaction activity is also included.

The technical scheme has good biocompatibility and does not influence the normal physiological function of cells.

The disulfide substance with high reaction activity used in the invention can be automatically absorbed by cells within a few days, thereby avoiding long-term influence on cell functions.

The invention can maintain the normal physiological function of the cell and simultaneously carry out multi-level regulation and control on the cell, including the aspects of proliferation, period, endocytosis efficiency and the like.

The invention also relates to the cell membrane engineered cell obtained by the method.

The use of protein materials is described in more detail as an example: through three-dimensional structure regulation and control of the protein, sulfydryl is exposed through reduction treatment, and a functional group with strong leaving capacity is modified on the sulfydryl, so that the sulfydryl activated protein is obtained. The activated protein can efficiently modify the cell surface by forming disulfide bonds with sulfydryl on various cell membranes, and can form disulfide bonds with the sulfhydrylated protein by utilizing residual activated groups for continuous and multiple-time assembly engineering. The feasibility of the whole method is proved, and the method is green in process, mild in condition, rapid in reaction and high in engineering efficiency.

Compared with the prior art, the invention is suitable for different cell types in the process of functionalizing the cell membrane, the whole process has high cell compatibility, and the used disulfide substance with high reaction activity can be automatically degraded after a period of time, thereby effectively avoiding the long-term influence on the cell function. The technology can temporarily regulate and control the functions of cells such as proliferation, period and endocytosis efficiency, and has wide application prospect in cell fate and function regulation and control.

Drawings

FIG. 1 is a scanning electron micrograph of the pre- (a) and post- (B) BSA engineered B16 cells used in the examples.

FIG. 2 is a confocal image of laser light after membrane engineering of B16 cell membranes at different times after labeling albumin with FITC, and cell nuclei were stained with DAPI in the examples.

FIG. 3 shows the efficiency of engineering to promote the entry of poorly entering substance (dextran) in the examples. The cell entry amount was analyzed by flow-based quantification of dextran.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

1. Bovine serum containing 1 free thiol and 17 disulfide bonds was added with a certain amount of SDS to promote protein exposure to the hydrophobic region, and then a proper amount of DTT was added to break the disulfide bonds to obtain albumin containing free thiol.

2. And (3) allowing a proper amount of activated protein to interact with the target cell-B16 for 20min to several hours.

3. Excess material was removed by centrifugation and after washing several times, cell membrane engineered cells were obtained.

The scanning electron microscope pictures before and after the bovine serum albumin engineered B16 cell used in this example are shown in FIG. 1, and it can be seen that the cell membrane of the B16 cell has obvious change.

4. And (3) analyzing the proliferation capacity: after CFSE labeling, when the cell is divided and proliferated, the cytoplasmic protein with fluorescence is evenly distributed to the second generation cell, so that the fluorescence intensity is reduced to half compared with the first generation cell; by analogy, the fluorescence intensity of the third generation cell obtained by division is weakened again compared with that of the second generation cell. This phenomenon can be detected and analyzed by flow cytometry under 488nm excitation light, and the cell division and proliferation conditions can be further analyzed by detecting the continuous decrease of the cell fluorescence intensity, and the result is shown in fig. 2. This principle can be used to detect the proliferation of cells.

5. The flow cytometry quantitative analysis cell engineering technology promotes the phagocytosis efficiency of cells. Taking DC2.4 as an example, DC2.4 cells in the logarithmic growth phase were subjected to centrifugal digestion, and the cell concentration was set at 5X 10⁴One per ml. According to 5X 10 per hole⁴Concentration of cells per ml, cells were plated into 24-well plates, 1ml per well. The cells were coated as described above. Adding fluorescence labeling glucan, incubating for different times, sampling and analyzing detection results by a FACSCalibur flow cytometer, and calculating the average fluorescence intensity of each group at different times, wherein the result is shown in figure 3.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A method for dynamically functionalizing a cell membrane is characterized in that three-dimensional structure regulation and control are carried out on a protein, a sulfhydrylation protein is obtained by reducing and exposing sulfhydryls, and a functional group with stronger leaving capacity is modified on the sulfhydrylation protein sulfhydryls to obtain a sulfhydryls activated protein;

under the in vitro physiological environment, the sulfhydryl activated protein and the sulfhydryl on the surface of the cell membrane generate high-efficiency exchange reaction, and the residual activating group of the sulfhydryl activated protein and the sulfhydryl protein form a disulfide bond to realize dynamic functionalization of the cell membrane and obtain the cell with the cell membrane engineered by the material.

2. The method of claim 1, wherein the cell membrane is derived from a human or animal tumor cell, stem cell or immune cell with surface rich thiol.

3. The method of claim 1, wherein the exchange reaction is performed within 2 hours.

4. The method of claim 1, further comprising removing the thiol-activated protein after the exchange reaction.

5. A cell membrane engineered cell obtained by the method of any one of claims 1 to 4.