CN114949259A - Gene delivery vector with aza-crown ether structure and preparation method and application thereof - Google Patents
Gene delivery vector with aza-crown ether structure and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of nano-drugs, in particular to a gene delivery vector with an aza-crown ether structure, and a preparation method and application thereof. The invention aims to provide a nano-carrier for a gene delivery system. The invention takes aza crown ether as raw material, and obtains a polymer carrier by Michael addition reaction and Polyethyleneimine (PEI) with molecular weight not more than 3500g/mol, and the polymer carrier has the structure shown in the following structural formula (I). Proved by verification, the polymer vector can be combined with plasmid DNA and can be effectively compressed into nanoparticles, and can be used as a non-viral vector. The polymer carriers with different modification degrees have low toxicity in normal cells and high cell transfection efficiency. Meanwhile, the aza crown ether has a PEG fragment structure, so that the crown ether polymer carrier has the potential of becoming a PEG substitute, and the application range of the aza crown ether structure derivative is widened.
Description
Technical Field
The invention relates to the technical field of nano-drugs, in particular to a gene delivery vector with an aza-crown ether structure, and a preparation method and application thereof.
Background
Cancer has always been the biggest health problem, and the general treatment strategies for cancer include chemotherapy, radiotherapy, tumor resection, cancer cell killing and the like, so that the immunity of the human body is reduced, and the harm is great. However, cells such as melanoma cannot be removed by surgery, and new treatments are sought. In recent years, gene therapy is still a promising therapeutic strategy for the treatment of genetic diseases and acquired diseases, and therefore, the development of gene vectors with high transfection efficiency and low cytotoxicity is one of the most critical loops in gene therapy.
The ideal gene vector is capable of targeting cells and successfully releasing and expressing nucleic acid drugs. Generally, the transfection efficiency of the viral vector is higher, but certain problems exist, such as higher cytotoxicity, clinical difficulty and the like; the safety of non-viral transfection vectors is high, but the efficiency of cell transfection is not ideal.
The cationic liposome is a potential nano-carrier, and can completely wrap macromolecular DNA with phosphate radical of nucleic acid through electrostatic interaction to form a lipid complex, so that the volume of the DNA is compressed, and the DNA is delivered to cells in a targeted manner to be released and expressed. Lipid gene vectors all have certain cytotoxicity, and the toxicity is determined by small molecular compounds, so the safety is extremely critical for the research of the lipid gene vectors.
Azacrown ethers are macrocyclic polyamine ligands with cavities that have been studied in the fields of catalysis, chemical and biological probes, separations, and cellular imaging due to their unique structural properties. In the research of the biological performance of the azacrown ether derivative, the azacrown ether derivative is proved to have the advantages of low cytotoxicity, reduction of multidrug resistance and the like.
Disclosure of Invention
The invention aims to provide a nano-carrier for a gene delivery system.
The technical scheme of the invention is that the gene delivery vector with aza crown ether structure has the structure shown in formula I,
wherein z is 0,1, 2; m, n and p are takenThe value range is 0 to 80.
The invention also provides a preparation method of the gene delivery vector, which comprises the following steps: the modified PEI modified cationic polymer is obtained by Michael addition reaction of a cationic monomer with a carbon-carbon double bond and PEI;
the cationic monomer is one of the following compounds:
n- (2- (3- ((2- (2- (1, 4, 7-trioxa-10-azacyclododec-10-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4-dioxa-7, 10-diazaododec-7-yl) acetamido) ethyl) amino) propionamide) ethyl) acrylamide;
10- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4-dioxa-7, 10-diazacyclododecane-7-carboxylic acid tert-butyl ester;
n- (2- (3- (2- (2- (1, 4, 7, 10-tetraoxa-13-azacyclopentadecan-13-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4, 10-trioxa-7, 13-diazacyclopentadecan-7-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
tert-butyl 13- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10-trioxa-7, 13-diazacyclopentadecane-7-carboxylate;
n- (2- (2- (2- (2- (2- (1, 4, 7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) ethyl) amino) propionamide) ethyl) acrylamide;
tert-butyl 16- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10, 13-tetraoxy-7, 16-diazacyclooctadecane-7-carboxylate.
Further, the molecular weight of the PEI is not more than 3500 g/mol.
Wherein, in the Michael addition reaction, the dosage of the cationic monomer and the PEI is in an equivalent unit, and the ratio is 0.9: 1, 1: 0.9, 1: 1 or 1: 1.1; adding a cationic monomer and PEI into a solvent, reacting for 7 days at 60 ℃ under the condition of stirring, concentrating the reaction solution, dialyzing at the molecular weight cutoff of 3500Da, and drying to remove the solvent.
Specifically, the solvent is methanol.
The invention also provides a gene delivery vector prepared by the method.
The invention also provides the application of the gene delivery vector in delivering plasmids.
Further, in the application, the mass ratio of the gene delivery vector to the plasmid is 0.2-1.6: 1.
Preferably, the mass ratio of the gene delivery vector to the plasmid is 0.2-1.0: 1.
In the invention, the functional groups and long chains which react with the polyethyleneimine are consistent, and the difference is that different cations are selected, and the azacrown ether has different cyclic azacrown ethers, and the azacrown ether does not directly react with the polyethyleneimine.
The invention discloses a preparation method and application of a crown ether polymer as a gene vector, and relates to the technical field of biological organic materials and biological medicines. The crown ether polymer is a polymer carrier obtained by adding aza crown ether serving as a raw material and Polyethyleneimine (PEI) with the molecular weights of 1800g/mol and 3500g/mol respectively through Michael addition reaction, and has the structure shown in the following structural formula (I). According to the specific embodiment of selecting the diaza-octadecanoyl hexaether polymer, the polymer carrier is proved to be capable of being combined with plasmid DNA and effectively compressed into nanoparticles and can be used as a non-viral carrier. The polymer carriers with different modification degrees have small toxicity in normal cells and high cell transfection efficiency. Meanwhile, the aza crown ether has a PEG fragment structure, so that the crown ether polymer carrier has the potential of becoming a PEG substitute, and the application range of the aza crown ether structure derivative is widened.
The invention has the beneficial effects that:
the invention synthesizes aza crown ether polymer material as gene carrier and its research in gene delivery. Azacrown ethers are macrocyclic polyamine ligands with cavities that have been studied in the fields of catalysis, chemical and biological probes, separations, and cellular imaging due to their unique structural properties. In the research of the biological performance of the aza crown ether derivative, the aza crown ether derivative is proved to have the advantages of low cytotoxicity, reduction of multidrug resistance and the like. Therefore, the inventors designed and synthesized crown ether polymer materials and studied them as gene vectors for gene delivery systems. The research result proves that the azacrown ether polymer carrier ACEx-bPEIy has low toxicity to normal cells (L02 cells), and cell transfection experiments show that ACEx-bPEIy containing 10% Fetal Bovine Serum (FBS) can also obtain higher transfection rate. The aza crown ether polymer carrier ACEx-bPEIy achieves the purpose of simulating polyethylene glycol fragments, and further intensive research is expected to be applied to clinic.
The azacrown ether polymer support is prepared by the Michael addition reaction. The feasibility of the aza crown ether polymer carrier for simulating the poly-PEG fragment is evaluated through an Ethidium Bromide (EB) intercalation test, particle size potential determination, a cytotoxicity test and a cell transfection test. The results of cytotoxicity and cell transfection experiments show that the ACEx-bPEIy/pDNA complex has low toxicity to L02 cells, high transfection efficiency is obtained in two serum-free cells, the fluorescence intensity is equivalent to that of 10% FBS cells, and the fluorescence intensity induced by part of cationic carrier complexes is higher than that of bPEI25k/pDNA complex. The structure design and the result show that the azacrown ether structure is feasible and has the potential of becoming a PEG substitute, so that the azacrown ether structure can be deeply applied to biomedicine and the application range of the materials in the field of chemical biology is expanded.
Drawings
FIG. 1 is a diagram of an azacrown ether polymer support structure.
FIG. 2 is a diagram of the aza crown ether polymer carrier ACEx-bPEIy 1 H NMR spectrum.
FIG. 3 is a graph of the ability of an azacrown ether polymer vector to bind to plasmid DNA. FIG. 3(a) is a graph of an electrophoretic gel retention assay for the binding of the azacrown ether polymer carrier ACEx-bPEIy to pDNA at various w/w ratios; FIG. 3 (b-c) is a graph of the binding of ACEx-bPEIy to pDNA in PBS buffer at various w/w ratios determined for ethidium bromide intercalation experiments.
FIG. 4 shows the average particle size and surface charge of the ACEx-bPEIy/pDNA complex. FIG. 4(a) ACEx-bPEI1.8k/pDNA; FIG. 4(b) shows the ACEx-bPEI3.5k/pDNA complex.
FIG. 5 is an SEM image of the ACEx-bPEIy/pDNA complex.
Fig. 6 shows the cytotoxic effects of L02 cells and a549 cells. a-b) toxic effects of ACEx-bPEIy/pDNA complex and bPEI25k/pDNA complex on L02 cells. c-d) toxic effects of ACEx-bPEIy/pDNA complex and bPEI25k/pDNA complex on A549 cells. All toxicity tests were done in triplicate.
FIG. 7 shows in vitro cell transfection assay. a-c), ACEx-bPEIy/pDNA complex and bPEI25k/pDNA complex in L02 and A549 cells transfected with and without FBS fluorescence images, respectively.
a).ACE 1 -bPEI1.8k/pDNA complex (I-III, w/ w 5, 10, 20), ACE 2 -bPEI1.8k/pDNA complex (IV-VI, w/ w 5, 10, 20), ACE 3 -bPEI1.8k/pDNA complex (VII-IX, w/w ═ 5, 10, 20), ACE 3 -bPEI3.5k/pDNA complex (X-XII, w/w ═ 2.5, 3, 3.5), ACE 4 -bpei3.5k/pDNA complex (xiii-xv, w/w ═ 2.5, 3, 3.5) and bPEI25k/pDNA complex (xvi, w/w ═ 1.4).
b).ACE 2 -bPEI1.8k/pDNA complex (I-III, w/ w 5, 10, 20), ACE 3 -bPEI1.8k/pDNA complex (IV-VI, w/ w 5, 10, 20), ACE 3 -bpei3.5k/pDNA complex (vii-ix, w/w ═ 2.5, 3, 3.5), ACE 4 -bpei3.5k/PDNA complex (x-xii, w/w ═ 2.5, 3, 3.5) and bPEI25k/PDNA complex (xiii, w/w ═ 1.4).
c).ACE 1 -bpei1.8k/pDNA complex (i-iii, w/ w 5, 10, 20) and bPEI25k/pDNA complex (iv, w/w 1.4).
d-f) transfection efficiency of ACEx-bPEIy/pDNA complex in L02 cells and A549 cells with/without FBS. All cell transfections were quantitated in triplicate.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The polyethyleneimine (Mw: 1800g/mol) in the following examples is a product of damas-beta, CAS: 9002-98-6.
The following examples are given for polyethyleneimine (Mw: 3500g/mol) as a product of Dow Aike reagent, Inc., CAS: 9002-98-6.
The following examples are given as polyethyleneimines (Mw 25000g/mol) from Shanghainefen chemical science and technology, CAS: 9002-98-6.
In the following examples, pDNA (pUC-19) was a product of BBI.
In the following examples, pEGFP and pGL3 were extracted gene products.
The L02 cells were obtained from Shanghai foil and applied Biotechnology Co.
The A549 cells are products of Shanghai wing and applied biotechnology limited.
Characterization in the examples:
an agarose gel electrophoresis experiment and an ethidium bromide intercalation experiment characterize the binding capacity of the aza crown ether polymer carrier and plasmid DNA; a 25 ℃ dynamic light scattering system characterizes the particle size and zeta potential of the aza crown ether polymer carrier/pDNA complex; scanning Electron Microscopy (SEM) characterizes the morphology of the azacrown ether polymer carrier/pDNA complex.
Example 1: preparation and characterization of Azacrorown Ether Polymer Supports
1. Azacarown ether polymer carrier ACEx-bPEIy (x represents the number of cationic monomers, y represents the molecular weight of the chosen PEI, b represents the synthesis of branched PEI)
Cationic monomer (1 equivalent; tert-butyl 16- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10, 13-tetraoxy-7, 16-diazacyclooctadecane-7-carboxylate) and polyethyleneimine (PEI; 0.9 equivalent) with molecular weights of 1800g/mol and 3500g/mol, respectively, were added to methanol and stirred at 60 ℃ for 7 days. After the reaction, the reaction solution was concentrated, dialyzed against 3500Da molecular weight cut-off dialysis bag for 3 days, and then the solvent was removed in a lyophilizer to obtain ACEx-bPEIy (ACE represents a cationic monomer, and x represents the amount of the cationic monomer, i.e., x ═ 1,2,3,4, 5; and y represents the molecular weight of the PEI selected, i.e., y ═ 1.8k or 3.5k) in the azacrown ether polymer carrier I. The actual reaction amounts are shown in table 1.
TABLE 1 Aza crown ether Polymer Carrier dosing Table
| Name (R) | Cationic monomer [ PEIn] | |
bPEIn/(mg) |
| ACE 1 - |
1 | 30.3 | 30.8 |
| ACE 2 - |
2 | 230.6 | 45.3 |
| ACE 3 - |
3 | 160.6 | 40.7 |
| ACE 3 - |
3 | 152.1 | 53.7 |
| ACE 4 - |
4 | 149.6 | 49.8 |
| ACE 5 - |
5 | 73.7 | 25.6 |
The synthesized aza crown ether polymer carrier ACEx-bPEIy is prepared by adopting a Bruker AVANCE III 600MHz type spectrometer 1 And H NMR test, measuring the chemical potential and number of hydrogen elements to determine the series, wherein the test value is very close to the theoretical value, and the obtained product is consistent with an expected synthetic structure as shown in the following formula.
1 H NMR(400MHz,D 2 O)δH:3.38(s),2.76(dd,J＝79.1,57.0Hz),1.36(s).
2. Preparation of Aza-crown Ether Polymer Carrier/pDNA Complex
0. mu.g, 0.025. mu.g, 0.05. mu.g, 0.075. mu.g, 0.10. mu.g, 0.125. mu.g, 0.15. mu.g, 0.175. mu.g and 0.20. mu.g of the azacrown ether polymer carrier, ACEx-bPEIy, was thoroughly mixed with 0.125. mu.g of DNA (pUC19) in w/w ratios of 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4 and 1.6 and incubated at room temperature for 30 min.
Selecting 6 aza crown ether polymer carriers (respectively ACE) prepared in step 1 1 -bPEI1.8k,ACE 2 -bPEI1.8k,ACE 3 -bPEI1.8k,ACE 3 -bPEI3.5k,ACE 4 -bPEI3.5k and ACE 5 -bpei3.5k), the preparation process and the times of the 6 supports are identical, except for the amounts of the reactants. According to the method, crown ether polymer carriers and compounds with DNA (w/w) of 0-1.6 are respectively assembled. The actual amounts of the complexes prepared are shown in Table 2.
TABLE 2 preparation of the amount of the complexes
| w/w | Azacarown ether Polymer Carrier/(μ g) | pUC19 DNA/(μg) |
| 0 | 0 | 0.125 |
| 0.2 | 0.025 | 0.125 |
| 0.4 | 0.05 | 0.125 |
| 0.6 | 0.075 | 0.125 |
| 0.8 | 0.1 | 0.125 |
| 1.0 | 0.125 | 0.125 |
| 1.2 | 0.15 | 0.125 |
| 1.4 | 0.175 | 0.125 |
| 1.6 | 0.2 | 0.125 |
3. Characterization of
(1) Binding ability of azacrown ether Polymer Carrier to DNA
And (3) performing agarose gel electrophoresis experiments and ethidium bromide intercalation experiments on the compound prepared in the step (2) to characterize the binding capacity of plasmid DNA (pDNA: pUC-19), and specifically operating as follows:
to 0.125. mu.g of pUC-19 solution, an appropriate amount of vector was added to prepare complexes at w/w ratios of 0,0.2,0.4,0.6,0.8,1.0,1.2,1.4 and 1.6. The complex was incubated at room temperature for 30 min. Then, 1 XTBE was used as a running buffer, 0.5% (W/V) agarose gel was used for electrophoresis at 80V for 40min, and the DNA was observed under an ultraviolet lamp of 312nm wavelength with Quantum-ST 41100/26M.
The quenching of ethidium bromide by the lipid complex was analyzed by fluorescence spectroscopy at room temperature (RF-6000 type spectrofluorometer, shimadzu, tokyo, japan). EB (10. mu.L, 0.025. mu.g/. mu.L) was placed in a quartz cuvette containing 200. mu.L of phosphate buffer solution (PBS; pH7.4), and the fluorescence intensity of EB was measured after shaking. Then, pUC-19 (20. mu.L, 0.025. mu.g/. mu.L) was mixed and added to the solution, and the fluorescence intensity of the interaction of DNA with EB was measured. Subsequently, a crown ether polymer carrier solution (0.05. mu.g/. mu.L, 1.0. mu.L each) was added to the above solution, and the fluorescence intensity thereof was measured. The excitation wavelength was 520nm and the emission wavelength was 620nm for all samples. The relative fluorescence intensity (% FI) of the sample was calculated according to the following formula:
in the formula, F EB Strong light intensity of pure EB solution, F 0 Is the fluorescence intensity after the interaction of EB and DNA.
The results are shown in FIG. 3. The results shown in FIG. 3(a) indicate that the crown ether polymer carrier ACEx-bPEIy can effectively bind to DNA (wrap DNA) and retard its electrophoretic mobility. Wherein, ACE 1 -bPEI1.8k/pDNA complex, ACE 2 -bPEI1.8k/pDNA complex and ACE 3 The bPEI1.8k/pDNA complex allows complete encapsulation of pDNA at w/w of 0.4, 1.2 and 0.4, respectively; ACE 3 -bPEI3.5k/pDNA complex, ACE 4 -bPEI3.5k/pDNA complex and ACE 5 The bPEI3.5k/pDNA complex can completely encapsulate DNA at w/w of 1.0, 0.4 and 0.6, respectively. The EB intercalation experiment was further used to determine the binding of the crown ether polymer carrier ACEx-bPEIy at different w/w ratios to pDNA in PBS buffer. FIG. 3(b, c) shows the results of EB intercalation analysis with relative fluorescence intensity as a function of w/w ratio in PBS solution at pH 7.4. As a result of experiments, the fluorescence intensity is gradually reduced along with the increase of w/w, because the fluorescence intensity is increased after the EB is inserted into the DNA, and the ACEx-bPEIy/pDNA compound is formed along with the addition of the crown ether polymer carrier ACEx-bPEIy to replace the insertion effect of the EB, so that the fluorescence intensity is reduced, and the latter has stronger DNA binding capacity. Wherein ACE 1 -bPEI.8/pDNA complex, ACE 3 -bPEI.8k/pDNA complex, ACE 3 -bPE3.5k/pDNA complex, ACE 4 -bPE3.5k/pDNA complex and ACE 5 -0 for the relative fluorescence intensity of the bPE3.5k/pDNA complexes below 10% w/w, respectively.7. 0.7, 1.4, 0.4, 1.0, 1.6 and 1.4, and when w/w is 1.6, ACE 2 The relative fluorescence intensity of the lowest quenching of the-bPEI.8k/pDNA complex was 12.7%. The results of agarose electrophoresis experiments and EB intercalation experiments show that the crown ether polymer carrier ACEx-bPEIy has good binding capacity with plasmid DNA.
(2) Particle size and particle size potential of the composite
The particle size and zeta potential of the crown ether polymer carrier/pDNA complex were measured at room temperature using a 25 ℃ dynamic light scattering system (NanoBrook 90PlusPALS, Brookhaven Instruments, Brookhaven, USA). The crown ether polymer carrier solution was mixed with 50. mu.L of DNA (0.025. mu.g/. mu.L) to prepare liposome particles in 1mL of ultrapure water.
The results are shown in FIG. 4, where the particle size of the composite decreases with increasing w/w ratio. The result shown in FIG. 4(a) indicates that 1-3 cationic monomers (tert-butyl 16- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10, 13-tetraoxy-7, 16-diazacyclooctadecane-7-carboxylate) are respectively modified on low molecular weight bPEI1.8k 1 -bPEI1.8k、ACE 2 -bPEI1.8k and ACE 3 of-bPEI1.8k, two modified ACEs 2 The particle size of the-bPEI1.8k/pDNA compound is smaller. ACE 2 -bpei1.8k/pDNA complex having a minimum particle size at a w/w of 20, the minimum particle size being 180 nm; and ACE 1 -bpei1.8k/pDNA complex having a minimum particle size at a w/w of 5, the minimum particle size being 270 nm; ACE 3 -bPEI1.8k/pDNA complex has a minimum particle size at a w/w of 10, the minimum particle size being 423 nm. Meanwhile, the Zeta potential range of the three crown ether polymer carriers is-0.9 mV- +0.3 mV. The result of FIG. 4(b) shows that 3-5 cationic monomers are modified to the cationic carrier ACE on bPEI3.5k 3 -bPEI3.5k、ACE 4 -bPEI3.5k and ACE 5 -nanoparticule size and Zeta potential of bpei3.5k. Wherein, ACE 3 -bPEI3.5k/pDNA complex and ACE 4 the-bPEI3.5k/pDNA compound has the minimum particle size of 286nm and 481nm when the w/w is 4, and the Zeta potential is positive charge and ranges from +0.31mV to +1.85 mV. And ACE 5 The nanoparticle size of the-bPEI3.5k/pDNA complex is higher, and the minimum particle size is obtained only when the w/w is 3.5The diameter is 553nm, and the Zeta range is-0.95 mV- +2.58 mV. According to the results of the nanometer particle size and the Zeta potential, the Zeta potential of the ACEx-bPEIy/pDNA compound is appropriate, but only ACE 2 -bPEI1.8k/pDNA complex having a nanoparticle size of less than 200 nm.
(3) SEM image of ACEx-bPEIy composite
After 6 crown ether polymer carriers and bPEI25k, respectively, were incubated with 50. mu.L (0.025. mu.g/. mu.L) of immobilized pDNA, the morphology of the complexes was observed with SEM.
The results are shown in FIG. 5. Compared with bPEI25k/pDNA complex, the complex formed by the PEI-based crown ether polymer carrier ACEm-bPEIn is found to agglomerate in solution, so that the nano size of the detected complex material is larger.
Example 2: application of crown ether polymer carrier compound presentation gene
1. Cell culture
L02 cells were cultured in 1640 medium containing 10% fetal bovine serum and 0.1% antibiotics (penicillin and streptomycin) at 37 deg.C with 5% CO 2 Incubation in a cell incubator in a humid environment.
2. CCK-8 cytotoxicity assay
Toxicity of the complexes to L02 cells was determined by the CCK-8 method. Will be 1 × 10 4 Ten thousand cells per well were seeded into a 96-well plate and placed in a 37 ℃ incubator for 24 hours. The bPEI25k complex (w/w:1.4) was used as a control, and then the cells were incubated in an incubator at 37 ℃ for 24 hours. Then 10 mul CCK-8 is added, the hole plate is lightly knocked and mixed, the mixture is put into an incubator at 37 ℃ for 2 hours, and the absorbance at 450nm is measured by a microplate reader.
The results are shown in FIG. 6, comparing the cytotoxicity of bPEI25k/pDNA complex with L02 cells and ACEx-bPEIy/pDNA complex. Both the ACEx-bPEIy/pDNA complex and bPEI25k/pDNA complex treated L02 cells with cell viability higher than 75% (FIG. 6 a-b). In A549 cells, the ACEx-bPEIy/pDNA complex and the bPEI25k/pDNA complex showed significant toxicity at high mass ratios. We speculate that the low molecular weight of the PEI precursor and the inherent properties of the cationic monomer may be responsible for the high cytotoxicity. Furthermore, by comparing the toxicity of normal cells and cancer cells, we can see that the crown ether polymer carrier complex is less toxic to normal cells.
3. In vitro transfection assay
Normal cells (L02) and human non-small cell lung carcinoma cells (A549) were seeded in 48-well plates at a density of 5X 10 4 Ten thousand per well, 0.25mL complete medium per well, 5% CO at 37 ℃ 2 Was cultured in the cell culture chamber for 24 hours. The old medium was removed before the transfection experiment and the transfected cells were washed 2 times with PBS, 0.2. mu.g of plasmid DNA (pEGFP DNA was used here) of the azacrown ether polymer carrier/pDNA complex was added to each well and the cells were incubated in the incubator for 4h with serum and serum free new medium at the desired w/w ratio. After 4h, the new medium containing serum was replaced and placed in the incubator for further 24h, and then the cells transfected with the pEGFP-containing complex were examined under a 40 Xinverted fluorescence microscope and recorded using the Cellsence Standard software. Luciferase assays were performed using lipofected cells containing pGL3 plasmid DNA. Luciferase assays were performed using the Picagene luciferase assay kit (Toyo Ink, Tokyo, Japan). Transfected cells were washed 3 times with PBS and lysed in cell lysis buffer. The lysate was centrifuged at 10000 x g for 2min at 4 ℃ and the supernatant was assayed for luciferase activity. Relative Luminescence Units (RLU) for chemiluminescence were measured using a luminometer (Turner Design, 20/20; Promega). bPEI25k/pDNA was used as a positive control.
The results are shown in FIG. 7. The transfection efficiency of ACEx-bPEIy/pDNA complex in L02 cells and A549 cells was determined and compared with bPEI25k/PDNA complex. In preliminary screening experiments, ACE 2 -bPEI1.8k/pDNA complex, ACE 3 -bPEI1.8k/pDNA complex, ACE 3 -bPEI3.5k/pDNA complex and ACE 4 The highest transfection efficiency of the bPEI3.5k/pDNA complex (FIGS. 7 a-c). Thus, ACE is selected 2 -bPEI1.8k/pDNA complex, ACE 3 -bPEI1.8k/pDNA complex, ACE 3 -bPEI3.5k/pDNA complex and ACE 4 The bPEI3.5k/pDNA complex was further investigated. As shown in FIGS. 7d-f, the ACEx-bPEIy/pDNA complex induced a high level of EGFP expression in L02 cells. Although the bPEI25k/pDNA complex induced a fluorescence density lower than that induced by the bPEI 25/pDNA complexThe fluorescence intensity induced by bPEI25k/pDNA complex under serum-free conditions, but the antiserum ability is one of the criteria for the judgment of non-viral gene vectors. The results show that the four compounds can mediate good gene transduction in the presence of serum, the fluorescence intensity of the expressed GFP can be even enhanced, and the w/w is 5ACE 3 -bPEI1.8k/pDNA complex and w/w 3.5ACE 4 The fluorescence intensity of the bPEI3.5k/pDNA complex is higher than that of the bPEI25k/pDNA complex, indicating that they have good serum tolerance.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be understood that it is capable of further modifications. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (9)
1. A gene delivery vector with aza crown ether structure is characterized in that the structure is shown as formula I,
wherein z is 0,1, 2; the value ranges of m, n and p are 0-80.
2. The method for preparing a gene delivery vector according to claim 1, comprising the steps of: the modified PEI is obtained by Michael addition reaction of a cationic monomer with a carbon-carbon double bond and PEI;
the cationic monomer is one of the following compounds:
n- (2- (3- ((2- (2- (1, 4, 7-trioxa-10-azacyclododec-10-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4-dioxa-7, 10-diazaodecan-7-yl) acetamido) ethyl) amino) propionamide) ethyl) acrylamide;
10- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4-dioxa-7, 10-diazacyclododecane-7-carboxylic acid tert-butyl ester;
n- (2- (3- (2- (2- (1, 4, 7, 10-tetraoxa-13-azacyclopentadecan-13-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4, 10-trioxa-7, 13-diazacyclopentadecan-7-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
tert-butyl 13- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10-trioxa-7, 13-diazacyclopentadecane-7-carboxylate;
n- (2- (2- (2- (2- (2- (1, 4, 7, 10, 13-pentaoxa-16-azacyclooctadecan-16-yl) acetylamino) ethyl) amino) propionamide) ethyl) acrylamide;
n- (2- (3- (2- (2- (1, 4, 10, 13-tetraoxa-7, 16-diazacyclooctadecan-7-yl) ethyl) amino) propionamide) ethyl) acrylamide;
tert-butyl 16- (2, 9, 14-trioxo-3, 6, 10, 13-tetraazahexadec-15-en-1-yl) -1, 4, 10, 13-tetraoxy-7, 16-diazacyclooctadecane-7-carboxylate.
3. The method of claim 2, wherein the PEI has a molecular weight of no more than 3500 g/mol.
4. The method of claim 2, wherein the cationic monomer and PEI are present in an equivalent amount in a ratio of 0.9: 1, 1: 0.9, 1: 1 or 1: 1.1 in a Michael addition reaction; adding a cationic monomer and PEI into a solvent, reacting for 7 days at 60 ℃ under the condition of stirring, concentrating the reaction solution, dialyzing at the molecular weight cutoff of 3500Da, and drying to remove the solvent.
5. The method of claim 4, wherein the solvent is methanol.
6. A gene delivery vector prepared by the method of any one of claims 2 to 5.
7. Use of the gene delivery vector of claim 1 or 6 in the delivery of plasmids.
8. The use of claim 7, wherein the mass ratio of the gene delivery vector to the plasmid is 0.2-1.6: 1.
9. The use of claim 8, wherein the mass ratio of the gene delivery vector to the plasmid is 0.2-1.0: 1.
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