Method for preparing egfr car-nk cells based on polypeptide gel droplet confined transfection technology and application thereof

Abstract

The application relates to the technical field of the cross of microfluidic technology, chemical synthesis and biomedicine, and discloses a method for preparing EGFR CAR-NK cells based on polypeptide gel droplet confined transfection technology and application, which comprises the following steps: (1) preparing collagen-mimic-peptide-modified alginate; (2) constructing a confined transfection system: adopting a droplet microfluidic chip, wrapping NK cells and slow virus carrying an EGFR CAR gene in the collagen-mimic-peptide-modified alginate droplet prepared in the step (1), and obtaining a gel droplet after gelation of the droplet; and (3) confined transfection and cell culture. The method can significantly reduce the dosage of slow virus and cell toxicity, greatly improve the transfection success rate of the CAR gene, the survival rate and functional activity of the NK cells, and finally obtain EGFR CAR-NK cell products with higher purity and stronger target killing capacity.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microfluidics, chemical synthesis and biomedicine, specifically to a method and application for preparing EGFR CAR-NK cells based on peptide gel droplet confined transfection technology. Background Technology

[0002] Glioblastoma (GBM), the most common and aggressive primary brain tumor, remains a major challenge in neuro-oncology. Existing therapies (surgery, radiotherapy, chemotherapy, and targeted drugs) have limited efficacy and poor patient outcomes, necessitating innovative treatments. Chimeric antigen receptor (CAR) engineered immunotherapy has shown transformative potential in hematologic malignancies, but in solid tumors (especially GBM), it faces bottlenecks such as tumor heterogeneity, target scarcity, and an immunosuppressive microenvironment.

[0003] Recent studies have revealed the potential application of CAR-NK cells targeting novel targets such as epidermal growth factor receptor (EGFR) in the treatment of some GBM cases. However, the traditional viral vector transfection method for preparing CAR-NK cells severely restricts their clinical translation. First, viral vectors are used in large quantities and are very expensive. Lentiviral transfection of NK cells requires extremely high MOI (usually >10), with vector costs accounting for over 60% of the total preparation cost, and the cost of a single batch of treatment can reach hundreds of thousands of yuan. Second, transfection efficiency is low and uneven. NK cells have low sensitivity to viral transfection, and the transfection efficiency of primary NK cells often hovers between 2% and 5%, resulting in a large number of unmodified cells in the product, diluting the efficacy. Finally, the process is complex and difficult to scale up, involving multiple steps such as virus production, cell activation, transfection, and expansion, with a long cycle (>2 weeks), making it difficult to meet the needs of large-scale and stable production of "off-the-shelf" products. Significant batch-to-batch differences, variations in virus production batches, and fluctuations in transfection process parameters make it difficult to standardize CAR expression levels, cell composition, and efficacy (FDA guidelines are a special concern). Therefore, developing novel, efficient, and safe lentiviral transfection technologies to construct EGFR CAR-NK cells offers a highly attractive strategy for overcoming the treatment challenges of GBM. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method and application for preparing EGFR CAR-NK cells based on peptide gel droplet confined transfection technology. By combining materials engineering, microfluidic technology, and virus transfection processes, and creating an optimized confined microenvironment, this invention systematically solves key problems in traditional preparation methods such as low virus utilization efficiency, significant cell damage, and poor product uniformity.

[0005] To achieve the above objectives, the technical solution of the present invention is: a method for preparing EGFR CAR-NK cells based on polypeptide gel droplet confined transfection technology, comprising the following steps: (1) Preparation of collagen mimic peptide (CMP) modified alginate: CMP was modified on the surface of alginate gel by carbodiimide coupling method to obtain CMP modified alginate. The CMP sequence (SEQ ID No:1) is GGYGGGPC(GPP)5GFOGER(GPP)5GPC, where O is hydroxyproline. (2) Construction of confined transfection system: NK cells and lentivirus carrying EGFR CAR gene were co-encapsulated in alginate droplets modified with collagen mimic peptides prepared in step (1) using droplet microfluidic chip, and gel droplets were obtained after gelation. (3) Confined transfection and cell culture: The gel droplets containing NK cells and lentivirus prepared in step (2) are cultured to transfect NK cells within the confined space of the droplets, thereby obtaining NK cells expressing EGFR CAR, namely EGFRCAR-NK cells.

[0006] Further; the modification method in step (1) is as follows: dissolve alginate in MES buffer, then add EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to the solution, stir and react for 20-24 hours at room temperature and in the dark to obtain an activated alginate solution, then add collagen mimic peptide to the activated alginate solution, continue stirring and react for 15-25 minutes, dialyze and freeze dry to obtain collagen mimic peptide modified alginate.

[0007] Furthermore, the alginate is sodium alginate, the MES buffer has a pH of 6.0, the dialysis bag has a molecular weight cutoff of 20 kDa, and the dialysis time is 40-60 h.

[0008] Further; the mass-to-volume ratio of sodium alginate to MES buffer is (1~2) g:100 mL, the mass ratio of sodium alginate, EDC, and NHS is 30:19:6, and the mass-to-volume ratio of collagen mimic peptide to activated alginate solution is 25 μg:1 mL.

[0009] Further; the droplet microfluidic chip in step (2) includes a chip substrate, on which a sample flow channel is formed. The sample flow channel includes a T-shaped region and a stable region. The T-shaped region is provided with an aqueous phase channel and an oil phase channel. The aqueous phase channel and the oil phase channel are vertically intersected and connected to the stable region.

[0010] Furthermore, the sample flow channel has a height of 40-60 μm and a width of 50-150 μm. The aqueous phase channel and the oil phase channel have the same length, both being 6-10 mm. The stable zone has a length of 40-50 mm. Alginate modified with collagen mimic peptides containing NK cells and lentiviruses carrying the EGFRCAR gene is introduced into the aqueous phase channel as the aqueous phase, and fluorocarbon oil is introduced into the oil phase channel as the oil phase. The aqueous phase flow rate in the aqueous phase channel is 10 μL / min, and the oil phase flow rate in the oil phase channel is 20 μL / min.

[0011] Furthermore, the gel droplets described in step (2) have an elastic modulus of 2500 MPa, a pore size of 5 μm, and a viscosity of 20 nN·s / m. 2 On average, a single gel droplet encapsulates one NK cell and five to six lentiviruses.

[0012] Further; the culture conditions described in step (3) are: incubation at 37°C and 5% CO2 in NK-92 complete culture medium for at least 7 days.

[0013] Another technical solution of the present invention is: EGFR CAR-NK cells prepared by the preparation method described above.

[0014] The next technical solution of the present invention is: the application of the EGFR CAR-NK cells in the preparation of a drug for treating glioblastoma.

[0015] The beneficial effects of this invention are as follows: By constructing a biomimetic cell-adaptive matrix using CMP-modified alginate gel, and employing droplet microfluidic technology, lentiviruses carrying the EGFR CAR gene and NK cells are co-encapsulated within the aforementioned gel droplets. Through precise control of the droplet's physicochemical microenvironment and utilizing its spatial confinement effect, the probability of local contact between the virus and cells and the transfection efficiency are significantly enhanced. This method significantly reduces the dosage of lentivirus and cytotoxicity while substantially improving the transfection success rate of the CAR gene, the viability and functional activity of NK cells, and ultimately yielding EGFR CAR-NK cell products with higher purity and stronger targeted killing ability. Attached Figure Description

[0016] Figure 1 It is a droplet microfluidic chip: (A) chip physical image, (B) sample flow channel structure diagram, (C) enlarged view of the intersection and convergence of aqueous phase channel and oil phase channel; Figure 2 Characterization of CMP-modified alginate gel: (A) Fourier transform infrared spectra of alginate gel and CMP-modified alginate gel, (B) XPS spectra of alginate gel and CMP-modified alginate gel; Figure 3The transfection efficiency on day 7 of conventional lentiviral transfection and peptide gel droplet-restricted transfection is as follows: (A) Flow cytometry images of negative control (NC), conventional lentiviral transfection (PC), and peptide gel droplet-restricted transfection (CHIP); (B) Transfection rate on day 7 of negative control, conventional lentiviral transfection, and peptide gel droplet-restricted transfection; (C) Cell viability on day 7 of negative control, conventional lentiviral transfection, and peptide gel droplet-restricted transfection. Figure 4 The following are the results of continuous expression of EGFR-CAR in NK-92 cells at 96 hours after 7 days of infection: (A) Flow cytometry images of continuous expression of EGFR-CAR in NK-92 cells after negative control, conventional lentivirus transfection, and peptide gel droplet-restricted transfection; (B) Statistical graph of expression of EGFR-CAR in NK-92 cells after negative control, conventional lentivirus transfection, and peptide gel droplet-restricted transfection; (C) Cell viability at 96 hours after 7 days after negative control, conventional lentivirus transfection, and peptide gel droplet-restricted transfection. Figure 5 The in vitro killing effect of EGFR CAR-NK cells prepared by peptide gel droplet-limited transfection on (A) U87 cells (human astrocytoma cells) and (B) GL261 (mouse glioma cells); Figure 6 This is a schematic diagram illustrating the principle of preparing EGFR CAR-NK cells by peptide gel droplet-limited transfection according to the present invention. Detailed Implementation

[0017] The present invention will be described below through specific embodiments. Unless otherwise specified, the methods used in the embodiments are conventional methods in the art.

[0018] Example:

[0019] A method for preparing EGFR CAR-NK cells based on peptide gel droplet confined transfection technology includes the following steps: (1) Preparation of CMP-modified sodium alginate: 600 mg of sodium alginate powder was added to 50 mL of pre-prepared 0.1 MMES buffer (pH 6.0). The solution was sonicated at room temperature for 30-60 minutes until a clear, viscous solution (i.e., alginate gel) was formed. The Fourier transform infrared spectroscopy and XPS analysis results are shown in [reference needed]. Figure 2Then, 380 mg EDC and 120 mg NHS were added sequentially to the clear, viscous solution. The mixture was stirred at 600 rpm for 20 hours at room temperature and in the dark to obtain an activated sodium alginate solution. 4 mL of the activated sodium alginate solution was taken, and 1 mL of 100 μg / mL CMP was added. The CMP sequence was GGYGGGPC(GPP)5GFOGER(GPP)5GPC, where O represents hydroxyproline. The mixture was stirred at 600 rpm for 20 minutes at room temperature and in the dark. Subsequently, 5 mL of the sample was transferred to a dialysis bag with a molecular weight cutoff of 20 kDa. Dialysis was performed at 4°C and in the dark for 48 hours to remove unreacted EDC, NHS, and free CMP. The dialysis result was a CMP-modified alginate gel (Fourier transform infrared spectroscopy and XPS analysis results are shown in [link to Fourier transform infrared spectroscopy]). Figure 2 ( ), freeze-dried into powder, to obtain CMP-modified sodium alginate.

[0020] (2) Constructing a droplet microfluidic chip: A sample flow channel is formed on the chip substrate. The sample flow channel includes a T-shaped region and a stable region. The T-shaped region is provided with an aqueous phase channel and an oil phase channel. The aqueous phase channel and the oil phase channel are vertically intersected and connected to the stable region. Figure 1 The sample flow channel height is 50 μm. The lengths of the aqueous and oil phase channels are the same, both 8 mm. The length of the stable zone is 46 mm. The width at the inlet of both the aqueous and oil phase channels is 120 μm, and the width at the intersection is 60 μm. The middle sections of both the aqueous and oil phase channels are tapered sections. The width of the stable zone gradually widens from 60 μm near the intersection to 120 μm.

[0021] (3) Preparation of aqueous and oil phases: Aqueous phase: Collect 1×10 6 100 μL of NK-92 cells (cell viability >90%, purchased from Shangen Biotechnology Co., Ltd.) were centrifuged, and the cells were resuspended in 90 μL of 0.5 wt% CMP-modified sodium alginate solution. 10 μL of lentivirus (titer = 1 × 10⁻⁶) was added. 8 TU / mL (purchased from Shanghai Jikai Gene Co., Ltd.), then add 2μL of P agent (Polybrene 25×, purchased from Shanghai Jikai Gene Co., Ltd.), mix well to obtain the aqueous phase.

[0022] Oil phase: 1 mL fluorocarbon oil (HFE-7500, purchased from Suzhou Zhongxin Qiheng Scientific Instruments Co., Ltd.).

[0023] (4) Restricted transfection and cell culture: Aqueous phase enters the aqueous phase channel through the aqueous phase inlet, and oil phase enters the oil phase channel through the oil phase inlet. The flow rate of the aqueous phase is 10 μL / min, and the flow rate of the oil phase is 20 μL / min. After the aqueous and oil phases converge, the aqueous phase is sheared into microdroplets by the oil phase (water-in-oil mode), generating uniform droplets with a diameter of approximately 100 μm. The droplets contain NK-92, lentivirus, and agent P. The droplets are collected at the stable region collection port, and an equal volume of 2wt% calcium chloride solution is added to gel the droplets. The physical parameters of the gelled droplets are: elastic modulus of 2500 MPa, pore size of 5 μm, and viscosity of 20 nN·s / m³. 2 Subsequently, the gelled droplets were transferred to NK-92 cell complete culture medium (purchased from Shangen Biotechnology Co., Ltd.) for further culture (37°C, 5% CO2) (see...). Figure 6 After 7 days of incubation, EGFR CAR-NK cells were collected, and infection efficiency was detected by flow cytometry. Figure 3 (A). Compared to conventional lentiviral transfection for EGFR CAR-NK cell preparation, the EGFR CAR-NK cells prepared by peptide gel droplet-limited transfection of this invention maintain a cell viability of 80% ( Figure 3 The transfection efficiency of the medium-sized C (C) method after 7 days was 34%, while that of the conventional method was 2%. Figure 3 (See B). Using peptide gel droplet-confined transfection, 96 hours after 7 days of transfection, 17% of NK-92 cells still continuously expressed EGFR-CAR (see B). Figure 4 (Among A), the cell viability remained at 80% (see Figure 4 (C)

[0024] The conventional lentiviral transfection method used in this invention to prepare EGFR CAR-NK cells is as follows: collect 1×10 6 100 μL of NK-92 cells (cell viability >90%, purchased from Shangen Biotechnology Co., Ltd.) were centrifuged, and the cells were collected and added to 90 μL of NK-92 complete cell culture medium (purchased from Shangen Biotechnology Co., Ltd.). 10 μL of lentivirus (titer = 1 × 10⁶) was added. 8 TU / mL (purchased from Shanghai Jikai Gene Co., Ltd.) was added, followed by 2 μL of P agent (Polybrene 25×, purchased from Shanghai Jikai Gene Co., Ltd.). The cells were then transferred to an NK-92 cell incubator for further culture (37℃, 5% CO2). After 7 days of incubation, cells were collected, and infection efficiency was assessed using flow cytometry. Figure 3 (A)

[0025] In vitro experiments EGFR CAR-NK cells prepared using the polypeptide gel droplet-limited transfection method of this invention were tested in vitro for their killing effect on GL261 (mouse glioma cells, Beina Biotechnology) and U87 (human astrocytoma cells, Beina Biotechnology): 100 μL of GL261 cell suspension (1 × 10⁶ cells per well) was added to each well of a 96-well plate. 4 (100 cells), place the well plate in a 37°C, 5% CO2 incubator overnight (approximately 15 hours) to allow the cells to adhere and grow to confluence (80-90% confluence). Culture U87 cells using the same steps, then add 100 μL of peptide gel droplets containing EGFR CAR-NK cells for restricted transfection to each well, with each 100 μL containing 5 × 10⁻⁶ cells. 4 Add 100 μL of NK-92 cell complete culture medium to each well, and incubate the plate at 37°C in a 5% CO2 incubator for 24 hours. Then discard the 100 μL NK-92 cell complete culture medium and add 10 μL of CCK-8 reagent to each well. Continue incubation for 2 hours, and measure the absorbance of each well at 450 nm using a microplate reader. Calculate the survival rate: Survival rate (%) = [(Treatment group OD - Blank OD) / (Control group OD - Blank OD)] × 100. Calculate the killing rate: Kill rate (%) = 100 - Survival rate (%), where blank refers to cell-free cells, 100 μL NK-92 cell complete culture medium + 10 μL CCK-8, and the control group uses EGFRCAR-NK cells prepared using conventional infection. Results are as follows. Figure 5 As shown, EGFR CAR-NK cells prepared by peptide gel droplet-limited transfection showed a 76% killing efficiency against GL261 gliomas and an 88% killing rate against U87 human astrocytoma cells within 24 hours in in vitro experiments. In contrast, EGFR CAR-NK cells prepared by conventional infection showed a 67% killing efficiency against GL261 gliomas and a 75% killing rate against U87 human astrocytoma cells within 24 hours.

[0026] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications may be made without departing from the technical solutions described in the claims.

Claims

1. A method for preparing EGFR CAR-NK cells based on peptide gel droplet confined transfection technology, characterized in that, Includes the following steps: (1) Preparation of collagen mimic peptide-modified alginate: Collagen mimic peptides were modified onto the surface of alginate gel by carbodiimide coupling method to obtain collagen mimic peptide-modified alginate. The collagen mimic peptide sequence is GGYGGGPC(GPP)5GFOGER(GPP)5GPC, where O is hydroxyproline. (2) Construction of confined transfection system: NK cells and lentivirus carrying EGFR CAR gene were co-encapsulated in alginate droplets modified with collagen mimic peptides prepared in step (1) using droplet microfluidic chip, and gel droplets were obtained after gelation. (3) Confined transfection and cell culture: The gel droplets containing NK cells and lentivirus prepared in step (2) are cultured to transfect NK cells within the confined space of the droplets, thereby obtaining NK cells expressing EGFR CAR, namely EGFRCAR-NK cells.

2. The method for preparing EGFR CAR-NK cells according to claim 1, characterized in that, The modification method in step (1) is as follows: dissolve alginate in MES buffer, then add EDC and NHS to the solution, stir and react for 20-24 hours at room temperature and in the dark to obtain an activated alginate solution, then add collagen mimic peptides to the activated alginate solution, continue stirring and reacting for 15-25 minutes, dialyze and freeze dry to obtain collagen mimic peptide-modified alginate.

3. The method for preparing EGFR CAR-NK cells according to claim 2, characterized in that: The alginate is sodium alginate, the MES buffer has a pH of 6.0, the dialysis bag has a molecular weight cutoff of 20 kDa, and the dialysis time is 40-60 h.

4. The method for preparing EGFR CAR-NK cells according to claim 3, characterized in that: The mass-to-volume ratio of sodium alginate to MES buffer is (1~2) g:100 mL, the mass ratio of sodium alginate, EDC, and NHS is 30:19:6, and the mass-to-volume ratio of collagen mimic peptide to activated alginate solution is 25 μg:1 mL.

5. The method for preparing EGFR CAR-NK cells according to claim 1, characterized in that, The droplet microfluidic chip in step (2) includes a chip substrate, on which a sample flow channel is formed. The sample flow channel includes a T-shaped region and a stable region. The T-shaped region is provided with an aqueous phase channel and an oil phase channel. The aqueous phase channel and the oil phase channel are vertically intersected and connected to the stable region.

6. The method for preparing EGFR CAR-NK cells according to claim 5, characterized in that: The sample flow channel has a height of 40-60 μm and a width of 50-150 μm. The aqueous phase channel and the oil phase channel have the same length, 6-10 mm. The stable zone has a length of 40-50 mm. Alginate modified with collagen mimic peptides containing NK cells and lentiviruses carrying the EGFR CAR gene is introduced into the aqueous phase channel as the aqueous phase, and fluorocarbon oil is introduced into the oil phase channel as the oil phase. The aqueous phase flow rate in the aqueous phase channel is 10 μL / min, and the oil phase flow rate in the oil phase channel is 20 μL / min.

7. The method for preparing EGFR CAR-NK cells according to claim 1, characterized in that: The gel droplets described in step (2) have an elastic modulus of 2500 MPa, a pore size of 5 μm, and a viscosity of 20 nN·s / m. 2 On average, a single gel droplet encapsulates one NK cell and five to six lentiviruses.

8. The method for preparing EGFR CAR-NK cells according to claim 1, characterized in that: The culture conditions described in step (3) are: incubation at 37°C and 5% CO2 in NK-92 complete culture medium for at least 7 days.

9. EGFR CAR-NK cells prepared by the preparation method according to any one of claims 1-8.

10. The use of the EGFR CAR-NK cells of claim 9 in the preparation of a medicament for treating glioblastoma.

Images