CN114480506A

CN114480506A - Method for improving transfection efficiency of stem cells difficult to transfect

Info

Publication number: CN114480506A
Application number: CN202210149539.8A
Authority: CN
Inventors: 阮刚; 丁万川; 温晓薇
Original assignee: NANTONG MATERIAL ENGINEERING TECHNOLOGY RESEARCH INSTITUTE OF NANJING UNIVERSITY
Current assignee: NANTONG MATERIAL ENGINEERING TECHNOLOGY RESEARCH INSTITUTE OF NANJING UNIVERSITY
Priority date: 2022-02-18
Filing date: 2022-02-18
Publication date: 2022-05-13

Abstract

The invention provides a method for improving the transfection efficiency of stem cells difficult to transfect. Our previous studies found that the low transfection efficiency of a class of mesenchymal stem cells (BMSCs) was mainly due to the difficulty of the ingested material escaping from its vesicles. We provide a method for solving the problem of difficult vesicle escape in BMSCs, further improve the transfection efficiency of the BMSCs, and can be widely applied to the fields of stem cell engineering, protein therapy, gene therapy and the like.

Description

Method for improving transfection efficiency of stem cells difficult to transfect

Technical Field

The invention relates to the technical field of in vitro cell gene transfection, in particular to a method for improving the transfection efficiency of stem cells difficult to transfect.

Background

The transfer of exogenous macromolecules in cells is of great significance to basic biological research and clinical application. However, due to the low transfection efficiency and reagent-related cytotoxicity, this intracellular delivery method is limited to certain cell types and is generally not suitable for cells that are difficult to transfect, including stem cells, suspension cells, and immune cells. And the underlying cause of low transfection efficiency is not clear. In previous work, we studied the cellular transport of exogenous substances in bone marrow mesenchymal stem cells (BMSCs), a difficult-to-transfect cell, and found that vesicle escape was one of the reasons for low transfection efficiency.

To address this particular intracellular delivery problem, we have attempted four screening methods to increase intracellular vesicle escape capacity, namely vesicle integrity disrupters (chloroquine at different concentrations), membrane fluidity enhancers (different organic solvents), large hydrophobic nanoscale surfaces (facilitating transmembrane) and transmembrane molecules (PDS).

The screening results show that compared with other methods, PDS can prevent the exogenous substances from being captured by vesicles. In addition, we used the mcherry plasmid to study transfection efficiency in BMSCs. We observed that higher transfection efficiencies resulted, especially compared to Lipo3000 on the market.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a method for improving the transfection efficiency of stem cells difficult to transfect, which effectively helps exogenous substances enter cells, avoids vesicle capture and further improves the transfection efficiency of the stem cells difficult to transfect.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for improving the transfection efficiency of stem cells difficult to transfect comprises the following steps:

1) synthesizing a polydithio Polymer (PDS);

2) coupling a PDS polymer to a support material;

3) the carrier material carries the target biomolecule;

4) adding the complex into stem cells to be transfected, incubating and culturing conventionally.

Further, the synthesized PDS polymer has a main chain composed of disulfide bonds (S-S), side chains composed of guanidine groups, and a polymer molecular weight in the range of 8000 to 15000.

Further, the carrier material used includes a silica mesoporous material, a polymer nanomaterial, a liposome, a metal organic framework, and the like.

Further, the biomolecules used include polypeptides, proteins, nucleic acids, and the like.

Further, the stem cells to be transfected include bone marrow mesenchymal stem cells (BMSCs), Adipose Mesenchymal Stem Cells (AMSCs), human umbilical cord mesenchymal stem cells (HMSCs) and the like.

Has the advantages that: compared with the prior art, the prepared PDS carrier material can directly bring biomolecules into cytoplasm of stem cells which are difficult to transfect, thereby improving the transfection efficiency, especially compared with commercially available Lipo 3000.

Drawings

FIG. 1 is a schematic diagram of four methods for improving the vesicle escape capacity of BMSCs, and the quantitative analysis and PDS synthesis of the BMSCs and the co-localization of the BMSCs and vesicle dyes.

FIG. 2 is a biological electron micrograph of wQD-PDS in BMSCs and single particle motion analysis.

FIG. 3 shows wQD-PDS co-localization quantitative analysis with membrane dye DiR in different stem cells.

FIG. 4 shows the transfection efficiency of different vector-carrying plasmids in BMSCs.

Detailed description of the invention

The technical solutions in the present invention will be described in further detail with reference to specific embodiments and drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined by the appended claims.

Example 1: a screening method for solving the problem of vesicle escape of BMSCs comprises the following specific operations:

1) water-soluble quantum dots (wQD) are prepared and attached with a cell-penetrating peptide (Tat) (biological targeting molecule that recognizes the nucleus) on their surface. Since the membrane-penetrating peptide Tat (the target position is cell nucleus) can help nanoparticles enter cells, but the nanoparticles are trapped in vesicles formed by invagination of cell membranes and are difficult to escape because the nanoparticles enter cells through endocytosis with the help of Tat.

2) Method 1, wQD-Tat was co-cultured with BMSCs for 8 hours, then 200. mu.M chloroquine was added for 0.5 hour, and finally DiR dye was added for 0.5 hour. And observing under a laser confocal microscope, and performing statistical analysis.

3) Method 2, wQD-Tat and 1% cosolvent are added into BMSCs at the same time for co-culture for 8 hours, then DiR dye is added for co-culture for 0.5 hour, and finally observation is carried out under a laser confocal microscope, and statistical analysis is carried out.

4) And 3, adding the QD-Tat with the 90% hydrophobic surface into the BMSCs for co-culture for 8 hours, then adding the DiR dye for co-culture for 0.5 hour, and finally observing under a laser confocal microscope and carrying out statistical analysis.

5) Method 4, Synthesis of PDS as shown in FIG. 1. wQD-PDS was prepared, then co-cultured for 8 hours with BMSCs, then co-cultured for 0.5 hours with DiR dye, and finally observed under a laser confocal microscope and subjected to statistical analysis.

As shown in fig. 1, it was found by the implementation of four methods that only wQD with PDS polymer molecules attached thereto could achieve vesicle-free capture.

Example 2: wQD-bioelectric microscopy of PDS in BMSCs and single particle motion analysis, specifically operating as follows:

1) after 8 hours of incubation of wQD-PDS in BMSCs, the cells were digested and centrifuged to remove the supernatant, mixed with 3% glutaraldehyde for 10 min, washed 2 times with deionized water, and acetone fractionated dehydrated. Cells were embedded and cut into 100 nm thick slices. The cells were then stained and finally TEM images were taken. As shown in fig. 2a, wQD was distributed in the cytoplasm and was not encapsulated in vesicles.

2) Using single particle tracking, 2,500 wQD motion trajectories were obtained in 30 cells. The trajectory of the fluorescent object is formed by connecting its positions at different points in time, which are determined by using a commercial rotating disk confocal microscope. For each trajectory, a quantitative relation of Mean Square Displacement (MSD) to duration (Δ t) is calculated, the motion is classified (including directional motion, normal diffusion, abnormal diffusion and angular diffusion) based on the MSD- Δ t relation, ratio columns of different motion types are determined, and finally a diffusion coefficient D value is calculated. As shown in FIG. 2b, wQD-PDS exhibited 88% of the diffusive motion in BMSCsWhile D is 0.111 + -0.001 μm²And/s, further illustrating that wQD-PDS is distributed predominantly in the cytoplasm without being trapped by the vesicles in BMSCs.

Example 3: wQD-PDS was quantitatively analyzed in different stem cells in co-localization with the membrane dye DiR, specifically by:

wQD-PDS was co-cultured with human umbilical cord mesenchymal stem cells (HMSCs), Adipose Mesenchymal Stem Cells (AMSCs) and bone marrow mesenchymal stem cells (BMSCs) for 8 hours, then added with DiR dye for co-culture for 0.5 hours, and finally observed under a confocal laser microscope and statistically analyzed.

As shown in FIG. 3, wQD-PDS has better vesicle-free capture ability for a class of stem cells difficult to transfect.

Example 4: the transfection efficiency of different carrier plasmids in BMSCs is specifically operated as follows:

1) preparing a silicon dioxide nano Material (MSN) with the aperture of about 12 nm, and respectively connecting the PDS and the Tat.

2) MSN-PDS, MSN-Tat and commercially available Lipo3000 were loaded with equal amounts of plasmid mcherry.

3) The cells were co-cultured with BMSCs for 48 hours, and finally observed under a laser confocal microscope and subjected to statistical analysis.

As shown in FIG. 4, the plasmid loaded with MSN-PDS has higher transfection efficiency than the other two.

The above examples show that a screening method provided by the present invention can obtain a method for improving the vesicle capture-free ability of exogenous substances in BMSCs and even in a class of stem cells difficult to transfect. Thereby improving the transfection efficiency of the BMSCs and being widely applied to the fields of stem cell engineering, protein therapy, gene therapy and the like.

Claims

1. A method for improving the transfection efficiency of stem cells difficult to transfect comprises the following steps:

1) synthesizing a polydithio Polymer (PDS);

2) coupling a PDS polymer to a support material;

3) the carrier material carries the target biomolecule;

2. The method for improving the transfection efficiency of the stem cells difficult to transfect according to claim 1, which is characterized in that: the PDS polymer synthesized in step 1 has a main chain composed of disulfide bonds (S-S), side chains composed of guanidine groups, and a polymer molecular weight in the range of 8000 to 15000.

3. The method for improving the transfection efficiency of the stem cells difficult to transfect according to claim 1, which is characterized in that: the carrier material used in the step 2 comprises a silicon dioxide mesoporous material, a polymer nanometer material, a liposome, a metal organic framework and the like.

4. The method for improving the transfection efficiency of the stem cells difficult to transfect according to claim 1, which is characterized in that: the biomolecules used in step 3 include polypeptides, proteins, nucleic acids, etc.

5. The method for improving the transfection efficiency of the stem cells difficult to transfect according to claim 1, which is characterized in that: the stem cells to be transfected in the step 4 comprise bone marrow mesenchymal stem cells (BMSCs), Adipose Mesenchymal Stem Cells (AMSCs), human umbilical cord mesenchymal stem cells (HMSCs) and the like.