Reduction sensitive type supramolecular gene vector as well as preparation method and application thereof

Abstract

A reductive degradation type adamantane-terminated polyethyleneimine derivative as shown in a formula I is constructed, then the surface of a compound is coated with poly-beta-cyclodextrin (PCD) under the action of a subject and an object, adamantane modified PEG (Ad-PEG) can be embedded into an unoccupied CD cavity, and therefore the reduction-sensitive and PEGylated supramolecular nucleic acid carrier is constructed. The nucleic acid vector can be used for expression, gene silencing and / or endocytosis of genes of tobaccos or other animals and plants, or breeding improvement of cells of animals and plants such as tobaccos or the like, or gene therapy, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biochemical engineering technology, and in particular to a reduction-sensitive supramolecular gene carrier, a preparation method and application thereof. Background Art

[0002] Tobacco, as an ideal model crop, can be used as a bioreactor to introduce anti-cancer, anti-AIDS and health-promoting genes of other crops into tobacco through the cationic polymer carrier, etc., so that they are fully expressed, and then extracted and processed by biotechnology to obtain corresponding products; on the other hand, some genes can be directly introduced into tobacco to construct transgenic tobacco, which can be used for tobacco breeding and improvement to obtain transgenic plants with excellent traits.

[0003] Tobacco transformation includes viral vector transformation and non-viral vector transformation. Non-viral vectors have attracted much attention in recent decades due to their weak immunogenicity, easy construction and relative safety. Non-viral vectors mainly include liposomes, cationic polymers, peptides, exosomes, etc. They deliver DNA or RNA into cells in different ways to achieve gene transfer and expression. At present, many cationic polymers have been widely studied and applied to non-viral gene vectors, such as polyethyleneimine (PEI), polydimethylaminoethyl methacrylate (PDMAEMA), polylysine (PLL) and chitosan and its derivatives. These cationic polymers deliver DNA or RNA to animal cells or plant cells; however, instability, cytotoxicity, transfection efficiency and insufficient targeting in vivo restrict their further development and application in gene therapy. Therefore, it is of great practical significance to develop nanogene vectors with low cytotoxicity, environmental selectivity, high stability and high introduction efficiency, and expand the library of nanogene vectors. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a reduction-sensitive supramolecular gene carrier, a preparation method and application thereof.

[0005] The present invention provides an adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL), which is prepared from adamantane-terminated PEI, AP-CI as shown in formula I, and BPPA-Cyst as shown in formula II;

[0006] Formula I ;

[0007] Formula II .

[0008] Furthermore,

[0009] The molar ratio of the adamantane-terminated PEI, the AP-CI shown in formula I and the BPPA-Cyst shown in formula II is (0.5~1.5): (0.5~1.5): (1~3); specifically, the molar ratio of the adamantane-terminated PEI, the AP-CI shown in formula I and the BPPA-Cyst shown in formula II is 1:1:2; in a specific embodiment of the present invention, 31.84 g of PEI-Ad4 was weighed and dissolved in 40 mL of chloroform, and the product after reacting with AP-CI (0.66 g, 3.39 mmol) was reacted with 0.768 mmol BPPA-Cyst to obtain the polyethyleneimine derivative (Ad4-PEI-SS-CL).

[0010] The PEI molecular chain structure contains Y adamantane end groups, wherein Y is an integer of 1 to 4.

[0011] Material transport Further, the adamantane-terminated polyethyleneimine derivative is prepared by the following steps:

[0012] Step 1: The adamantane-terminated PEI reacts with the AP-CI shown in formula I to generate Ad X -PEI-(N3)4;

[0013] Step 2: Ad X -PEI-(N3)4 reacts with BPPA-Cyst shown in formula II to obtain the polyethyleneimine derivative with the adamantane end group;

[0014] Wherein, X is an integer from 1 to 4.

[0015] In the present invention, in the adamantane-terminated PEI (PEI-Ad), PEI is purchased and has a molecular weight of 1 kDa-25 kDa, specifically 1.8 kDa. PEI is terminated with adamantane to obtain adamantane-terminated PEI; the PEI molecular chain structure contains Y adamantane end groups, wherein Y is an integer of 1-4.

[0016] Further, the synthesis of adamantane-terminated PEI (PEI-Ad) (see references Liu J, HenninkW E, van Steenbergen MJ, et al. 2016. Versatile supramolecular gene vectorbased on host-guest interaction[J]. Bioconjugate Chemistry, 27(4): 1143~1152.) Its typical preparation steps are: reacting PEI with 1-adamantane acyl chloride to obtain adamantane-terminated PEI; different amounts of 1-adamantane acyl chloride added result in different numbers of adamantane end groups, and the number of adamantane end groups affects the transfection efficiency of the nucleic acid vector of the polymer formed by Ad-PEI and Ad4-PEI-SS-CL. Specifically, the number of adamantane end groups affects the number of host-guest binding sites of Ad4-PEI-SS-CL and PCD. If the number is small, the number of binding sites is small, and the structure of the formed complex is not stable enough, so the ability to load nucleic acids is weak, and the corresponding transfection ability is also weak; because Ad is hydrophobic, too much of it will cause Ad-PEI and Ad4-PEI-SS-CL to be insoluble in water, and thus cannot function as a carrier.

[0017] Specifically, in the preparation method of adamantane-terminated PEI, the molar ratio of PEI to 1-adamantane acid chloride is 1:(1-4), specifically 1:4;

[0018] More specifically, the preparation method of the adamantane-terminated PEI comprises the following steps:

[0019] Step 1, dissolve 1.8 kDa PEI (1.0 g, 0.56 mmol) in 20 mL of chloroform and cool in an ice bath to obtain a PEI solution;

[0020] Step 2, 441.7 mg (2.22 mmol) of 1-adamantane acid chloride was dissolved in chloroform, and the mixture was added dropwise to the PEI solution. After stirring and reacting overnight, the solvent was removed to obtain a solid residue.

[0021] Step 3, dissolve the residue in 10 mL of water, centrifuge at 5100 rpm / min for 30 minutes to remove the undissolved residue, and obtain the supernatant;

[0022] Step 4: The product was dialyzed in water (MWCO cutoff: 1000 Da) for 24 h and freeze-dried to obtain PEI-Ad.

[0023] The present invention provides a carrier, which comprises the polyethyleneimine derivative with adamantane end group described in the present invention and poly-β-cyclodextrin;

[0024] Furthermore,

[0025] The molecular weight of the poly-β-cyclodextrin is 15.0 kDa to 35.0 kDa, specifically 28.6 kDa;

[0026] In the present invention, the synthesis of poly-β-cyclodextrin (PCD, which is linear poly-β-cyclodextrin) is described in Liu J, HenninkW E, van Steenbergen MJ, et al. 2016. Versatile supramolecular gene vectorbased on host-guest interaction[J]. Bioconjugate Chemistry, 27(4): 1143~1152.

[0027] Poly-β-cyclodextrin is made by polymerization of β-cyclodextrin. Its typical synthesis method is: using β-cyclodextrin (β-CD) as raw material and epichlorohydrin (ECH) as a cross-linking agent, β-cyclodextrin polymer microspheres (β-CDP) are synthesized by the reverse emulsion method.

[0028] Specifically, the steps include:

[0029] Take 5g of β-cyclodextrin (β-CD) and dissolve it in 8mL of sodium hydroxide solution (15wt%), heat it at 35℃ for 2h, add 1mL of toluene, and stir overnight. Then, add 1.38mL of epichlorohydrin and stir at 35℃ for 3h. Subsequently, the formed polymer is precipitated in 200mL of isopropanol and filtered. The residue obtained by filtration is dissolved in 20mL of water, neutralized with 2M hydrochloric acid, and dialyzed in water. The dialysis cutoff MWCO: 12~14 kDa, and then freeze-dried to obtain white solid poly β-cyclodextrin.

[0030] The molar ratio of the adamantane end group of the polyethyleneimine derivative with the adamantane end group to the β-cyclodextrin in the poly-β-cyclodextrin is (1-3): (0.5-1.5), specifically 2:1; different ratios have different polymerization ability, transfection and cell endocytosis ability for nucleic acids. Experimental results show that the polymerization ability, transfection and cell endocytosis ability for nucleic acids are best when the molar ratio of the adamantane end group polyethyleneimine derivative with the poly-β-cyclodextrin is 1:2.

[0031] The carrier of the present invention also includes PEG with adamantane terminal group.

[0032] Specifically, there is one adamantane terminal group.

[0033] In the present invention, adamantane-terminated PEG (Ad-PEG) (synthesis see reference Liu J, Hennink WE, vanSteenbergen MJ, et al. 2016. Versatile supramolecular gene vector based on host-guest interaction [J]. Bioconjugate Chemistry, 27(4): 1143-1152.) is prepared by reacting mPEG and 1-adamantane carboxyl chloride, wherein the mass ratio of 1-adamantane carboxyl chloride to mPEG is (1-3): (4-6); specifically 2:5. The classical preparation method thereof comprises the following steps:

[0034] Step 1, dissolve 1-adamantanecarbonyl chloride in 50 mL of dry dichloromethane to obtain 1-adamantanecarbonyl chloride solution (2.0 g, 10.06 mmol);

[0035] Step 2: mPEG (5.0 g) and triethylamine (1.1 g, 10.87 mmol) were dissolved in 100 mL of dry dichloromethane and mixed with the 1-adamantanecarbonyl chloride solution obtained in step 1, immersed in an ice bath for 2 hours, and then stirred at room temperature overnight.

[0036] Step 3: remove dichloromethane and triethylamine under reduced pressure to obtain a residue, dissolve the residue in 20 ml of water, centrifuge at 5100 rpm for 30 minutes, remove 1-adamantanecarboxylic acid (precipitate), dialyze the supernatant in water for 48 hours, and obtain Ad-PEG after MWCO of 3.5 kDa.

[0037] Among them, mPEG (mPEG, lot number LOT: BCBG9781 V, Mn: 4677 Da, PDI: 1.13) was purchased from Sigma-Aldrich.

[0038] The molar ratio of the β-cyclodextrin to the adamantane-terminated PEI is (2-20):1.

[0039] In the present invention, 1 mol of β-cyclodextrin can be combined with 1 mol of adamantane. The present invention ensures that the molar ratio of β-cyclodextrin to adamantane in the poly-β-cyclodextrin is 2:1, so that half of the cavity of the poly-β-cyclodextrin is embedded in the polyethyleneimine derivative with the adamantane end group; the other half of the cavity can be used to embed the adamantane-terminated PEG, and the entire half is embedded as 100% PEG, at which time the molar ratio of β-cyclodextrin in the poly-β-cyclodextrin to the adamantane-terminated PEI can be 2:1; one-half of the half is embedded as 50% PEG, at which time the molar ratio of β-cyclodextrin in the poly-β-cyclodextrin to the adamantane-terminated PEI can be 4:1; 30% of the half is embedded as 30% PEG, at which time the molar ratio of β-cyclodextrin in the β-cyclodextrin to the adamantane-terminated PEI can be (6-7):1, specifically 6.66:1;

[0040] The present invention provides the use of at least one of the following A) to C) as a carrier in carrying and / or carrying a substance:

[0041] A), the polyethyleneimine derivative with adamantane end group of the present invention;

[0042] B), a polyethyleneimine derivative with an adamantane end group obtained by the preparation method of the present invention;

[0043] C), the vector of the present invention.

[0044] Furthermore, the substance comprises nucleic acid, and is used for expression and / or silencing of the nucleic acid;

[0045] Further, the molar concentration ratio of the nucleic acid to the carrier is 1:(10-50);

[0046] In the application of the present invention, the source of the nucleic acid includes plant or animal cells, the plant cells include tobacco cells; the animal cells include 293T cells, HeLa cells and / or COS7 cells;

[0047] In the application of the present invention, the vector carries and / or carries substances into a host; the host may be a plant or animal cell, the plant cell includes a tobacco cell; the animal cell includes a 293T cell, a HeLa cell and / or a COS7 cell;

[0048] Tobacco cells, as plant cells, obtain protoplasts after removing the cell wall. The structure of protoplasts is similar to that of animal cells, and plant tissue culture technology often uses plant protoplasts to cultivate new plants. In the specific embodiment of the present invention, animal cells are used as the research object to verify the performance of the vector, which provides a basis for the subsequent development of tobacco vectors.

[0049] In the present invention, the nucleic acid and the vector form a complex for expression and / or silencing of the nucleic acid;

[0050] The expression includes high expression, low expression and / or partial expression, which is not limited in the present invention;

[0051] The nucleic acid of the present invention can be DNA, RNA, cDNA or PNA. In an embodiment of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA.

[0052] The nucleic acid described in the present invention may also include a plasmid vector carrying the nucleic acid, which is not limited in the present invention; the nucleic acid and the vector form a complex for the expression and / or silencing of the nucleic acid by transforming, transfecting and / or endocytosing the nucleic acid in the form of a plasmid, virus and / or polymer;

[0053] The plasmid refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence or element that is essential for the expression of an operably linked coding gene in a specific host organism. In this specification, "plasmid" and "vector" can sometimes be interchanged, because plasmid is the most commonly used vector form. However, the present invention is intended to include such other forms of expression vectors, which play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genome inserts.

[0054] The transformation includes chemical transformation and electrotransformation; the transfection includes calcium phosphate coprecipitation, artificial liposome method, and viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc., which are not limited in the present invention.

[0055] The cell endocytosis is the process of transporting extracellular substances into cells through the deformation movement of the plasma membrane. The cell endocytosis includes phagocytosis, pinocytosis and / or receptor-mediated endocytosis, which is not limited in the present invention.

[0056] In the present invention, the complex composed of the nucleic acid and the vector includes various specific examples:

[0057] A complex of a nucleic acid and an adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL); and / or

[0058] A complex of a nucleic acid, an adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL) and poly-β-cyclodextrin (PCD); and / or

[0059] Complexes composed of nucleic acids, adamantane-capped polyethyleneimine derivatives (Ad4-PEI-SS-CL), poly-β-cyclodextrin (PCD), and adamantane-capped PEI (Ad-PEG).

[0060] Ad4-PEI-SS-CL is a reduction-responsive gene vector. In a reducing environment, the disulfide bonds are broken and the cell is degraded into low molecular weight Ad4-PE.

[0061] When the molar ratio (N / P) of Ad4-PEI-SS-CL to nucleic acid is higher than 2, Ad4-PEI-SS-CL can effectively bind DNA and prevent its migration in the electric field; the transfection efficiency of Ad4-PEI-SS-CL as a nucleic acid carrier first increases with the increase of N / P ratio, and reaches the highest transfection rate when the N / P ratio is 10, 20 or 30 (among which 293T cells reach the highest when the N / P ratio is 20; HeLa cells reach the highest when the N / P ratio is 10; COS7 cells reach the highest when the N / P ratio is 30), and the transfection efficiency decreases with the increase of N / P ratio after reaching the highest value, and the particle size of the complex formed by Ad4-PEI-SS-CL and nucleic acid is also the smallest when the N / P ratio is 30, reaching 105.58nm;

[0062] The ability of PCD / Ad4-PEI-SS-CL to bind DNA after PCD and Ad4-PEI-SS-CL combined was reduced, and the particle size became larger, but still below 200nm (about 180.766nm). It can only bind effectively when the N / P ratio is higher than 3. However, the cytotoxicity after binding with PCD was reduced, the anti-protein adsorption ability was increased, and it still had good reduction response ability;

[0063] At a relatively low N / P ratio (30 or below), the transfection efficiency and / or endocytosis efficiency of the PCD / Ad4-PEI-SS-CL / pDNA complex vector is lower than that of Ad4-PEI-SS-CL. However, at a relatively high N / P ratio (40 or 50, the highest at 50), the transfection efficiency and / or endocytosis efficiency of the PCD / Ad4-PEI-SS-CL complex vector is higher than that of Ad4-PEI-SS-CL. In general, the transfection efficiency of the Ad4-PEI-SS-CL / pDNA complex with an N / P ratio of 30 is higher than that of the PCD / Ad4-PEI-SS-CL / pDNA complex. However, PCD / Ad4-PEI-SS-CL is less affected by cell types and culture conditions, has a more stable transfection efficiency, is less restricted by conditions, and has a wider range of applications.

[0064] After PCD is combined with Ad4-PEI-SS-CL, PCD / Ad4-PEI-SS-CL / pDNA can be effectively improved after PEGylation, and the salt resistance, stability, and anti-protein adsorption ability of the complex can be improved. The complex still has a high reduction responsiveness and maintains a high transfection efficiency and cell endocytosis efficiency.

[0065] The advantages of the polyethyleneimine derivative (Ad4-PEI-SS-CL) and / or the vector of the present invention compared with the existing nucleic acid vectors include: the entire vector is modularized, and has a main module PCD for improving the molecular weight and biocompatibility of the complex, a guest module adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL) for loading compressed genes, and a guest module Ad-PEG for improving the stability and biocompatibility of the vector. According to the needs of the present invention, the corresponding functional modules can be added to realize the corresponding functions, which is very simple and easy; and it has reduction sensitivity, good salt stability and anti-protein adsorption ability, and good biocompatibility; and the PEGylation ratio can be freely adjusted according to actual needs, which is simple and easy; compared with other existing biological vectors, it has high transfection, transformation or endocytosis efficiency, good stability, anti-protein adsorption ability and safety, and has high transfection, transformation or endocytosis efficiency and stability in different cell culture systems (different culture media), has broad application prospects, and expands the vector library for cells under different culture conditions.

[0066] The present invention provides the use of at least one of the following a) to c) as a carrier in preparing a product that carries and / or carries a substance;

[0067] a), the polyethyleneimine derivative with adamantane end group of the present invention;

[0068] b) a polyethyleneimine derivative having an adamantane terminal group obtained by the preparation method of the present invention;

[0069] c) The vector of the present invention.

[0070] The present invention provides a product for carrying and / or carrying a substance, comprising auxiliary materials and at least one of the following I) to III):

[0071] I), the polyethyleneimine derivative with adamantane end group of the present invention;

[0072] II), a polyethyleneimine derivative with an adamantane end group obtained by the preparation method of the present invention;

[0073] III), the vector of the present invention.

[0074] In a specific embodiment of the present invention, the substance is nucleic acid, and the product can be used for expression and / or silencing of nucleic acid.

[0075] Furthermore, the auxiliary material is a solid substance that maintains the activity or state of the substance, or a buffer, culture medium, preservative, stabilizer and / or antibiotic that assists the substance in exerting its function, and the present invention is not limited to this.

[0076] In the embodiments of the present invention, the nucleic acid contained in the product is a plasmid DNA from any source, which is not limited in the present invention; plasmid DNA is a conventional form of nucleic acid and is arbitrarily selectable. In a specific experiment, the replaceable sequence therein can be replaced with a specific target gene;

[0077] The target gene may be a tobacco gene or a tobacco-derived gene or a gene from other animals or plants, wherein the tobacco-derived gene includes a tobacco gene that has been modified, mutated, truncated, codon-optimized or otherwise altered;

[0078] Further, the product may also include a product using tobacco or animal cells as a host, wherein the complex of nucleic acid and vector is introduced into tobacco or other animal or plant cells to prepare transgenic transformants.

[0079] The protoplasts obtained after the cell walls of tobacco cells are removed have a structure similar to that of animal cells. The specific embodiments of the present invention use animal cells as research objects to verify the performance of the vector, providing a basis for the subsequent development of tobacco vectors.

[0080] In a specific embodiment of the present invention, the plasmid DNA carries genes encoding green fluorescent protein and luciferase.

[0081] The present invention provides a method for carrying and / or carrying a substance, which comprises carrying and / or carrying the substance by using at least one of the following i) to iv):

[0082] i), the polyethyleneimine derivative with adamantane end group of the present invention;

[0083] ii), a polyethyleneimine derivative with an adamantane end group obtained by the preparation method of the present invention;

[0084] iii), the vector of the present invention;

[0085] iv) The product of the present invention.

[0086] In the present invention, the substance is nucleic acid; the nucleic acid may be derived from plants, animals, bacteria, fungi, mycoplasma, chlamydia and / or viruses, which is not limited in the present invention;

[0087] Furthermore, the plants include but are not limited to model organisms such as tobacco and Arabidopsis thaliana, and can be used to construct tobacco and / or Arabidopsis thaliana transgenic plants.

[0088] The present invention constructs a reduction-degradable adamantane-terminated polyethyleneimine derivative as shown in formula I, and then utilizes the host-guest effect to coat poly-β-cyclodextrin (PCD) on the surface of the complex, wherein the remaining CD cavity can be embedded with adamantane-modified PEG (Ad-PEG), thereby constructing a reduction-sensitive and PEGylated supramolecular nucleic acid vector. The nucleic acid vector can be used for the expression, gene silencing and / or cellular endocytosis of tobacco or other animal and plant genes, or for breeding and improvement of tobacco and other animal and plant cells, or for gene therapy, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Schematic diagram showing the self-assembly of nanocarriers for gene delivery driven by host-guest interactions among PCD, Ad4-PEI-SS-CL and Ad-PEG;

[0090] Figure 2 Shown is the synthetic route of Ad4-PEI-SS-CL polymer;

[0091] Figure 3 1H NMR spectra are shown, wherein A is the 1H NMR spectrum of Ad4-PEI-(N3)4; B is the 1H NMR spectrum of Ad4-PEI-SS-CL in heavy water (D2O);

[0092] Figure 4 FTIR spectra of BPPA-Cyst, Ad4-PEI-(N3)4, and Ad4-PEI-SS-CL are shown;

[0093] Figure 5 2D NMR (D2O, 300 MHz) of PCD and Ad4-PEI-SS-CL host-guest inclusion complex at a ratio of 2:1.

[0094] Figure 6 Figure 2 shows the agarose gel electrophoresis blocking test of DTT on PCD / Ad4-PEI-SS-CL / pDNAAd4-PEI-SS-CL / pDNA complex at different N / P ratios, where A is in the presence of 10 mM DTT; B is in the absence of DTT;

[0095] Figure 7 Shows the particle size and DLS zetaA potential of the Ad4-PEI-SS-CL matrix complex, where A is the particle size; B is the DLS zeta potential, and the error is the standard deviation (3 treatments);

[0096] Figure 8 Figure 5 shows the TEM images of Ad4-PEI-SS-CL (N / P 20) complexes under DTT treatment and untreated conditions, where A is the image of Ad4-PEI-SS-CL / pDNA complex without DTT treatment; B is the image of PCD / Ad4-PEI-SS-CL / pDNA complex without DTT treatment; C is the image of 100% PEG / PCD / Ad4-PEI-SS-CL / pDNA without DTT treatment; D is the image of Ad4-PEI-SS-CL / pDNA treated with 10 mM DTT; E is the image of PCD / Ad4-PEI-SS-CL / pDNA treated with 10 mM DTT; F is the image of Ad4-PEI-SS-CL polymer (N / p20) of 100% PEG / PCD / Ad4-PEI-SS-CL / pDNA treated with 10 mM DTT. Scale bars in A~C are 200 nm, and scale bars in D~E are 2 μm.

[0097] Fig. 9 The effect of DTT on the complex is shown; A is the SEC trace of Ad4-PEI-SS-CL in the absence and presence of DTT (1.8 kDa PEI is the control); B is the size of the Ad4-PEI-SS-CL (N / P 20) based complex measured by DLS before and after the addition of 10 mmol DTT;

[0098] Fig.10 Figure 4 shows the cytotoxicity test of PCD, Ad4-PEI-SS-CL and control 25 kDa PEI after cell incubation for 48 h, where A is the cytotoxicity test of HeLa cells; B is the cytotoxicity test of 293T cells (mean±SD, n = 4);

[0099] Fig.11 The transfection efficiency of the complex is shown, where A is the luciferase expression of the Ad4-PEI-SS-CL-based complex in 293T, HeLa and COS7 cells at different N / P ratios; B is the fluorescence field image of the EGFP expression of the Ad4-PEI-SS-CL-based complex in 293T cells, with the 25 kDa PEI (N / p is 10) complex as a control; the magnification is 40×;

[0100] Fig.12The siRNA knockdown efficiency of the supramolecular complex is shown in Figure 1, where A is the expression of green fluorescent protein; B is the fluorescence image and relative average fluorescence intensity of HeLa-GFP cells treated with Ad4-PEI-SS-CL and siRNA complex (N / P 20 and 40); scale bar 100 μm, data are expressed as mean ± SD (n = 3);

[0101] Fig.13 The figure shows the cellular engulfment of the PEGylated complex characterized by confocal laser scanning microscopy (CLSM), where YOYO-1d stains the plasmid in green, Hoechst 33258 stains the cell nucleus in blue, and the merged image is merge; scale bar 50 μm;

[0102] Fig.14 The figure shows the anti-protein adsorption capacity of PCD and PEG gene carrier systems, where A is the particle size of the polymer (N / p20) in Hepes and HBS buffers; B is the size of the polymer (N / p20) with different PEGylation ratios determined by DLS in HBS buffer at room temperature; C is the adsorption of free carrier to BSA protein carrier / DNA polymer (N / P is 20); BSA adsorption capacity is the ratio of BSA adsorption to carrier;

[0103] Fig.15 The size of the complex of PCD / Ad4-PEI-SS-CL / 100% PEG with N / P ratio of 20 and pDNA in the presence and absence of DTT is shown;

[0104] Fig.16 The figure shows the effect of the presence or absence of 10% FBS on the luciferase expression of Ad4-PEI-SS-CL / pDNA complex in 293T cells at different N / P ratios and different PEGylation ratios, wherein A is the absence of 10% FBS; B is the presence of 10% FBS;

[0105] Fig.17 Figure 3 shows the potential changes of PCD / Ad4-PEI-SS-CL / PEG and pDNA polymers at different PEGylation ratios when the N / P ratio is 20. DETAILED DESCRIPTION

[0106] The present invention provides a reduction-sensitive supramolecular gene carrier, a preparation method and an application thereof. A person skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0107] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0108] Example 1 Construction of reduction-sensitive supramolecular gene vector

[0109] Compared with traditional synthesis methods, the host-guest interaction can more simply construct multiple functional modules in the same carrier system. The present invention synthesized a reducible and degradable PEI derivative (Ad4-PEI-SS-CL) (Figure 1) with adamantane end group to compress DNA. Subsequently, poly-β-cyclodextrin (PCD) was coated on the surface of the complex by using the host-guest interaction, and the remaining CD cavity could be embedded with adamantane-modified PEG (Ad-PEG), thereby constructing a reduction-sensitive and PEGylated supramolecular gene carrier. Finally, we systematically studied the physicochemical properties, cytotoxicity, transfection and endocytosis efficiency of this carrier system.

[0110] 1. Experimental Methods

[0111] 1. Reagents

[0112] Poly-β-cyclodextrin (PCD), adamantane-modified PEI (Ad4-PEI), adamantane-terminated polyethylene glycol (Ad-PEG, 3500 Da), bis(propargylcarbamate ethyl) disulfide (BPPA-Cyst), and azidopropyl carbonyl imidazole (AP-CI) were synthesized in previous work (J. Liu, WE Hennink, MJ van Steenbergen, R. Zhuo and X. Jiang, Bioconjugate Chem., 2016, 27, 1143–1152) and used directly. 1.8 kDa branched polyethyleneimine (PEI) and 25 kDa PEI were purchased from Alfa Aesar, and N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (Hepes), DL-dithiothreitol (DTT), and bovine serum albumin (BSA) were purchased from Sigma Aldrich. N,N'-dimethylformamide (DMF) was purchased from Shanghai Chemical Reagent Co., Ltd. and used after redistillation. Plasmid pcDNA3-Luc (5 mg / mL, dissolved in TE buffer) was purchased from Plasmid Factory, Germany. Plasmid pEGFP and anti-GFP siRNA and negative control siRNA were purchased from Sigma Aldrich.

[0113] 2. Synthesis

[0114] 2.1 Synthesis of adamantane and azide-modified polyethyleneimine (Ad4-PEI-(N3)4)

[0115] Synthesis of adamantane and azide-modified polyethyleneimine (Ad4-PEI-(N3)4) (Reference: Liu J, Xu L, Wang X, et al. 2010. Novel reduction-responsive cross-linked polyethylenimine derivatives by click chemistry for nonviral gene delivery[J]. BioconjugateChemistry, 21(10): 1827~1836.) 31.84 g of PEI-Ad4 was dissolved in 40 mL of chloroform, and AP-CI (0.66 g, 3.39 mmol) was dissolved in 10 mL of chloroform and added dropwise to the above solution. Then, the reaction was refluxed at 75°C for 10 h. After the reaction was completed, the chloroform was removed under reduced pressure to obtain a yellow viscous liquid, and the product was directly characterized by NMR.

[0116] 2.2 Synthesis of polyethyleneimine cross-linked polymer containing disulfide bonds and adamantyl groups (Ad4-PEI-SS-CL) by click reaction

[0117] Azide and adamantane modified polyethyleneimine (Ad4-PEI-(N3)4, 0.384mmol) and BPPA-Cyst (0.768mmol) were dissolved in DMF, and the oxygen in the system was removed by a vacuum-nitrogen circulation system. CuBr (1.536mmol) was added as a catalyst and reacted at 50℃ for 48h. After the reaction was completed, the solution was transferred to a 3500Da dialysis bag for dialysis, first dialyzed in a 5% EDTA solution at pH 6.5 for 2 days, then dialyzed in ultrapure water for 4 days, and finally freeze-dried to obtain Ad4-PEI-SS-CL. The structure of Ad4-PEI-SS-CL was characterized by nuclear magnetic resonance hydrogen spectrum and infrared spectrum. The molecular weight and distribution were measured by size exclusion chromatography-multi-angle dynamic light scattering system (SEC-MALLS).

[0118] 3. Structural characterization

[0119] The H NMR spectrum was measured using a Mercury VX-300 MHz NMR spectrometer with deuterated chloroform and deuterated water as solvents. The Fourier transform infrared spectrum was measured using a Perkin-Elmer Spectrum One spectrometer. The molecular weight and distribution of the polymer were measured using a size exclusion chromatography-multi-angle dynamic light scattering system (SEC-MALLS), which consisted of a Waters 2690D pump, a Waters 2414 differential refractive index detector, and a Wyatt DAWN EOS MALLS multi-angle dynamic light scattering detector. The chromatographic separation column consisted of a pre-column Shodex SB-G, Shodex OHpak SB-803, and SB-802.5. To determine the molecular weight of PCD, a mixed solvent of 80% 0.1 M NaNO3 and 20% acetonitrile (V / V) was prepared as the mobile phase. The molecular weight of Ad4-PEI-SS-CL was determined by preparing a mixture of 0.3 M sodium acetate solution at pH 4.4 and acetonitrile (V / V = 7 / 3) as the mobile phase at a flow rate of 0.5 mL / min. The molecular weight and distribution data were processed and analyzed using Astra software (Wyatt Technology).

[0120] The molecular weight and distribution of the degradation product of polymer Ad4-PEI-SS-CL after reduction were tested using SEC-MALLS: Ad4-PEI-SS-CL was dissolved in 0.9 mL of mobile phase, and then 0.1 mL of 1.0 mol / L DTT solution was added, incubated at 37°C for 2 hours, and then tested using the SEC-MALLS system.

[0121] The interaction between cyclodextrin polymer PCD and Ad4-PEI-SS-CL and Ad-PEG was characterized by two-dimensional nuclear magnetic resonance NOESY spectra, and the test was performed using a Mercury VX-300 MHz spectrometer. Deuterated water was used as the solvent to test the interaction between PCD and Ad4-PEI-SS-CL, and PCD and Ad-PEG, respectively. The concentration of all polymers was 5 mg / mL, and the polymer solution was allowed to stand at room temperature for 30 minutes before the test.

[0122] 4. Cell culture

[0123] Human embryonic kidney cells 293T, human cervical cancer cells HeLa, African green monkey fibroblast kidney cells COS7 and human cervical cancer cells stably expressing green fluorescent protein HeLa-GFP were cultured in a 5% CO2 incubator at 37°C in high-glucose DMEM medium containing 10vt% fetal bovine serum (FBS) and penicillin / streptomycin (100U / mL).

[0124] 5. Preparation of cationic polymer / pDNA complexes and PEGylation

[0125] PCD, Ad4-PEI-SS-CL and Ad-PEG were dissolved in Hepes (20mM, pH 7.4), HBS (20mM, 130mMNaCl, pH 7.4) or HBG (20mM, 5% glucose, pH 7.4) buffer solution to prepare a 1mg / mL solution. According to different N / P ratios, different volumes of PCD and Ad4-PEI-SS-CL (to ensure that the molar ratio of cyclodextrin to adamantane was 2) were added to 40μL pcDNA3-Luc solution (50μg / mL, 20mM Hepes), vortexed for 5s and incubated at room temperature for 30min to obtain PCD / Ad4-PEI-SS-CL / pDNA complexes. Using the prepared PCD / Ad4-PEI-SS-CL / pDNA complex, different volumes of Ad-PEG solution were added according to the PEGylation ratio, vortexed for 5 seconds and incubated at room temperature for 30 minutes to obtain the PEGylated complex.

[0126] 6. Agarose gel electrophoresis analysis

[0127] According to different N / P ratios, 25 μL of complex electrophoresis samples (each sample contains 100 ng DNA) were prepared. While incubating, 1% (w / v) agarose gel containing 5 μL SYBR® Safe DNA gel stain dye (dissolved in Tris-acetate solution) was prepared. 5 μL Loading Buffer was added to the electrophoresis sample of the complex, mixed, and added to the gel holes. In Tris-acetate buffer (TAE), run at 90 V for 90 min, and then use a gel imager (Bio-Rad, USA) to image and take pictures under UV (254 nm) irradiation.

[0128] Agarose gel electrophoresis was used to analyze the reduction degradation of the complex under DTT conditions and the release of DNA. 0.9 mL of PCD / Ad4-PEI-SS-CL / pDNA complex was prepared with N / P (N / P represents the molar ratio of N atoms (nitrogen atoms) in the complex to P atoms (phosphorus atoms) in DNA) ratios ranging from 1 to 8, and then 0.1 mL of 0.1 M DTT solution was added, incubated at 37°C for 30 min, and then tested using the above method.

[0129] 7. Determination of the particle size and zeta potential of the complex

[0130] The particle size and ζ potential of the complex were measured by Nano-ZS ZEN3600 (Malvern Instruments). According to different N / P ratios, a series of complex HBS solutions were prepared according to the method in "5". Then they were placed in a four-sided square glass cuvette for particle size measurement. Similarly, a series of complex HBG solutions were prepared according to the method in 5 and placed in a potential sample cell for ζ potential measurement.

[0131] 8. Transmission electron microscopy (TEM) observation

[0132] The morphologies of Ad4-PEI-SS-CL / pDNA, PCD, d4-PEI-SS-CL / pDNA, 100% PEG / PCD / Ad4-PEI-SS-CL / pDNA complexes and their morphologies after being treated with 10mM DTT were observed by transmission electron microscopy (Tecnai Spirit, 120kV). The copper mesh with Formvar membrane was soaked in the sample solution and then taken out. After a few minutes of deposition, the excess solution was removed and the sample was placed under a transmission electron microscope for observation after drying.

[0133] 9. Study on the stability of the complex

[0134] 9.1 Study on the stability of complex salts

[0135] The complexes were prepared in Hepes (20 mM Hepes, pH 7.4) and HBS (20 mM Hepes, 130 mM NaCl, pH 7.4) solutions, and their particle size changes over time were tested by dynamic light scattering (DLS), with 25 kDa PEI / pDNA (N / P 10) complex as a reference.

[0136] 9.2 Testing of the ability of the complex to resist protein adsorption

[0137] Dissolve the carrier material (1 mg / mL) and the carrier material / pDNA complex under the optimal N / P ratio in pure water. Use bovine serum albumin (BSA) as a model protein and prepare it into a 2 mg / mL aqueous solution. Mix 1 mL of BSA solution with 1 mL of carrier or complex solution and react at 37°C for 30 min. Then, centrifuge the sample at 8000 rpm and take the supernatant to test the ultraviolet absorption at a wavelength of 280 nm. Calculate the mass of BSA that can be adsorbed by a unit carrier material or complex through the standard curve of BSA aqueous solution and define it as: (BSA) adsorbed = ([BSA]0–[BSA] s ) / W polymer , where [BSA]0 represents the total amount of BSA in the initial mixture (mg), [BSA] s represents the content of BSA in the supernatant (mg), W polymer is the content of carrier material in the mixed solution (mg).

[0138] 10. Study on reduction sensitivity of complex

[0139] Ad4-PEI-SS-CL / pDNA and PCD / Ad4-PEI-SS-CL / pDNA(N / P 20) complexes were incubated in Hepes solution containing 10mM DTT and without DTT, respectively. 100% PEG / PCD / Ad4-PEI-SS-CL / pDNA(N / P20) complexes were incubated in HBS solution containing 10mM DTT and without DTT, respectively. The changes in particle size of the above complexes over time were measured by dynamic light scattering (DLS), and their morphological changes were characterized by TEM.

[0140] 11. In vitro toxicity test

[0141] The in vitro cytotoxicity of the polymers was evaluated by detecting the relative survival rate of 293T and HeLa cells (MTT method). 293T and HeLa cells were seeded in 96-well cell culture plates at a density of 6000 cells / well and cultured in a 5% CO2, 37 ℃ incubator for 24 hours. 100 μL of fresh DMEM medium was replaced before use. PCD, Ad4-PEI-SS-CL, PCD / Ad4-PEI-SS-CL, 25 kDa PEI and 1.8 kDa PEI were dissolved in DMEM medium, filtered and sterilized, and diluted in equal proportions with culture medium to form solutions of different concentrations, and 100 μL of material solution was added to each well. Two columns of blank wells were used as blank controls, and two columns of wells with only culture medium added were used as 100% survival controls. After the material solution was added, the cells were cultured for 48 hours, and then 200 μL of fresh DMEM medium was replaced in each well, and 20 μL of MTT (5 mg / mL, dissolved in PBS buffer) was added, and incubated in a 37 ℃ incubator for 4 hours. The medium and MTT solution were removed with a syringe, and 150 μL of DMSO was added and gently shaken for 1 minute at room temperature to dissolve the purple formazan crystals. The absorbance was tested at 570 nm using a Microplate Reader (BIO-RAD 550). The relative survival rate of cells = [OD (sample) - OD (blank background)] / [OD (cells without material) - OD (blank background)] × 100%. Each concentration of the polymer was tested in 4 independent parallel samples, and the test results are the mean ± standard deviation (SD).

[0142] 12. In vitro transfection experiment

[0143] 12.1 Luciferase Assay

[0144] 293T, HeLa and COS7 cells were selected as cell lines for in vitro transfection experiments, and pcDNA3-Luc was used as a reporter gene to study the in vitro transfection efficiency of the complex. 4The cells were seeded at a density of 100 μL / well in a 96-well plate and cultured at 37°C for 24 h in a 5% CO2 incubator. Before transfection, 100 μL of fresh serum-free or 10 vt% FBS-containing culture medium was replaced. According to the method of "5" of the present invention, the polymer / pDNA complex (25 μL, N / P ratio from 10 to 50) was prepared and added to the cells and co-cultured with the cells for 4 h. Then the culture medium containing the complex was aspirated, replaced with 100 μL of fresh DMEM culture medium containing 10 vt% FBS, and cultured for another 44 h. After transfection, the culture medium was removed, 100 μL of lysis solution (Promega, diluted to 1× with deionized water) was added, and the cell lysate was obtained by freeze-thawing 3 times in a -80°C refrigerator. 20 μL of the cell lysate was mixed with 100 μL of luciferase substrate (Promega), vortexed for 25 s, and measured in a chemiluminometer (Lumat LB9507, EG&G Berthold, Germany). The protein concentration in the cell lysate was measured using a BCA protein assay kit (Pierce), and the luciferase activity was expressed as RLU / mg Protein.

[0145] 12.2 Green Fluorescent Protein Detection

[0146] 293T cells were selected as the experimental cell line and pEGFP was used as the reporter gene. The cells were cultured at 2×10 4 The cells were seeded at a density of 100 μL / well in a 24-well plate and cultured at 37°C in a 5% CO2 incubator for 24 h. Before transfection, 0.4 mL of fresh serum-free or 10vt% FBS-containing culture medium was replaced. Polymer / pDNA complexes (100 μL, N / P ratio from 10 to 50) were prepared according to the method of "5" of the present invention, added to the cells, and co-cultured with the cells for 4 h. The culture medium containing the complex was then aspirated, replaced with 0.5 mL of fresh DMEM culture medium containing 10% FBS, and cultured for another 44 h. After transfection, the cells were directly observed using a fluorescent inverted microscope (Olympus IX 70) and photographed using CoolSNAP-Pro software.

[0147] 12.3 In vitro siRNA complex silencing efficiency test

[0148] Human cervical cancer cells HeLa-GFP stably expressing green fluorescent protein were used to study the silencing efficiency of siRNA complexes on green fluorescent protein. Cells were seeded at 40,000 / well on a 24-well plate and cultured for 24 hours. Then 0.4 mL of fresh culture medium was replaced in each well, and 0.1 mL of siRNA complex solution (25 pmol siRNA) was added, and cultured at 37°C for 4 hours. Subsequently, the culture medium containing the complex was aspirated, replaced with fresh culture medium, and cultured for another 44 hours. After transfection, a fluorescence inverted microscope (Olympus IX 71) was used to observe and take pictures directly, and then the cells were digested and collected, and the mean fluorescence intensity (MFI) of the cells was tested using a flow cytometer (Apogee A50-Micro), and the silencing results were expressed as relative fluorescence intensity (Relative MFI).

[0149] Relative fluorescence intensity (Relative MFI) is defined as follows: Relative MFI = (MFI sample -MFI negative ) / (MFI control -MFI negative )×100% where MFIsample represents the mean fluorescence intensity of cells interacting with the complex, MFI control Represents the mean fluorescence intensity of blank cells, MFI negative The mean fluorescence intensity of cells treated with negative control is shown.

[0150] 13. Confocal laser scanning microscopy (CLSM) characterization of cell phagocytosis efficiency

[0151] The phagocytosis of the complex in HeLa cells was characterized by confocal laser scanning microscopy. Plasmid pcDNA3-Luc was labeled with YOYO-1 (10 μM in PBS, green molecular probes), and cell nuclei were labeled with 0.01 mg / mL Hoechst 33258 (blue molecular probes). HeLa cells were seeded in a 6-well plate at a density of 60,000 per well and cultured at 37°C for 24 h. Fresh medium was then replaced, and 0.1 mL of the complex solution (1 μg pcDNA3-Luc) was added to each well. After 4 h of phagocytosis, the medium containing the complex was aspirated, washed twice with PBS, and then the cells were fixed with 4% paraformaldehyde solution, washed three times with PBS, and 500 μL of Hoechst33258 solution was added for staining for 10 min. Finally, the solution was aspirated, washed three times with PBS, and 500 μL of fresh medium was added. The cells were observed and photographed under a confocal laser scanning microscopy (Nikon C1-si TE2000, Japan).

[0152] 2. Results and Discussion

[0153] 1. Synthesis and characterization of polymers

[0154] 1.1 Ad4-PEI-SS-CL was successfully prepared

[0155] The synthesis and characterization of the host polymer poly-β-cyclodextrin (PCD) and the guest polymer adamantane-terminated polyethylene glycol (Ad-PEG) have been reported in previous laboratory work (Liu J, Hennink WE, van Steenbergen M J, et al. 2016. Versatile supramolecular gene vector based on host-guest interaction [J]. Bioconjugate Chemistry, 27 (4): 1143~1152.), and are directly used in the present invention. The weight average molecular weight of PCD used in the present invention was measured by SEC-MALLS to be 28.6 kDa, as shown in Table 1. The guest cationic polymer-polyethyleneimine cross-linked polymer containing disulfide bonds and adamantyl groups (Ad4-PEI-SS-CL) was obtained by a two-step reaction, as shown in Figure 2 First, Ad4-PEI-(N3)n was obtained by reacting Ad4-PEI with azidopropyl carbonyl imidazole (AP-CI). Figure 3 A in the figure shows that on average, four azide groups are attached to each Ad4-PEI molecule. Then, through click chemistry reaction, using bis(propargyl carbamate ethyl) disulfide (BPPA-Cyst) as a crosslinker and cuprous bromide as a catalyst, Ad4-PEI-(N3)4 is crosslinked to form a high molecular weight, reducible and degradable adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL). The structure of Ad4-PEI-SS-CL was characterized by hydrogen nuclear magnetic resonance and infrared spectroscopy. By comparing the hydrogen nuclear magnetic spectra of Ad4-PEI-SS-CL and Ad4-PEI-(N3)4 ( Figure 3 ), it can be seen that the new peak at the chemical shift of 8.1ppm is the proton peak on the triazole ring produced by the click chemistry reaction. And in the infrared spectrum ( Figure 4 ), 2099 cm -1 (azido) and 2126 cm -1The absorption peak at (alkyne) was very weak in Ad4-PEI-SS-CL, indicating that the alkyne and azide groups were effectively subjected to click chemistry reaction. In addition, the molecular weight and distribution of Ad4-PEI-SS-CL were characterized by SEC-MALLS. As shown in Table 1, the molecular weight of the cationic polymer Ad4-PEI-SS-CL increased to about 10 times that of the raw material Ad4-PEI. The above results show that the guest cationic polymer-high molecular weight reductively degradable adamantane-terminated polyethyleneimine derivative (Ad4-PEI-SS-CL) was successfully synthesized.

[0156] Table 1. Molecular weight of polymers used in this application

[0157]

[0158] 1.2 Host-guest interactions between PCD and adamantane-modified polymers

[0159] In order to verify that the cavity of PCD can be intercalated with adamantane through host-guest interaction, PCD and Ad4-PEI-SS-CL were dissolved in deuterated heavy water (ensuring that the molar ratio of CD to Ad was 2:1) and characterized using 2D NOESY nuclear magnetic resonance hydrogen spectroscopy. In aqueous solution, through host-guest interaction, adamantane enters the cavity of cyclodextrin, and the protons on adamantane and the protons (H3, H5 and H6) in the hydrophobic cavity inside cyclodextrin approach each other and interact, and corresponding cross-peaks will appear in the NOESY spectrum. In heavy water, the chemical shift of adamantane protons is 1.6~2.1 ppm, and the chemical shift of cyclodextrin protons is 11 3.5~4.1ppm, such as Figure 5 As shown in Figure 2, there are obvious cross peaks in the above region of PCD / Ad4-PEI-SS-CL. Therefore, 2D NOESY shows that PCD and Ad4-PEI-SS-CL can form a complex in aqueous solution through the host-guest interaction between adamantane and cyclodextrin. PCD and Ad-PEG can also achieve effective host-guest chimerism.

[0160] 1.3 Agarose gel electrophoresis analysis

[0161] The ability of the Ad4-PEI-SS-CL-based vector to bind DNA was tested by gel electrophoresis. As shown in Figure 6A, when the N / P ratio was higher than 2, the cationic polymer Ad4-PEI-SS-CL could effectively bind DNA and prevent its migration in the electric field. After the addition of the main polymer PCD, the ability of the supramolecular complex PCD / Ad4-PEI-SS-CL to bind DNA was reduced. It was only effective when the N / P ratio was higher than 3. This may be because PCD, as a polysaccharide polymer, can shield the charge of the complex to a certain extent ( Figure 7 B) This weakens the complex's ability to compress and bind DNA.

[0162] 1.4 Determination of the particle size and zeta potential of the complex

[0163] As an effective gene delivery system, it is expected that the carrier material can compress DNA into nanoparticles with appropriate size and surface potential. Figure 7 , with 25kDA PEI and 1.8kDA PEI as controls). It can be seen that the cationic polymer Ad4-PEI-SS-CL can compress DNA to form slightly positively charged (about +20mV) nanoparticles below 200nm. After adding PCD, it was found that at the same N / P ratio, the particle size of the PCD / Ad4-PEI-SS-CL / pDNA complex was slightly larger than that of the Ad4-PEI-SS-CL / pDNA complex, but still below 200nm. This may be because PCD, as a polysaccharide polymer, can shield the charge of the complex to a certain extent ( Figure 7 B) thus weakening the ability of the complex to compress and bind DNA ( Figure 6 A in the figure), which eventually leads to the loosening of nanoparticles and the enlargement of their size. However, both the cationic polymer Ad4-PEI-SS-CL and the PCD / Ad4-PEI-SS-CL complex can effectively complex with DNA to form nanoparticles with appropriate size and surface charge, which is very conducive to the engulfment of gene vectors by tumor cells, thereby improving transfection efficiency.

[0164] 1.5 Transmission electron microscopy (TEM) observation

[0165] The morphology of the complex at the optimal N / P ratio was further observed by transmission electron microscopy (TEM), as shown in Figure 8. Ad4-PEI-SS-CL, PCD / Ad4-PEI-SS-CL, and 100% PEG / PCD / Ad4-PEI-SS-CL can form relatively regular spherical particles with a size of less than 200 nm after complexing with DNA, which is consistent with the DLS results.

[0166] 1.6 Cationic polymers

[0167] Reduction sensitivity test of Ad4-PEI-SS-CL and its complex DTT was used as a reducing agent to simulate the intracellular reducing environment, and agarose gel electrophoresis, SEC-MALLS, DLS and TEM were used to characterize the reduction degradation behavior of the cationic polymer Ad4-PEI-SS-CL and its complex.

[0168] Fig. 9 A in the figure shows the SEC differential curve of the polymer. It can be seen that the retention time of the cationic polymer Ad4-PEI-SS-CL is significantly shorter than that of 1.8kDa PEI when there is no DTT. After 2 hours of action with 10 mM DTT, the peak shape changed significantly, the retention time increased, and the curve moved significantly toward the low molecular weight direction. The molecular weight of Ad4-PEI-SS-CL decreased from 24.4 kDa to 2.37 kDa, which is very close to the molecular weight of Ad4-PEI (about 2.34 kDa). The results of SEC-MALLS show that the disulfide bond of Ad4-PEI-SS-CL breaks in a reducing environment and degrades into low molecular weight Ad4-PEI.

[0169] like Figure 6 As shown in Figure B, after the Ad4-PEI-SS-CL / pDNA and PCD / Ad4-PEI-SS-CL / pDNA complexes were treated with 10mM DTT for half an hour, a large amount of DNA escaped from the complexes under the action of the electric field in the agarose gel electrophoresis test, indicating that the complexes can effectively release DNA in a reducing environment.

[0170] The DLS test shows the change of the particle size of the composite under a reducing environment, such as Fig. 9 As shown in B, the particle size of Ad4-PEI-SS-CL / pDNA and PCD / Ad4-PEI-SS-CL / pDNA complexes rapidly increased after the addition of 10 mM DTT, indicating that Ad4-PEI-SS-CL was degraded under the action of the reducing agent, causing the complex particles to become looser and larger in size.

[0171] The changes in the morphology of the composite particles under a reducing environment were characterized by TEM. Figure 8 As shown in Figures D to E, after a period of action with DTT, the composite particles underwent serious aggregation, changing from regular spherical nanoparticles to irregular clusters, and their size also changed to micrometer level. This is consistent with the above test results, indicating that the composite nanoparticles are indeed reduction-responsive.

[0172] 1.7 Cytotoxicity analysis

[0173] HeLa and 293T cells were selected, and 25 kDa PEI was used as a control. The cytotoxicity of the main polymer PCD and the guest cationic polymer Ad4-PEI-SS-CL used in this system was evaluated by the MTT method. As shown in Figure 10, even at very high concentrations, PCD still had no toxicity, while the cationic polymer Ad4-PEI-SS-CL showed lower toxicity compared with 25 kDa PEI, especially under low concentration conditions. The toxicity of the PCD / Ad4-PEI-SS-CL complex is between PCD and Ad4-PEI-SS-CL. The disulfide-crosslinked cationic polymer Ad4-PEI-SS-CL showed low toxicity, which may be due to the fact that after being engulfed by cells, the relatively high concentration of glutathione (GSH) in the cells reduces and degrades Ad4-PEI-SS-CL into low-toxic small molecule products. After compounding with PCD, the toxicity is further reduced, which may be attributed to two reasons: first, cyclodextrin itself has good biocompatibility. A large number of studies have found that modifying cationic polymers with polysaccharides (such as cyclodextrin, dextran, hyaluronic acid, etc.) can reduce toxicity; in addition, since PCD and Ad4-PEI-SS-CL are compounded through host-guest interaction, PCD can shield part of the positive charge carried by Ad4-PEI-SS-CL, thereby reducing the toxic effect on cells.

[0174] 1.8 Analysis of in vitro gene transfection efficiency

[0175] 1.8.1 Luciferase expression analysis

[0176] 293T, HeLa and COS7 cells were selected as transfected cells, and the luciferase plasmid pcDNA3-Luc was used as a reporter gene to analyze the transfection efficiency of the complex. Fig.10A in the figure shows that for Ad4-PEI-SS-CL vector, its transfection efficiency first increases with the increase of N / P ratio and reaches the highest value when the N / P ratio is 20 or 30, and then the transfection efficiency decreases with the increase of N / P ratio. This is because as the concentration of Ad4-PEI-SS-CL increases, the cytotoxicity increases and causes cell death, which leads to a decrease in transfection efficiency. Interestingly, as the N / P ratio increases, at a relatively low N / P ratio (below 30), the transfection efficiency of the PCD / Ad4-PEI-SS-CL complex vector is lower than that of Ad4-PEI-SS-CL, but when it reaches a relatively high N / P ratio (40 or 50), the transfection efficiency of the PCD / Ad4-PEI-SS-CL complex vector is higher than that of Ad4-PEI-SS-CL. The reason for this phenomenon may be that at low N / P ratios, the complex of PCD and Ad4-PEI-SS-CL shields part of the positive charge of the system, thereby reducing the mutual binding between the complex and the cells; at high N / P ratios, cytotoxicity becomes the main factor affecting transfection efficiency. Due to the introduction of PCD, the toxicity of the complex is reduced ( Fig.10 In summary, the Ad4-PEI-SS-CL-based vector system showed better transfection efficiency at most N / P ratios compared with the control 25 kDa PEI.

[0177] 1.8.2 Analysis of green fluorescent protein expression

[0178] 293T cells were selected as transfected cells, and the green fluorescent protein plasmid pEGFP was selected as the reporter gene. The expression of green fluorescent protein was directly observed under a fluorescence microscope. The transfection effect based on the Ad4-PEI-SS-CL vector and the control group 25kDa PEI was as follows Fig.11 As shown in B, compared with the control group, the Ad4-PEI-SS-CL / pDNA complex at relatively low N / P ratios (20 and 30) and the PCD / Ad4-PEI-SS-CL / pDNA complex at relatively high N / P ratios (40 and 50) expressed more fluorescent proteins with stronger intensity, which is consistent with the luciferase transfection results.

[0179] 1.8.3 Study on the efficiency of siRNA gene silencing

[0180] In addition to testing the transfection efficiency of plasmid DNA, the possibility of using this vector system for siRNA delivery was also explored. Anti-GFP siRNA was selected as the silencing gene, and HeLa-GFP cells that can stably express green fluorescent protein were selected as research cells to analyze the silencing efficiency. Fig.12As shown, although the silencing efficiency of the Ad4-PEI-SS-CL vector is lower than that of the commercial transfection reagent Lipofectamine, the vector system of the present invention still shows better silencing efficiency compared with the control 25kDa PEI. The results of siRNA silencing efficiency are also consistent with those of plasmid DNA. Therefore, all the above transfection results show that the vector system based on Ad4-PEI-SS-CL has good gene transfection ability.

[0181] 1.9 Endocytosis Research

[0182] The efficiency of endocytosis is very important for an effective gene delivery system. Laser scanning confocal microscopy (CLSM) was used to evaluate the endocytosis efficiency of different complexes. pDNA was labeled with the green fluorescent dye YOYO-1, and the cell nucleus was labeled with the blue fluorescent dye Hoechst 33258. Fig.11 As shown in the figure, at a relatively low N / P ratio (N / P 20), the internalization efficiency of the Ad4-PEI-SS-CL / pDNA complex is higher than that of the PCD / Ad4-PEI-SS-CL / pDNA complex. At a high N / P ratio (N / P 40), the internalization efficiency of the Ad4-PEI-SS-CL / pDNA complex is lower than that of the PCD / Ad4-PEI-SS-CL / pDNA complex. The results of cell phagocytosis efficiency also further explain the results of gene transfection ( Fig.11 , FIG12 ). The high cellular phagocytosis efficiency of the vector system used in the present invention indicates that the vector system is very promising.

[0183] 1.10 PEGylation via host-guest interaction and its effects

[0184] As is known to all, during the blood circulation process, due to the presence of high concentrations of salt and negatively charged proteins, the complex is prone to aggregation, so it is important to improve the stability of the complex. In the present invention, the PEGylation of the complex is very simple through the host-guest interaction, and the PEG / PCD / Ad4-PEI-SS-CL / pDNA complex is constructed, and their salt resistance, protein adsorption resistance and transfection efficiency are investigated. First, the particle size of the complex (N / P is 20) in Hepes (20mM, pH 7.4) and HBS (20mM, 130mM NaCl, pH 7.4) was tested respectively, and 25kDa PEI was used as a control. Fig.14As shown in A in Figure 1, compared with 25kDa PEI, the particle size of the carrier system based on Ad4-PEI-SS-CL is less affected by salt, and this effect becomes smaller after PEGylation, and decreases with the increase of PEGylation ratio. When the PEGylation ratio reaches 100%, the particle size remains basically the same in Hepes and HBS. This shows that PEGylation can indeed effectively improve the salt resistance of the complex. Subsequently, the change of the complex particle size over time in the salt solution HBS was tested, as shown in Figure 1. Fig.14 As shown in Figure B, PCD / Ad4-PEI-SS-CL / pDNA complexes and 25 kDa PEI are not stable in HBS, and their particle sizes increase to more than 400 nm in a very short time. After the addition of Ad-PEG, the complex becomes more stable, and the stability increases with the increase of PEGylation ratio. When the PEGylation ratio is 100%, the particle size of the complex can be kept below 150 nm during the test time. The above results show that compared with 25 kDa PEI, the carrier system based on Ad4-PEI-SS-CL has better salt resistance, especially after PEGylation.

[0185] Serum proteins can bind to cationic polymer / pDNA complexes non-specifically due to electrostatic attraction between negatively charged proteins and positively charged complexes, which leads to aggregation of the complexes. Here, bovine serum albumin (BSA) was used as a model protein to simulate the adsorption of non-specific proteins by the carrier. Fig.14 As shown in Figure C, the control group 25kDaPEI has the weakest anti-protein adsorption ability, while the carrier based on Ad4-PEI-SS-CL shows better anti-protein adsorption ability, especially after compounding PCD and different proportions of PEG. On the one hand, cyclodextrin derivatives and cyclodextrin modifications can enhance the stability of the complex and hinder the adsorption of the complex and protein. On the other hand, PEG, as a highly water-soluble neutral polymer, can form a protective hydrophilic layer on the surface of the carrier, providing a "steric stabilization" effect, thereby reducing the adsorption of proteins. Therefore, this gene carrier system with PCD and PEG has good anti-protein adsorption ability.

[0186] Moreover, PEGylation does not have much effect on the reduction responsiveness of this vector system. The particle size change of 100% PEG / PCD / Ad4-PEI-SS-CL / pDNA complex before and after the addition of DTT was tested. Although a large amount of PEG covering the surface of the complex may hinder the disassembly of the complex, it still shows a certain reduction responsiveness - the particle size increases to a certain extent, which is very important for the rapid release of DNA. Interestingly, PEGylation does not affect the transfection efficiency of the system too much through host-guest interaction. Fig.16 As shown in the figure, in the serum-free medium, the transfection efficiency of the PEGylated complex was slightly reduced, but still similar to that of 25 kDa PEI. In the medium containing 10% FBS, the advantage of PEGylation was prominent, and the transfection efficiency of the PEGylated complex was significantly higher than that of 25 kDa PEI. The cellular phagocytosis of the PEGylated complex was characterized by confocal laser scanning microscopy (CLSM) ( Fig.13 ), the phagocytic efficiency did not decrease much, which further explains the transfection results ( Fig.16 ). Although, PEGylation leads to a decrease in the potential of the complex ( Fig.17 ), which is not conducive to cell endocytosis. However, PEGylation provides a hydrophilic protective layer for the complex, which can hinder the nonspecific protein adsorption of the complex and keep the complex stable ( Fig.14 ), which will make the PEGylated complexes have a higher chance of reaching the cell surface and being phagocytosed. In addition, the delivery silencing efficiency of anti-GFP siRNA at different N / P ratios of complexes with different ratios of PEGylated was also tested ( Fig.12 ), the results were similar to those of plasmid DNA transfection and did not show much difference.

[0187] 2.4 Conclusion

[0188] In the present invention, reduction-sensitive cationic polymer Ad4-PEI-SS-CL, poly-β-cyclodextrin (PCD) and adamantane-modified polyethylene glycol Ad-PEG were synthesized, and a gene carrier system was constructed by self-assembly of the three in aqueous solution. This carrier system can effectively compress DNA to form nanoparticles, and exhibits reduction sensitivity, good salt stability and anti-protein adsorption ability. More importantly, compared with the control 25kDa PEI, this carrier system exhibits lower cytotoxicity and higher transfection efficiency. In summary, the present invention constructs a gene carrier with reduction sensitivity, good biocompatibility, simplicity and high efficiency through host-guest interaction, which will have great potential for application in future disease treatment.

[0189] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polyethyleneimine derivative having an adamantane terminal group, characterized in that: Made of adamantane-terminated PEI, such as AP-CI shown in Formula I and BPPA-Cyst shown in Formula II; Formula I ; Formula II .

2. The adamantane-terminated polyethyleneimine derivative according to claim 1, characterized in that: The molar ratio of the adamantane-terminated PEI, the AP-CI shown in formula I and the BPPA-Cyst shown in formula II is (0.5-1.5): (0.5-1.5): (1-3).

3. The adamantane-terminated polyethyleneimine derivative according to claim 1 or 2, characterized in that: The PEI molecular chain structure contains Y adamantane end groups; Wherein, Y is an integer from 1 to 4.

4. The adamantane-terminated polyethyleneimine derivative according to any one of claims 1 to 3, characterized in that Prepared by the following steps: Step 1: The adamantane-terminated PEI reacts with the AP-CI shown in formula I to generate Ad X -PEI-(N3)4; Step 2: Ad X -PEI-(N3)4 reacts with BPPA-Cyst shown in formula II to obtain the polyethyleneimine derivative with the adamantane end group; Wherein, X is an integer from 1 to 4.

5. A carrier, characterized in that The invention comprises the polyethyleneimine derivative with adamantane end group as claimed in any one of claims 1 to 4 and poly-β-cyclodextrin.

6. The carrier according to claim 5, characterized in that The molar ratio of the adamantane end group of the adamantane end group polyethyleneimine derivative to the β-cyclodextrin in the poly-β-cyclodextrin is (1-3): (0.5-1.5).

7. The carrier according to claim 5 or 6, characterized in that Also included are adamantane terminated PEIs.

8. The carrier according to claim 7, characterized in that The molar ratio of β-cyclodextrin in the poly-β-cyclodextrin to the adamantane-terminated PEI is (2-10):

1.

9. Use of at least one of A) to B) as a carrier in carrying and / or carrying a substance; A), the adamantane-terminated polyethyleneimine derivative according to any one of claims 1 to 4; B), the vector according to any one of claims 5 to 8.

10. The use according to claim 9, characterized in that: The substance includes nucleic acid; The molar ratio of nitrogen atoms in the carrier to phosphorus atoms in the nucleic acid to the carrier is (10-50):1.

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