Polyethyleneimine low-toxicity nano-carrier capable of being used for delivering DNA molecules as well as preparation method and application of polyethyleneimine low-toxicity nano-carrier

Abstract

The invention discloses a polyethyleneimine low-toxicity nano-carrier capable of being used for delivering DNA (deoxyribonucleic acid) molecules as well as a preparation method and application of the polyethyleneimine low-toxicity nano-carrier, and the polyethyleneimine low-toxicity nano-carrier can be used for accurately controlling surface charges and particle sizes of PEI nano-particles by adjusting parameters such as molecular weight, N / P ratio and the like of PEI, so that an ideal delivery effect is achieved, components and structures of a compound are reasonably designed, and gene delivery performance is optimized; a safe, efficient and reusable DNA delivery solution is provided for the fields of scientific research and medical treatment, and the method is of great significance in promoting gene therapy, vaccine development and other leading-edge scientific and technological progress.

Description

Technical Field

[0001] The present invention relates to a low-toxic polyethyleneimine nanocarrier and its preparation method and application. More specifically, it relates to a low-toxic polyethyleneimine nanocarrier that can be used to deliver DNA molecules, its preparation method and application, belonging to the field of bioengineering. Background Art

[0002] Nucleic acid drugs, especially DNA and RNA molecules, have shown great potential in the treatment of genetic diseases, cancer therapies, and the development of novel vaccines. However, the instability of nucleic acid molecules themselves and the physical property of being difficult to penetrate cell membranes pose great challenges to the delivery of nucleic acid drugs. Although traditional viral vectors have high delivery efficiency, the accompanying cytotoxicity, immunogenicity, and gene integration risks limit their widespread application. Therefore, the development of safe, stable, and efficient non-viral delivery vectors has become the focus of current research.

[0003] Polyethyleneimine (PEI) is a commonly used non-viral vector material and has attracted attention due to its high transfection efficiency. However, the high cytotoxicity of PEI limits its promotion in practical applications. To overcome this problem, researchers have tried to reduce the cytotoxicity of PEI by methods such as chemical modification, changing the molecular weight, and binding with auxiliary lipids, while maintaining its high gene delivery ability. Summary of the Invention

[0004] To solve the above-mentioned problems in the prior art, the present invention provides a low-toxic polyethyleneimine nanocarrier that can be used to deliver DNA molecules, its preparation method and application, which have the technical features of being able to solve the technical difficulties of the cytotoxicity and instability of the PEI carrier, and realizing a new low-toxic and highly efficient delivery carrier for gene therapy and vaccine delivery.

[0005] To achieve the above object, the present invention is realized by the following technical solutions:

[0006] A low-toxic polyethyleneimine nanocarrier that can be used to deliver DNA molecules according to the present invention is characterized in that the nanocarrier is made by mixing branched polyethyleneimine bPEI, dioleoyl phosphatidylethanolamine DOPE, and cholesterol, the molecular weight of the branched polyethyleneimine bPEI includes 1.2 kDa and 25 kDa, and the particle size range of the nanocarrier is 200 - 300 nm, and the N / P molar ratio is 3 or 5.

[0007] A method for preparing a low-toxic polyethyleneimine nanocarrier that can be used to deliver DNA molecules according to the present invention includes the following steps:

[0008] Step a. Branched polyethylenimine bPEI with molecular weights of 1.2 kDa and 25 kDa was respectively mixed with dioleoyl phosphatidylethanolamine DOPE and cholesterol to prepare mixed nanoparticles P / LNP;

[0009] Step b. PEI / DNA complexes with different N / P molar ratios were prepared by microfluidic technology;

[0010] Step c. The initially obtained PEI / DNA complex solution was diluted and concentrated with PBS;

[0011] Step d. The particle size and Zeta potential of the concentrated PEI / DNA complexes were measured;

[0012] Step e. PEI / DNA complexes with a particle size range of 200 - 300 nm and an N / P ratio of 3 or 5 were screened and obtained.

[0013] Preferably, in step c, the initially obtained PEI / DNA complex solution was diluted with PBS to more than 10 times the volume, the diluted solution was transferred to a 50KD ultrafiltration tube, and centrifuged at 4°C and 3000g until the solution was concentrated.

[0014] Preferably, in step d, a dynamic light scattering DLS system was used to measure the physicochemical properties of the PEI / DNA complexes.

[0015] Preferably, the physicochemical properties include particle size and Zeta potential.

[0016] Preferably, step e includes: placing the complex solution in a Y-shaped micro cuvette, setting the temperature, scattering angle, and working voltage to 25°C, 90°, and 100 mV respectively, measuring each sample three times, and reporting the average value. Finally, PEI / DNA complexes with a particle size range of 200 - 300 nm and an N / P ratio of 3 or 5 were screened.

[0017] Preferably, in step b, pCMV-N-EGFP and pSV40Luc were used as the delivery contents.

[0018] Application of the nanocarrier described in the present invention in delivering DNA molecules in vitro and in vivo.

[0019] Preferably, the application includes gene therapy and vaccine delivery.

[0020] Beneficial effects: The surface charge and particle size of the PEI nanoparticles of the present invention are precisely controlled by adjusting parameters such as the molecular weight of PEI and the N / P ratio, so as to achieve ideal particle size and potential results; the present invention optimizes its gene delivery performance by reasonably designing the composition and structure of the complex, and provides a safe, effective, and reusable DNA delivery carrier system. Description of the Drawings

[0021] Figure 1 It is the average particle size diagram of the NA-PEI nanoparticles of the present invention.

[0022] Figure 2 It is the average ζ potential diagram of the NA-PEI nanoparticles of the present invention.

[0023] Figure 3 It is the dispersion index diagram of the DNA-PEI nanoparticles of the present invention.

[0024] Figure 4 It is the comparison diagram of the cytotoxicity of 1.2 kDa PEI and 1.2 kDa PEI-DOPE-Chol of the present invention at different N / P ratios.

[0025] Figure 5 It is the comparison diagram of the cytotoxicity of 25 kDa PEI and 25 kDa PEI-DOPE-Cho of the present invention at different N / P ratios.

[0026] Figure 6 It is the diagram of the expression of pCMV-N-eGFP plasmid DNA delivered by nanoparticles prepared with two different PEIs, 25KDa PEI and 1.2KDa PEI, observed by fluorescence microscopy in HEK 293T cells of the present invention.

[0027] Figure 7 It is the diagram of the expression of DNA-PEI nanoparticles with a phosphorus molar ratio of 5 by in vivo imaging after intramuscular injection in mice of the present invention.

[0028] Among them, Figure 6 the N / P ratios in are 1, 3, 5, and 9 respectively. Detailed implementation manners

[0029] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0030] The present invention provides a cationic polymer-lipid hybrid nanoparticle (P / LNP) carrier based on polyethyleneimine and lipid complex, which can be used for efficient and low-toxic in vitro and in vivo delivery of DNA molecules.

[0031] The specific steps of the present invention are as follows:

[0032] Step 1: Preparation of hybrid nanoparticles

[0033] First, branched polyethylenimine (bPEI) with molecular weights of 1.2 kDa and 25 kDa was mixed with dioleoyl phosphatidylethanolamine (DOPE) and cholesterol respectively to form mixed nanoparticles (P / LNP). pCMV-N-EGFP and pSV40Luc were selected as the delivery contents, and the conditions were precisely controlled by microfluidic technology to prepare PEI / DNA complexes with different N / P molar ratios.

[0034] Step 2: Solution Dilution and Concentration

[0035] The initially obtained PEI / DNA complex solution was diluted with PBS to more than 10 times its volume, and then the diluted solution was transferred to a 50KD ultrafiltration tube and centrifuged at 3000g at 4°C until the solution was concentrated to an appropriate volume, and the volume confirmation could be set according to actual needs.

[0036] Step 3: Physicochemical Property Determination

[0037] The particle size and Zeta potential of the concentrated PEI / DNA complex were measured using a dynamic light scattering (DLS) system (such as Malvern Zetasizer Nano ZS).

[0038] Step 4: Sample Measurement

[0039] The complex solution was placed in a Y-shaped microcuvette, and the temperature was set at 25°C, the scattering angle was 90°, and the working voltage was 100 mV. Each sample was measured three times and the average value was reported.

[0040] Step 5: Screening and Optimization

[0041] PEI / DNA complexes with a particle size range of 200 - 300 nm and N / P ratios of 3 and 5 were screened. The results are as Figures 1-3 shown. Through specific experiments, it can be known that the above parameters are considered the ideal size and charge state (0.1, showing high uniformity) of the particles for gene delivery.

[0042] Innovation points of the present invention: By adjusting parameters such as the molecular weight of PEI and the N / P ratio, the surface charge and particle size of PEI nanoparticles are precisely controlled to achieve an ideal delivery effect. The composition and structure of the complex are reasonably designed to optimize the gene delivery performance, providing a safe, effective, and reusable DNA delivery vector system.

[0043] Experiments were conducted on the technical solution of this application

[0044] 1. Detect the toxicity of PEI / DNA nanocarrier particles in HEK293T cells:

[0045] To evaluate the cell viability of different molecular weight PEI lipid polymers modified with DOPE and cholesterol, as well as the N / P molar ratio on HEK 293t cells, a CCK-8-based cell viability assay was used and compared with 1.2 kDa and 25 kDa PEI without co-lipid modification. HEK 293 cells (8000 cells / well) were seeded into 96-well plates and allowed to adhere for 12 h. 100 μL of DMEM complete medium containing PEI / DNA complexes of different groups (complex: 0.2 μg pCMV-N-EGFP) was added to each well, and a total of 6 replicates were set for each concentration gradient. Thereafter, the 96-well plates were returned to the cell culture incubator and cultured for another 24 h. Thereafter, the lipid polymer solution was replaced with 100 μL of a sterile-filtered CCK-8 stock solution in 0.1 mg / mL phosphate buffered saline (PBS) and incubated at 37 °C for another 1 h. After incubation, the 96-well plates were taken out, the color change of the solution in the 96-well plates was observed, and the OD value of each sample at 490 nm was detected using an ELISA plate reader. The cell viability was confirmed using equation (3).

[0046] Cell viability = (ODtreatment / ODcontrol) × 100%, (3) where OD is the optical density.

[0047] As can be seen Figures 4-5 from the results, compared with the control group, at different N / P ratios, the cytotoxicity of the PEI lipid polymers after adding DOPE and cholesterol was lower than that of the naked PEI cationic polymer.

[0048] This indicates that the addition of DOPE and cholesterol co-lipids helps to stabilize the bilayer membrane structure and reduce the toxicity of the cationic component, thus enabling the PEI lipid polymer / DNA complex to exhibit lower cytotoxicity.

[0049] 2. Detect the in vitro transfection of PEI / DNA nanocarrier particles:

[0050] After screening the particle size and potential results of the polyethyleneimine lipid polymer / DNA complex, N / P ratios of 3 and 5 were selected for in vitro transfection experiments. To verify that the lipid polymer can deliver the pCMV-N-eGFP plasmid into mammalian cells and successfully express, 293T cells were seeded into 12-well plates at a density of 2.5 × 105, and after the cells adhered for 12 h, 10 μg of the pCMV-N-eGFP plasmid was mixed with 25 kDa and 1.2 kDa PEI lipid polymers (N / P molar ratios of 3 and 5) by microfluidic technology. The PEI / pEGFP mixture was added to 1 ml of serum-containing DMEM and pipetted and mixed well. 100 μl of the complex was taken to transfect HEK 293t cells, and the transfection was observed using an inverted fluorescence microscope 24 and 48 h after transfection.

[0051] Figure 6 The results in showed that all four groups of lipid polymers prepared could successfully deliver DNA into cells and achieve gene expression. At the same N / P ratio, the transfection efficiency of 25 kDa PEI was higher than that of 1.2 kDa PEI, and when the N / P ratio was 5, the transfection efficiency of 25 kDa PEI was comparable to that of the positive control group. The experiment also verified that the ζ potential of the complex must be positive to observe obvious fluorescence expression, that is, effective transfection. Compared with the control group, no fluorescence was observed after transfection of 293t cells with complexes with N / P ratios of 1 (negative potential) and 9 (particle size greater than 400 nm). For the four groups of complexes with N / P ratios of 3 and 5 (ζ potential was positive and the particle size range was between 200 - 300 nm), fluorescence could always be detected. According to the above experimental results, 1.2 kDa and 25 kDa PEI / DNA complexes with N / P of 5 were selected for in vivo delivery studies in subsequent experiments.

[0052] 3. Detect the in vivo transfection of PEI / DNA nanocarrier particles:

[0053] Mix 10 μg of pSV40Luc plasmid with a predetermined amount of 25 kDa and 1.2 kDa PEI (such that the N / P molar ratio is 5) using a microfluidic device. After ultrafiltration of the mixture solution, take 50 μl of the supernatant solution and inject it into the thigh muscle of 6 - to 8-week-old male BALB / c mice. On the third day after injection, anesthetize the mice with isoflurane and intraperitoneally inject 200 μl of enalapril (20 mg / ml), and measure the luciferase activity in vivo through the IVIS system. As Figure 7 The results showed that both of the two complexes prepared (1.2 kDa PEI / DNA and 25 kDa PEI / DNA) could successfully deliver the luciferase reporter gene into mice, and the protein could still be successfully expressed on the third day after injection. The transfection efficiency of the 25 kDa PEI / DNA complex was much higher than that of the 1.2 kDa PEI / DNA complex.

[0054] In summary, the experimental verification and effects are as follows:

[0055] (1) Cytotoxicity detection: Evaluate the effects of PEI lipid polymers with different molecular weights and N / P molar ratios on the cell viability of HEK293T cells through CCK-8 cell viability assay. The results showed that the cytotoxicity of PEI lipid polymers after adding DOPE and cholesterol was lower than that of naked PEI cationic polymers, indicating that the addition of auxiliary lipids helped to stabilize the bilayer membrane structure and reduce toxicity.

[0056] (2) In vitro transfection experiment: Complexes with N / P ratios of 3 and 5 were selected for in vitro transfection experiments. The transfection was observed using an inverted fluorescence microscope, and the results showed that at the same N / P ratio, the transfection efficiency of 25kDa PEI was higher than that of 1.2kDa PEI, and when the N / P ratio was 5, the transfection efficiency of 25kDa PEI was equivalent to that of the positive control group.

[0057] (3) In vivo transfection experiment: The prepared complexes were injected into the thigh muscles of BALB / c mice, and the luciferase activity in vivo was measured by IVIS system. The results showed that both complexes could successfully deliver the luciferase reporter gene into mice and successfully express the protein on the third day after injection, and the transfection efficiency of the 25kDa PEI / DNA complex was much higher than that of the 1.2kDa PEI / DNA complex.

[0058] Therefore, the lipid nanocarrier designed and prepared based on polyethyleneimine provided by the present invention can deliver DNA molecules into the body and outside the body with low toxicity and high efficiency and successfully express proteins, providing a new and promising delivery carrier system for gene therapy and vaccine delivery.

[0059] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A low-toxic polyethyleneimine nanocarrier that can be used to deliver DNA molecules, characterized in that: The nanocarrier is prepared by mixing branched polyethyleneimine bPEI, dioleoylphosphatidylethanolamine DOPE and cholesterol. The molecular weight of the branched polyethyleneimine bPEI includes 1.2kDa and 25kDa. The particle size of the nanocarrier ranges from 200 to 300nm, and the N / P molar ratio is 3 or 5.

2. A method for preparing the polyethyleneimine low-toxic nanocarrier for delivering DNA molecules according to claim 1, comprising the following steps: Step a. mixing branched polyethyleneimine bPEI with molecular weights of 1.2 kDa and 25 kDa respectively with dioleoylphosphatidylethanolamine DOPE and cholesterol to prepare mixed nanoparticles P / LNP; Step b. preparing PEI / DNA complexes with different N / P molar ratios by microfluidic technology; Step c. diluting the initially obtained PEI / DNA complex solution with PBS and concentrating; Step d. Determining the particle size and Zeta potential of the concentrated PEI / DNA complex; Step e. Screening to obtain PEI / DNA complexes with a particle size range of 200-300 nm and an N / P ratio of 3 or 5.

3. The method of preparing a polyethyleneimine low-toxic nanocarrier for delivering DNA molecules according to claim 2, characterized in that: In step c, the PEI / DNA complex solution initially obtained is diluted with PBS to a volume of 10 times or more, and the diluted solution is transferred to a 50KD ultrafiltration tube and centrifuged at 4°C and 3000g until the solution is concentrated.

4. The method of preparing a polyethyleneimine low-toxic nanocarrier for delivering DNA molecules according to claim 2, characterized in that: In step d, a dynamic light scattering (DLS) system is used to measure the physicochemical properties of the PEI / DNA complex.

5. The method of preparing a polyethyleneimine low-toxic nanocarrier for delivering DNA molecules according to claim 4, characterized in that: Physicochemical properties include particle size and zeta potential.

6. The method for preparing a polyethyleneimine low-toxic nanocarrier for delivering DNA molecules according to claim 2, wherein step e comprises: The complex solution was placed in a Y-shaped micro-cuvette, and the temperature, scattering angle and operating voltage were set to 25°C, 90° and 100 mV, respectively. Each sample was measured three times and the average value was reported. Finally, PEI / DNA complexes with a particle size range of 200-300 nm and an N / P ratio of 3 or 5 were screened.

7. The method of claim 2 for delivering a low-toxic polyethyleneimine nanocarrier of DNA molecules, characterized in that: In step b, pCMV-N-EGFP and pSV40Luc were used as delivery contents.

8. Use of the nanocarrier according to claim 1 in delivering DNA molecules in vivo or in vitro.

9. The use according to claim 8, characterized in that: Such applications include gene therapy and vaccine delivery.

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