Preparation method of single cell surface starch / CMC self-assembled shell material
Through the electrostatic layer-by-layer assembly of starch and CMC and the solidification of alginate, a stable single-cell shell material is formed, which solves the problem of strong cytotoxicity and maintains the high activity and proliferation ability of cells in adverse environments.
Patent Information
- Application Number
- CN202310151497.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-22
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2043-02-22
AI Technical Summary
Existing layer-by-layer self-assembled polyelectrolyte materials have strong cytotoxicity during the cell embedding process and are difficult to maintain high cell activity in adverse environments.
Starch and carboxymethyl cellulose (CMC) are assembled layer by layer on the cell surface through electrostatic interaction to form a multilayer shell material. Quaternized ammonium starch (Q+) and CMC are alternately coated on the cell surface, and a stable shell is finally formed by compounding alginate (ALG) with CMC and solidifying with Ca2+.
It maintains high cell activity in adverse environments, delays cell division and proliferation, and does not affect cell proliferation ability after release.
Smart Images
Figure CN116334063B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of composite material preparation, and relates to a method for preparing a single cell surface starch / CMC self-assembled shell material. Background Art
[0002] Bioethanol, which converts various biomasses into fuel alcohol through microbial fermentation, holds great promise for development. However, the fermentation environment is complex, and maintaining high microbial activity during the fermentation process is crucial to maintain bioethanol yield. Cell encapsulation technology can address this issue by constructing a shell on the cell surface. Preparation methods include covalent grafting, non-covalent grafting, and layer-by-layer self-assembly. Layer-by-layer self-assembly offers milder conditions and can better maintain cell activity. However, the polyelectrolyte materials currently used are mostly synthetic and have high cytotoxicity.
[0003] Starch and carboxymethyl cellulose offer advantages such as a wide range of raw material sources, good biocompatibility, and easy degradation. Carboxymethyl cellulose carries a negative charge, while starch, through quaternization, introduces nitrogen methyl groups, giving it a positive charge. Through electrostatic interactions, these materials assemble layer by layer onto the cell surface, encapsulating the cells within the material and protecting them from environmental factors that adversely affect their survival. To maximize the utilization rate of encapsulated cells, continuous stirring is performed during the encapsulation reaction, ensuring that the polyelectrolyte material is fully deposited on the surface of the single cell through electrostatic adsorption, thus achieving single-cell encapsulation. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for preparing a single cell surface starch / CMC self-assembled shell material, wherein the shell material prepared by the method can ensure high cell activity in an environment that is not conducive to cell survival.
[0005] The technical solution adopted by the present invention is a method for preparing a single cell surface starch / CMC self-assembled shell material, which specifically includes the following steps:
[0006] Step 1, preparation of cationic starch Q + ;
[0007] Step 2: Place the cells in PBS buffer and then add the cells according to the mass ratio of cells:Q + =1:2 add Q + , stirring continuously for 30 to 40 minutes at room temperature, after centrifugal washing, the supernatant was poured out to obtain a surface coating of Q + cell / Q + ;
[0008] Step 3: Disperse the yeast cells prepared in step 2 in PBS, add CMC according to the mass ratio of cells: CMC = 1:0.5, stir continuously for 30 to 40 minutes at room temperature, centrifuge and wash, and pour off the supernatant to obtain a surface coated with a layer of Q + and a layer of CMC cells / Q + / CMC;
[0009] Step 4: Disperse the cells prepared in step 3 in PBS and then add PBS according to the mass ratio of cells:Q + =1:2 add Q + , stirring continuously for 30 to 40 minutes at room temperature, after centrifugal washing, the supernatant was poured out to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + cell / Q + / CMC / Q + ;
[0010] Step 5, processing the cells obtained in step 4 to obtain.
[0011] The present invention is also characterized in that:
[0012] The specific process of step 1 is:
[0013] 2,3-Epoxypropyltrimethylammonium chloride GTA and NaOH were dissolved in deionized water respectively, then the GTA solution and NaOH solution were mixed, and the mixture was dripped dropwise into a round-bottom flask containing amylopectin and deionized water, and stirred evenly; the final reaction solution was dialyzed with deionized water for 2-3 days, and quaternized ammonium starch Q was obtained by freeze-drying. + .
[0014] The specific process of step 5 is:
[0015] The cells prepared in step 4 were dispersed in PBS, and CMC was added according to the mass ratio of cells: CMC = 1:0.5. The mixture was stirred for 30 to 40 minutes at room temperature. After centrifugation and washing, the supernatant was discarded to obtain a surface coated with a layer of Q + , one layer of CMC, one layer of Q + and a layer of CMC cells / Q + / CMC / Q + / CMC.
[0016] The specific process of step 5 is: the cell surface obtained in step 4 forms three layers of Q + / CMC / Q + Finally, the outermost layer was adsorbed and embedded with alginate ALG and CMC in a mass ratio of 1:0.4, and finally Ca 2+The outermost layer is cured to obtain the product.
[0017] The specific process of step 5 is:
[0018] Step 5.1: Disperse the cells prepared in step 4 in PBS, add CMC-ALG according to the mass ratio of cells: CMC-ALG = 1:0.5, stir continuously for 30-40 minutes at room temperature, wash by centrifugation, and discard the supernatant to obtain a surface coated with a layer of Q + , one layer of CMC, one layer of Q + and a layer of CMC-ALG cell / Q + / CMC / Q + / CMC-ALG;
[0019] Step 5.2: Disperse the cells prepared in step 5.1 in anhydrous CaCl2 and continue stirring for 15 to 20 minutes. After centrifugation and washing, discard the supernatant to obtain the cell / Q after the shell solidification. + / CMC / Q + / CMC-ALG Ca 2+ .
[0020] The beneficial effect of the present invention is that the method of the present invention is used to prepare a single cell surface coated with two Q + The double-layered CMC shell and the cured shell ensure that cells retain high activity after being embedded, improving the cells' tolerance to unfavorable environments. This shell can delay cell division and proliferation to a certain extent, but does not affect the cells' inherent proliferation ability after being released from the shell. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is a flow chart of preparing multilayer starch / CMC embedded cells in Example 1 of the method for preparing the single cell surface starch / CMC self-assembled shell material of the present invention;
[0022] Figures 2(a) and (b) are Fourier transform infrared images of the multilayer starch / CMC shell on the surface of a single cell prepared in Example 1 of the method for preparing the starch / CMC self-assembled shell material on the surface of a single cell of the present invention;
[0023] Figure 3 This is the Zeta potential diagram of the multilayer starch / CMC shell on the surface of a single cell prepared in Example 1 of the method for preparing the starch / CMC self-assembled shell material on the surface of a single cell of the present invention;
[0024] Figure 4 This is a diagram of cell proliferation with different numbers of encapsulation layers and free cells prepared in Example 1 of the method for preparing the single cell surface starch / CMC self-assembled shell material of the present invention. DETAILED DESCRIPTION
[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
[0026] The method for preparing the single cell surface starch / CMC self-assembled shell material of the present invention specifically comprises the following steps:
[0027] Step 1, preparation of cationic starch, 5.5-6.5g of 2,3-epoxypropyltrimethylammonium chloride (GTA) and 1.6-2.0g of NaOH are dissolved in 40mL and 10mL of deionized water, respectively. Then, the GTA and NaOH solutions are mixed, and the mixture is slowly dripped into a round-bottom flask containing 5-6g of amylopectin and 200ml of deionized water. Finally, the flask is stirred at room temperature for 24 hours. The final reaction solution is dialyzed with deionized water for 2-3 days, and finally freeze-dried to obtain quaternized starch (Q + );
[0028] Step 2: Take cells (cell wet weight 0.2-0.3 g) and dissolve them in 50 mL PBS (phosphate buffered saline) buffer. Then, add the cells according to the mass ratio of cells:Q + =1:2 add Q + , stirred at room temperature for 30 to 40 minutes, washed by centrifugation 3 times, and the supernatant was discarded to obtain a layer of Q + cells (cell / Q + ). Zeta potential and Fourier transform infrared spectroscopy test results show that Q + Successfully coated on the cell surface.
[0029] Step 3: Cell / Q prepared in step 2 + Disperse in 50mL PBS, then add CMC according to the mass ratio of cells: CMC = 1:0.5, stir at room temperature for 30-40min, wash three times by centrifugation, and discard the supernatant to obtain a surface coated with a layer of Q + and a layer of CMC cells (cell / Q + / CMC).
[0030] Step 4: Cell / Q prepared in step 3 + / CMC were dispersed in 50mL PBS and then the cells were mixed according to the mass ratio of cells:Q + =1:2 add Q + , stirred at room temperature for 30 to 40 minutes, washed by centrifugation 3 times, and the supernatant was poured out to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + cells (cell / Q + / CMC / Q + ).
[0031] Step 5: Cell / Q prepared in step 4 + / CMC / Q + Disperse in 50mL PBS, then add CMC according to the mass ratio of cells: CMC = 1:0.5, stir at room temperature for 30-40min, wash three times by centrifugation, and discard the supernatant to obtain a surface coated with a layer of Q + , one layer of CMC, one layer of Q + and a layer of CMC cells (cell / Q + / CMC / Q + / CMC). Zeta potential and Fourier transform infrared images show that the multilayer Q + / Successful construction of CMC on the cell surface.
[0032] Step 6: Disperse the cells prepared in step 4 in 50 mL of PBS, add CMC (ALG) at a mass ratio of cells: CMC (ALG) = 1:0.5, stir continuously for 30 to 40 minutes at room temperature, wash three times by centrifugation, and discard the supernatant to obtain a surface coated with a layer of Q + , one layer of CMC, one layer of Q + and a layer of CMC (ALG) cells (cell / Q + / CMC / Q + / CMC(ALG)).
[0033] Step 7: The cells prepared in step 6 were dispersed in 50 mL of 1 M anhydrous CaCl2 and stirred for 15 to 20 min. The cells were washed by centrifugation 3 times, and the supernatant was discarded to obtain the cells with solidified shell (cell / Q + / CMC / Q + / CMC(ALG) Ca 2+ ).
[0034] Example 1
[0035] 2,3-Epoxypropyltrimethylammonium chloride (GTA) 5.5g and NaOH 1.6g are dissolved in 40mL and 10mL deionized water respectively.Then, GTA and NaOH solution are mixed, and the mixture is slowly dropped into the round-bottom flask containing 5g amylopectin and 200ml deionized water.Finally, flask is stirred at room temperature for 24 hours.The final reaction solution is dialyzed with deionized water for 2 days, and finally lyophilized to obtain quaternized starch (Q + ).
[0036] The preparation process of the shell material is shown in the following figure: Figure 1Yeast cells (wet weight 0.2 g) were taken and placed in 50 mL PBS (0.1 M, PH = 6.58) buffer, and then the mixture was stirred at a mass ratio of yeast cell:Q + =1:2 add Q + , stirred at room temperature for 30 min, washed 3 times by centrifugation (5500 rpm, 3 min), and the supernatant was discarded to obtain a surface coating of Q + Yeast cell (yeast cell / Q + ), the Fourier infrared test results are shown in Figure 2(a), indicating that Q + It was successfully adsorbed on the surface of yeast cells. It was then redispersed in 50 mL of PBS buffer, and CMC was added according to the mass ratio of yeast cell:CMC=1:0.5. It was stirred for 30 minutes under warm conditions, centrifuged and washed 3 times, and the supernatant was discarded to obtain a surface coated with 1 Q + / CMC double layer yeast cell (yeast cell / Q + / CMC), the Fourier infrared test results are shown in Figure 2(b), indicating that CMC was successfully adsorbed on the surface of yeast cells. It was then redispersed in 50mL PBS buffer and then + =1:2 add Q + , stirred at room temperature for 30 min, washed by centrifugation 3 times, and the supernatant was poured off to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + Yeast cell (yeast cell / Q + / CMC / Q + ), and finally redispersed in 50mL PBS buffer, and then CMC was added according to the mass ratio of yeast cell:CMC=1:0.5, stirred for 30min under warm conditions, and washed by centrifugation 3 times. After the supernatant was discarded, the surface was coated with 2Q + / CMC double layer yeast cell (yeast cell / Q + / CMC / Q + / CMC).
[0037] In Figure 2(a), 3387cm -1 and 2930cm -1 The extremely broad bands at 1159, 1084, and 993 cm-1 are attributed to O–H stretching and C–H stretching vibrations, respectively. -1 It's Q + The C–O stretching vibration characteristics, while Q + At 1482cm -1 The characteristic vibration peak at the coating layer Q +It also appears in the cells, and with the Q + The increase in the amount of + Successfully coated on the cell surface. In Figure 2(b), 3326cm -1 The increase in the band at 2939cm is due to the -OH stretching in CMC, and the intensity increases with the increase of CMC content. -1 The vibration peak at 1606 cm is related to CH stretching. -1 The band at 1655cm is the carboxyl group on CMC. The band at 802 is related to the COS group of sulfate ester. It is because sulfuric acid is hydrolyzed in the production process of CMC and sulfate ester exists on the surface of CMC. -1 and 1541cm -1 The bands at the cell / Q are the amide I band and the amide II band of the cell. + After a layer of CMC was coated on the surface, the characteristic peak of CMC appeared at the corresponding position, and the peaks of amide I and amide II bands unique to the cells were also detected, indicating that CMC was successfully coated on the outer surface of the cells.
[0038] Yeast cell surface Q + Zeta potential of CMC layer-by-layer assembly Figure 3 As shown, Figure 3 The Zeta potential of free cells is -18mV. + =Q of 1:2 + After coating, the measured potential value was +2.13mV. After centrifugal washing and purification, CMC with a mass ratio of cell:CMC=1:0.5 was added for coating, and the measured potential value was -2.73mV. The positive and negative changes in the potential value can preliminarily prove that Q + and CMC were successfully coated on the cell surface. + After embedding with CMC at different ratios, the results in the figure show that cell:Q + =1:2, cell:CMC=1:0.5 is more suitable.
[0039] The proliferation curves of yeast cells after multilayer embedding and free yeast cells at 30℃ are as follows Figure 4 As shown, Figure 4 The results show that 1, 2, and 3 double layers (Q + The results showed that multilayer encapsulation can effectively slow down cell proliferation and has no adverse effect on the cell proliferation process when the cells resume proliferation, nor does it affect the activity of the encapsulated cells.
[0040] Example 2
[0041] 2,3-Epoxypropyltrimethylammonium chloride (GTA) 6.5g and NaOH 2.0g are dissolved in 40mL and 10mL deionized water respectively.Then, GTA and NaOH solution are mixed, and the mixture is slowly dripped into the round-bottom flask containing 6g amylopectin and 200ml deionized water.Finally, flask is stirred at room temperature for 24 hours.The final reaction solution is dialyzed with deionized water for 3 days, and finally lyophilized to obtain quaternized starch (Q + ).
[0042] Take Escherichia coli (EC) (cell wet weight 0.3g) in 50mL PBS (0.1M, pH = 6.58) buffer, and then add EC:Q + =1:2 add Q + , stirred at room temperature for 40 min, washed 3 times by centrifugation (5500 rpm, 3 min), and the supernatant was poured out to obtain a surface coating of Q + Escherichia coli (EC / Q + ); then redispersed in 50 mL PBS buffer, and then added CMC according to the mass ratio of EC:CMC = 1:0.5, stirred at room temperature for 40 min, washed by centrifugation 3 times, and the supernatant was poured off to obtain a surface coated with 1 Q + / CMC bilayer of Escherichia coli (EC / Q + / CMC); then redispersed in 50mL PBS buffer and then + =1:2 add Q + , stirred at room temperature for 40 minutes, washed by centrifugation 3 times, and the supernatant was poured out to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + Escherichia coli (EC / Q + / CMC / Q + ), and then redispersed in 50 mL of PBS buffer, and then CMC (ALG) was added according to the mass ratio of EC: CMC (ALG) = 1:0.5, stirred at room temperature for 40 minutes, and washed by centrifugation 3 times. After the supernatant was discarded, the Escherichia coli (EC / Q + / CMC / Q + / CMC(ALG)); finally, it was dispersed in 50mL 1M anhydrous CaCl2 and stirred for 20min. After centrifugation and washing three times, the supernatant was discarded to obtain Escherichia coli (EC / Q + / CMC / Q + / CMC(ALG) Ca 2+ ).
[0043] Example 3
[0044] 2,3-Epoxypropyltrimethylammonium chloride (GTA) 6.0g and NaOH 1.8g are dissolved in 40mL and 10mL deionized water respectively.Then, GTA and NaOH solution are mixed, and the mixture is slowly dripped into the round-bottom flask containing 5.5g amylopectin and 200ml deionized water.Finally, flask is stirred at room temperature for 24 hours.The final reaction solution is dialyzed with deionized water for 3 days, and finally lyophilized to obtain quaternized starch (Q + ).
[0045] Yeast cells (wet weight 0.25 g) were taken and dissolved in 50 mL PBS (0.1 M, pH = 6.58) buffer solution, and then the mixture was stirred at a mass ratio of yeast cell:Q + =1:2 add Q + , stirred at room temperature for 35 min, washed three times by centrifugation (5500 rpm, 3 min), and the supernatant was discarded to obtain a surface coating of Q + Yeast cells (cell / Q + ); then redispersed in 50 mL PBS buffer, and then added CMC according to the mass ratio of yeast cells: CMC = 1:0.5, stirred at room temperature for 35 minutes, washed by centrifugation 3 times, and the supernatant was discarded to obtain a surface coated with 1 Q + / CMC double layer yeast cell (yeast cell / Q + / CMC); then redispersed in 50mL PBS buffer, and then according to the mass ratio of yeast cells: Q + =1:2 add Q + , stirred at room temperature for 35 minutes, washed by centrifugation 3 times, and the supernatant was poured out to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + Yeast cell (yeast cell / Q + / CMC / Q + ), and finally redispersed in 50 mL PBS buffer, and then CMC was added according to the mass ratio of yeast cells: CMC = 1:0.5, stirred at room temperature for 35 minutes, and washed by centrifugation 3 times. After the supernatant was discarded, the surface of the sample was coated with 2 Q + / CMC double layer yeast cell (yeast cell / Q + / CMC / Q + / CMC).
Claims
1. A method for preparing a single cell surface starch / CMC self-assembled shell material, characterized by: The specific steps include: Step 1, preparation of cationic starch Q + ; The specific process of step 1 is: 2,3-Epoxypropyltrimethylammonium chloride (GTA) and NaOH were dissolved in deionized water, respectively. Then, the GTA solution and the NaOH solution were mixed and the mixture was dripped dropwise into a round-bottom flask containing amylopectin and deionized water and stirred evenly. The final reaction solution was dialyzed with deionized water for 2-3 days and freeze-dried to obtain quaternized starch Q. + ; Step 2: Place the cells in PBS buffer and then add the cells according to the mass ratio of cells:Q + =1:2 add Q + , stirring continuously for 30-40 min at room temperature, after centrifugal washing, the supernatant was poured out to obtain a Q coating on the surface. + cell / Q + ; Step 3: The cells prepared in step 2 were dispersed in PBS, and CMC was added according to the mass ratio of cells: CMC = 1:0.
5. The mixture was stirred for 30-40 min at room temperature. After centrifugation and washing, the supernatant was discarded to obtain a surface coated with a layer of Q + and a layer of CMC cells / Q + / CMC; Step 4: Disperse the cells prepared in step 3 in PBS and then add PBS according to the mass ratio of cells:Q + =1:2 add Q + , stirring continuously for 30-40 min at room temperature, after centrifugal washing, the supernatant was poured out to obtain a surface coated with a layer of Q + , a layer of CMC and a layer of Q + cell / Q + / CMC / Q + ; Step 5, treating the cells obtained in step 4 to obtain; The cells prepared in step 4 were dispersed in PBS, and CMC was added according to the mass ratio of cells: CMC = 1:0.
5. The mixture was stirred for 30-40 min at room temperature. After centrifugation and washing, the supernatant was discarded to obtain a surface coated with a layer of Q + , one layer of CMC, one layer of Q + and a layer of CMC cells / Q + / CMC / Q + / CMC; the cells are yeast cells.