Acid-responsive gene vector, construction method and application thereof

Abstract

The present application relates to the technical field of biological chemical industry, and particularly relates to an acid-responsive gene carrier, a construction method and application thereof.The present application provides acid-responsive supramolecular nanoparticles SNs based on star cationic polymer beta-CD-PDMAEMA and Pasp-benzoic imine-Ad host-guest interaction of adamantane modified polyaspartamide, and experimental results show that the mixing ratio (Ad / CD) of Pasp-benzoic imine-Ad and beta-CD-PDMAEMA, and the mixing ratio (N / P) of SNs and DNA can affect cell toxicity, transfection efficiency and phagocytosis efficiency, wherein the complex has high cell transfection endocytosis efficiency and stability when the N / P ratio is 20 and the Ad / CD is 2 or 4; based on this, the carrier can be used for expression, gene silencing and / or intracellular endocytosis of tobacco or other plant and animal genes, or for breeding improvement of tobacco and other plant and animal cells, or for gene therapy, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, and particularly relates to an acid-responsive gene vector, a construction method and application thereof. BACKGROUND

[0002] Tobacco is an important model organism and one of the most valuable research materials. Many pioneering researches in botany and biotechnology originate from tobacco. On the one hand, tobacco is widely used as a biological reactor in many gene expressions, including genes related to human health such as anti-cancer and anti-AIDS genes, and structural and functional analysis of various other source genes. The genes are introduced into tobacco by means of gene transformation, transfection or transduction to obtain corresponding products. On the other hand, as an important economic crop, the development of tobacco plants with multiple purposes has important significance in the full use of tobacco resources, the stable income of tobacco farmers, and the improvement of economic and social value.

[0003] The improvement of tobacco plants and the introduction of external genes both need the help of genetic engineering. At present, non-viral vectors are widely used in genetic engineering due to their weak immunogenicity, easy construction and relative safety. For polycation vectors, high molecular weight and sufficient positive charge density are the prerequisites for effective gene encapsulation, but these will lead to high toxicity and hinder intracellular release of genes. Therefore, many new strategies have been developed, such as the cationization of some biocompatible polymers to construct new structural vectors. Among them, polyamino acid-based polypeptides have attracted people's attention because they can be easily degraded into non-toxic small molecules and excreted from the body. Biodegradable polyaspartamide derivatives can be easily derived from polysuccinimide (PSI), and their side chains can be further modified to introduce reduction or pH-sensitive groups. Therefore, a series of intelligent materials based on polyaspartamide have been widely studied for their application in gene vectors. However, gene vectors need to balance stability, specificity, low cytotoxicity and transformation efficiency. Therefore, it is of great research value and application prospect to develop gene vectors with high stability, specificity, low cytotoxicity and high transformation efficiency, and to expand the gene vector library. SUMMARY

[0004] Therefore, the present application aims to provide an acid-responsive gene vector, a construction method and application thereof.

[0005] The present application provides a composition comprising adamantane end group polyaspartamide derivative (Pasp-benzoicimine-Ad) and star cationic polymer (β-CD-PDMAEMA).

[0006] The adamantane end group polyaspartamide derivative is prepared by the following steps:

[0007] Step 1, open ring reaction of one end Boc-protected ethylenediamine with polysuccinimide (PSI) as shown in formula I and removal of Boc protecting group to obtain Pasp-EDA;

[0008] Step 2, reaction of Pasp-EDA with adamantane acid benzaldehyde ester (ACB) as shown in formula II to obtain adamantane-modified polyaspartamide derivative;

[0009] The star cationic polymer is prepared from dimethylaminoethyl methacrylate (DMAEMA) monomer under the condition of β-CD-Br4 as shown in formula III as initiator;

[0010] Further, the star cationic polymer is prepared from DMAEMA monomer under the condition of brominated β-cyclodextrin as shown in formula III as initiator;

[0011] Formula I ;

[0012] Formula II ;

[0013] Formula III .

[0014] Further,

[0015] The synthesis of the star cationic polymer comprises the following steps:

[0016] Step 1, β-CD and bromoisobutyryl bromide BIBB to obtain β-CD-Br4 initiator;

[0017] Step 2, synthesis of the star cationic polymer from DMAEMA monomer under the condition of brominated β-cyclodextrin as shown in formula III as initiator and CuBr / PMDETA as catalytic system;

[0018] Among them, the molar ratio of β-CD-Br4 initiator, DMAEMA, PMDETA and CuBr is (0.1~0.3): (10~15): (0.4~0.7): (0.4~0.7); specifically 0.23:13.87:0.55:0.46; more specifically, in the present application, the addition amounts are 0.23 mmol: 13.87 mmol: 0.55 mmol: 0.46 mmol;

[0019] The number average molecular weight of the adamantane-modified polyaspartamide derivative is 30 Kda ~50 Kda; specifically 43 Kda;

[0020] The number average molecular weight of the star cationic polymer is 5.38 Kda~13.27Kda; specifically, 8.35kDa.

[0021] In a specific embodiment of the present application, a combination of molecular exclusion chromatography and multi-angle laser light scattering method (SEC-MALLS) and 1 The number average molecular weight (Mn) of adamantane-modified polyaspartamide derivative and star cationic polymer is determined by H NMR. n Specifically, the relative molecular weight of the adamantane-modified polyaspartamide derivative is 43 Kda as determined by SEC-MALLS; the relative molecular weight of the star cationic polymer is 8.45kDa. 1 The molecular weight of the star cationic polymer is 8.78kDa as determined by H NMR.

[0022] In the present application, the molar ratio of adamantane end group in the adamantane end group polyaspartamide derivative to β-cyclodextrin in the star cationic polymer is (2~8):1.

[0023] Further, when the molar ratio of the adamantane-modified polyaspartamide derivative to the star cationic polymer is 4, the polymer has small particle size, high stability, high anti-protein adsorption capacity, highest transfection efficiency and endocytosis efficiency.

[0024] In the specific embodiments of the application, the molar ratio of beta-CD-PDMAEMA and Pasp-benzoic imine-Ad as gene carriers is optimized; the particle size of the beta-CD-PDMAEMA mixed with DNA is very large (about 500 nm); after the addition of Pasp-benzoic imine-Ad, the particle size is significantly reduced (below 200 nm), especially when the Ad / CD (molar ratio of adamantane end group in adamantane end group-containing polyaspartamide derivative and beta-cyclodextrin in star cationic polymer) ratio is 4 and 8, the SN particle size is reduced to about 100 nm, and the morphology and size are more uniform, and the particle size is the smallest when the Ad / CD is 8; the stability of Pasp-benzoic imine-Ad / beta-CD-PDMAEMA in a salt solution first increases and then decreases, and the stability is the highest when the Ad / CD is 4; the anti-protein adsorption capacity of Pasp-benzoic imine-Ad / beta-CD-PDMAEMA increases with the increase of the Ad / CD ratio, and the anti-protein adsorption capacity is the best when the Ad / CD is 8, but there is no significant difference with the Ad / CD being 4; the beta-CD-PDMAEMA / pDNA complex shows low cytotoxicity, after the addition of the polymer Pasp-benzoic imine-Ad, the cytotoxicity of the SNs increases at the same N / P ratio, and the cytotoxicity is the largest when the Ad / CD is 8, and the cytotoxicity is smaller at the same N / P (molar ratio of nitrogen atoms in the carrier to phosphorus atoms in the nucleic acid) ratio when the Ad / CD is 4 or 2; at the same N / P ratio, the SNs of Pasp-benzoic imine-Ad / beta-CD-PDMAEMA have the highest transfection efficiency and endocytosis efficiency when the Ad / CD molar ratio is 4.

[0025] The application provides application of the composition as a carrier in substance carrying and / or carrying.

[0026] Further, the substance includes nucleic acid.

[0027] Further,

[0028] The molar ratio of nitrogen atoms in the carrier to phosphorus atoms in the nucleic acid is (5-20):1.

[0029] In the application, the adamantane-modified polyaspartamide derivative Pasp-benzoic imine-Ad has acid degradability, depolymerization occurs under acidic conditions, which is beneficial to the release of DNA in cells and promotes the improvement of transfection efficiency.

[0030] In the present application, adamantane modified polyaspartamide derivative (Pasp-benzoic imine-Ad) and star cationic polymer (β-CD-PDMAEMA) are mixed to be used as nucleic acid carrier for expression and silencing of nucleic acid.

[0031] In the embodiment of the present application, the molar ratio (N / P) of the complex of the polymer formed by β-CD-PDMAEMA and Pasp-benzoic imine-Ad and pDNA is optimized, and the experimental results show that the transfection efficiency and endocytosis efficiency are the highest when the molar ratio of the complex of the polymer formed by β-CD-PDMAEMA and Pasp-benzoic imine-Ad and pDNA is 20.

[0032] In the present application, the complex of nucleic acid and carrier is used for expression and / or silencing of nucleic acid.

[0033] In the application of the present application, the source of the nucleic acid includes plant or animal cells, the plant cells include tobacco cells, and the animal cells include 293T cells, HeLa cells and / or COS7 cells.

[0034] In the application of the present application, the carrier carries and / or carries substances into the host, and the host can be plant or animal cells, the plant cells include tobacco cells, and the animal cells include 293T cells, HeLa cells and / or COS7 cells.

[0035] Tobacco cells, as plant cells, are obtained by removing cell walls to obtain protoplasts, and the protoplasts are similar in structure to animal cells, and plant tissue culture technology often uses plant protoplasts to cultivate new plants; in the embodiment of the present application, animal cells are used as research objects to verify the performance of the carrier, thereby providing a basis for subsequent development of tobacco carriers.

[0036] The expression includes high expression, low expression and / or partial expression, which is not limited in the present application.

[0037] The nucleic acid in the present application can be DNA, RNA, cDNA or PNA; in the embodiment of the present application, the nucleic acid is in the form of DNA or RNA; the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.

[0038] The nucleic acid in the present application can also include a plasmid carrier carrying nucleic acid, which is not limited in the present application; the complex of the nucleic acid and the carrier for expression and / or silencing of nucleic acid is in the form of plasmid, virus and / or polymer, which is realized by transformation, transfection and / or endocytosis in cells.

[0039] The plasmid refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. In the present specification, "plasmid" and "vector" can sometimes be used interchangeably because plasmid is the most commonly used form of vector at present. However, the present application is intended to include such other forms of expression vectors which perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genomic inserts.

[0040] The transformation includes: chemical transformation and electroporation; the transfection includes calcium phosphate coprecipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection and the like, and the present application does not limit this.

[0041] The endocytosis is a process of transporting extracellular material into cells through the deformation movement of the plasma membrane. The endocytosis mode includes phagocytosis, pinocytosis and / or receptor-mediated endocytosis, and the present application does not limit this.

[0042] The present application provides the use of the composition in the preparation of a substance carrying and / or carrying product.

[0043] The present application provides a substance carrying and / or carrying product, which comprises an excipient and the composition of the present application.

[0044] Further, the excipient is a solid that maintains the activity or state of the substance, or a buffer, a culture medium, a preservative, a stabilizer and / or an antibiotic that assists the substance to play a role, and the present application does not limit this.

[0045] In the embodiments of the present application, the product contains plasmid DNA of any source, and the present application does not limit this; the plasmid DNA is a conventional form of nucleic acid, which is any optional, and in specific experiments, the replaceable sequence therein can be replaced by a specific target gene;

[0046] The target gene can be a tobacco gene or a tobacco-derived gene or a gene derived from other animals or plants, and the tobacco-derived gene includes a gene modified, mutated, truncated, codon-optimized or otherwise changed from a tobacco gene;

[0047] Further, the product can also include a product with tobacco or animal cells as a host, and a nucleic acid and a carrier complex are introduced into tobacco or other animal or plant cells to prepare a transgenic transformant.

[0048] After removing the cell wall from tobacco cells, the resulting protoplasts have a structure similar to that of animal cells. In a specific embodiment of the present invention, animal cells were used as the research object to verify the performance of the vector, providing a foundation for the subsequent development of tobacco vectors.

[0049] This invention provides the application of the product in the expression and / or silencing of nucleic acids.

[0050] The present invention provides a method for carrying and / or transporting matter, comprising carrying and / or transporting matter using the composition described in the present invention.

[0051] In this invention, the substance is nucleic acid; the nucleic acid may be derived from plants, animals, bacteria, fungi, mycoplasma, chlamydia and / or viruses, and this invention does not limit the source.

[0052] Furthermore, the plants include, but are not limited to, model organisms such as tobacco and Arabidopsis thaliana, which can be used to construct transgenic tobacco and / or Arabidopsis thaliana plants.

[0053] This invention relates to the field of biochemical technology, and particularly to acid-responsive gene vectors, their construction methods, and applications. This invention provides acid-responsive supramolecular nanoparticles (SNs) based on the host-guest interaction of the star-shaped cationic polymer β-CD-PDMAEMA and adamantane-modified polyasparagine, specifically Pasp-benzoic imine-Ad. Experimental results show that the mixing ratio of Pasp-benzoic imine-Ad to β-CD-PDMAEMA (Ad / CD) and the mixing ratio of SNs to DNA (N / P) both affect cytotoxicity, transfection efficiency, and phagocytosis efficiency. Specifically, the complex with an N / P ratio of 20 and an Ad / CD ratio of 2 or 4 exhibits both stability and high cell transfection and endocytosis efficiency. Based on this, the vector of this invention can be used for gene expression, gene silencing, and / or endocytosis in tobacco or other plants and animals, for breeding and improving tobacco and other plant and animal cells, or for gene therapy, demonstrating broad application prospects. Attached Figure Description

[0054] Figure 1 A schematic diagram showing the formation of supramolecular polymerization / pDNA nanocomplexes and the intracellular pH-stimulated degradation process;

[0055] Figure 2 The synthesis processes of Pasp-benzoic imine-Ad and β-CD-PDMAEM are shown, where A is the synthesis of Pasp-benzoic imine-Ad and B is the synthesis of β-CD-PDMAEM.

[0056] Figure 3 Show 1H NMR spectra, where A is Boc-EDA (solvent is CDC13); B is Pasp-g-EDA-Boc (solvent is DMSO-d6); C is Pasp-g-EDA (solvent is DMSO-d6); D is β-CD-Br (solvent is DMSO-d6);

[0057] Figure 4 Figure 2 shows the 1H NMR spectra of Pasp-benzoic imine-Ad and β-CD-PDMAEMA, where A is Pasp-benzoic imine-Ad; B is β-CD-PDMAEMA; 1 H NMR spectra, where A is Pasp-benzoic imine-Ad; B is β-CD-PDMAEMA;

[0058] Figure 5 Figure 3 shows the 2D1H NOESY NMR spectra of Pasp-benzoic imine-Ad / β-CD- PDMAEMA (solvent is D2O), the concentration of Pasp-benzoic imine-Ad / β-CD- PDMAEMA is 10 mg / mL, Ad / CD molar ratio = 4;

[0059] Figure 6 Figure 4 shows the effect of the ratio of Ad and CD on the particle size of SNs, A is the agarose gel electrophoresis retardation test of SNs with different Ad / CD ratios under different N / P ratios, where a~d are incubated in pH 7.4 PBS, c' is incubated in pH 5.0 acetic acid buffer for 4h when Ad / CD = 4; B is the particle size and potential of SNs with different Ad / CD ratios under different N / P ratios, where a is the particle size, b is the potential; C is the TEM images of SNs with different Ad / CD ratios under N / P ratio of 20, where a~d are incubated in pH 7.4 PBS, b'~d' are incubated in pH 5.0 acetic acid buffer for 4h; where the scale bar, a~d is 500 nm, inset is 100 nm, b'~d' is 2 μm;

[0060] Figure 7 Figure 5 shows the 1H NMR spectra of Pasp-benzoic imine-Ad before and after incubation in pH 5.0 acetic acid buffer (solvent is D2O); 1 H NMR spectra and FTIR spectra, where A is 1 H NMR spectra and FTIR spectra, where A is

[0061] Figure 8Figure 1 shows the stability and anti-protein adsorption ability test of different Ad / CD ratio nanocomplexes (N / P = 20) in HBS buffer (20 mM HEPES, 130 mM NaCl, pH 7.4) for more than 6 days, where A is the particle size of different Ad / CD ratio nanocomplexes (N / P = 20) in HBS buffer (20 mM HEPES, 130 mM NaCl, pH 7.4) for more than 6 days; B is the quantification of BSA protein adsorbed by different Ad / CD ratio nanocomplexes at N / P ratio of 20; where the relative adsorption ratio of BSA is defined as the amount of adsorbed BSA per milligram, *** p < 0.001, t test, data represented as mean ± SD, n = 3;

[0062] Figure 9 Figure 2 shows the cell viability of different Ad / CD ratio polycation / pDNA nanocomplexes at different N / P ratios, where A is 293T cells; B is B16-F10 cells; C is CHO-K1 cells; data represented as mean ± SD, n = 3;

[0063] Figure 10 Figure 3 shows the comparison of in vitro expression of luciferase protein mediated by PEI / pDNA nanocomplexes in serum medium and different Ad / CD ratio polycation / pDNA nanocomplexes at different N / P ratios, where A is 293T cells; B is B16-F10 cell line; C is CHO-K1 cell line; data represented as mean ± SD, n = 3;

[0064] Figure 11 Figure 4 shows the in vitro EGFP expression of 293T treated with different Ad / CD ratio nanocomplexes; where a1 is the picture of EGFP expressing cells, scale bar 100 nm; a2 is the ratio of EGFP positive cells; a3 is the mean fluorescence intensity (MFI);

[0065] Figure 12 Figure 5 shows the in vitro EGFP expression of B16-F10 treated with different Ad / CD ratio nanocomplexes; where b1 is the picture of EGFP expressing cells, scale bar 100 nm; b2 is the ratio of EGFP positive cells; b3 is the mean fluorescence intensity (MFI);

[0066] Figure 13 Figure 6 shows the in vitro EGFP expression of CHO-K1 treated with different Ad / CD ratio nanocomplexes; where c1 is the picture of EGFP expressing cells, scale bar 100 nm; c2 is the ratio of EGFP positive cells; c3 is the mean relative fluorescence intensity (MFI);

[0067] Figure 14FIG. 1 shows the EGFP fluorescence images and relative mean fluorescence intensity of HeLa-GFP cells treated with different Ad / CD ratio nanocomplexes (N / P is 20), wherein A is the EGFP fluorescence image; B is the relative mean fluorescence intensity, the scale is 100 nm;

[0068] Figure 15 FIG. 3 shows the laser confocal scanning images and cell counting chart of 293T cells co-incubated with different Ad / CD ratio complexes for 4 hours, wherein A is the laser confocal scanning image, B is the endocytosis efficiency; C is the mean relative fluorescence intensity (MFI); plasmid, nucleus and cell membrane are stained with TOTO-3 (red), Hoechst 33342 is used to stain the nucleus, which is blue, WGA-488 is used to stain the cell membrane, which is green, the scale is 20 μm. DETAILED DESCRIPTION

[0069] The acid-responsive gene carrier, the construction method and the application thereof are provided in the present application, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and the application of the present application have been described by the preferred embodiments, and the related personnel can obviously make changes or appropriate changes and combinations to the method and the application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0070] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The present application is further described below in combination with embodiments:

[0071] Embodiment 1 Construction and application of acid-responsive gene carrier

[0072] The present application provides a host-guest interaction based on β-CD-PDMAEMA and adamantane-modified polyaspartamide (adamantane units are connected to the polymer main chain through an acid-sensitive benzoyl imide bond) to construct acid-responsive supramolecular nanoparticles (SNs), as shown in FIG. 1. Figure 1 The present application adjusts the mixing ratio of Ad and CD to control the properties of the particles, and the effects of Ad / CD on the physicochemical properties, cytotoxicity, transfection efficiency and phagocytosis efficiency of SNs are studied in detail, and the Ad / CD molar ratio is optimized to obtain a SNs gene carrier with excellent biocompatibility and high transfection efficiency.

[0073] 1. Experimental procedure

[0074] 1.1 Reagents Poly succinimide (PSI, Mn = 17.8 kDa, PDI = 1.46) and adamantane benzaldehyde (ACB) were synthesized in previous work (Yu H, Sun J, Zhang Y, et al. 2015. pH- and β-cyclodextrin-responsive micelles based on polyaspartamide derivatives as drug carrier [J]. Journal of Polymer Science Part A: Polymer Chemistry, 53(11): 1387-1395.) and used directly. Di-tert-butyl dicarbonate (Boc anhydride), ethylenediamine, β-cyclodextrin (β-CD, 99%), N, N'-carbonyldiimidazole (CDI) were purchased from China National Pharmaceutical Group. Bromoisobutyryl bromide (BIBB, 98%), trifluoroacetic acid were purchased from Aladdin. Branched polyethylenimine (PEI, 25 kDa), thiazolyl blue (MTT), Lipofectamine® RNAiMAX transfection reagent were purchased from Sigma-Aldrich. TOTO-3, WGA Alexa Fluor 488 (WGA488) and Hoechst 33342 were purchased from Life Technologies. Plasmid pcDNA3-Luc, pEGFP, anti-GFP siRNA and negative control siRNA were purchased from Sigma.

[0075] 1.2 Synthesis of moieties

[0076] 1.2.1 Synthesis of polyaspartamide derivative containing benzoimine linkage and adamantane modification (Pasp-benzoicimine-Ad) The synthesis was based on the previous report in our lab (Yu H, Sun J, Zhang Y, et al. 2015. pH- and β-cyclodextrin-responsive micelles based on polyaspartamide derivatives as drug carrier [J]. Journal of Polymer Science Part A: Polymer Chemistry, 53(11): 1387-1395.) and mainly consisted of the following steps.

[0077] 1.2.1.1 Synthesis of Boc-EDA

[0078] Take 10 mL of ethylenediamine (EDA) dissolved in 80 mL of dichloromethane, and 50 mL of dichloromethane is dissolved in 8.1 g of Boc anhydride, and then placed in a constant pressure dropping funnel. Under ice bath conditions, Boc anhydride is added dropwise to ethylenediamine, and the reaction is continued at room temperature for 12 h. After the reaction is completed, the solvent is spin-dried and a proper amount of water is added to stir and filter, collect the filtrate and add NaCl to saturation, then extract with ethyl acetate (80 mL x 3), spin-dry the organic phase, dissolve in chloroform and filter to remove NaCl, finally add anhydrous MgSO4 to dry, filter and spin-dry to obtain a colorless viscous liquid, which is characterized by nuclear magnetic resonance hydrogen spectrum.

[0079] 1.2.1.2 Synthesis of Pasp-EDA

[0080] Take 3.2 g of Boc-EDA dissolved in 10 mL of DMF, add it to 10 mL of DMF solution containing 1.0 g of PSI, react in a 70°C oil bath for 24 h, precipitate the reaction product in ether, filter to obtain the product, remove the ether with N2, and dry at room temperature under vacuum for 24 h to obtain white solid Pasp-EDA-Boc. Dissolve the white solid in 15 mL of trifluoroacetic acid (TFA) at room temperature for 3 h, precipitate in ether, and dry the precipitate under vacuum at room temperature to obtain white solid, which is characterized by nuclear magnetic resonance hydrogen spectrum.

[0081] 1.2.1.3 Synthesis of polymer Pasp-benzoicimine-Ad

[0082] Take 0.8 g of Pasp-EDA, 0.43 g of ACB dissolved in 10 mL of DMF, and add a small amount of NaOH as catalyst, and react in a 50°C oil bath for 24 h, precipitate with ether and centrifuge, dry under vacuum at room temperature for 24 h to obtain light yellow product, which is characterized by nuclear magnetic resonance hydrogen spectrum.

[0083] 1.2.2 Synthesis of star-shaped cationic polymer β-CD-PDMAEMA

[0084] Refer to the previous literature report synthesis (Xu F, Zhang Z, Ping Y, et al. 2009. Star-Shaped Cationic Polymers by Atom Transfer Radical Polymerization from β-Cyclodextrin Cores for Nonviral Gene Delivery [J]. Biomacromolecules, 10 (2): 285-293), which mainly consists of the following two steps.

[0085] 1.2.2.1 Synthesis of four-arm ATRP initiator (β-CD-Br4)

[0086] β-CD (2.27 g, 2 mmol) was dissolved in 12 mL DMF, and stirred under nitrogen protection, then cooled to 0°C in an ice bath, 1 mL BIBB dissolved in DMF was added dropwise, 1 h dropwise addition was completed, and then stirred for 2 h in an ice bath, and then stirred at room temperature for 24 h. After the reaction was completed, the solvent was concentrated under reduced pressure, precipitated in excess ether, washed with acetone and distilled water, and dried at 40°C under vacuum for 12 h to obtain a white solid, which was characterized by nuclear magnetic resonance hydrogen spectrum.

[0087] 1.2.2.2 Synthesis of four-arm star cationic polymer β-CD-PDMAEMA

[0088] The synthesis of β-CD-PDMAEMA was carried out by ATRP reaction, using β-CD-Br4 as initiator, CuBr / PMDETA as catalyst system, and reacting at 25°C to obtain: β-CD-Br4 (0.4 g, 0.23 mmol), DMAEMA (2.7 g, 13.87 mmol) and PMDETA (120 mg, 0.55 mmol) were dissolved in 8 mL mixed solvent methanol / water (V / V, 20 / 1), and the oxygen in the system was removed by vacuum-nitrogen circulation system, then the catalyst CuBr (82 mg, 0.46 mmol) was added, and the reaction was carried out at 25°C for 1 h. The reaction was terminated by adding excess THF, and the CuBr was removed by passing the reaction solution through a neutral alumina column. Then the solvent was concentrated, and the final product β-CD-PDMAEMA was precipitated three times in excess ether, and characterized by nuclear magnetic resonance hydrogen spectrum.

[0089] 1.2.3 Structural characterization

[0090] NMR spectra were recorded on a Varian INOVA-400 MHz NMR spectrometer using deuterated chloroform, deuterated dimethylsulfoxide and deuterated water as solvents. NMR was used to characterize the structure of Pasp-benzoicimine-Ad before and after degradation. Fourier transform infrared spectroscopy (FT-IR) was performed on a Spectrum one spectrometer (Perkin-Elmer). FT-IR was used to characterize the infrared absorption difference of Pasp-benzoicimine-Ad before and after degradation. Two-dimensional nuclear magnetic NOESY spectra were used to characterize the interaction between Pasp-benzoicimine-Ad and β-CD-PDMAEMA. The Varian INOVA-600 MHz NMR spectrometer was used to test the samples, with deuterated water (D2O) as the solvent, and all polymer concentrations were 5 mg / mL. The polymer solution was allowed to stand at room temperature for 30 min before testing. The molecular weight and its distribution of the polymers were measured by size exclusion chromatography-multiple angle laser light scattering analyzer (SEC-MALLS). The instrument used a dual detector system, including a multiple angle laser light scattering signal detector (DAWN EOS, Wyatt Technology, laser wavelength of 690 nm) and a differential refractive index detector (Optilab DSP, Wyatt Technology). The chromatographic separation column consisted of Styragel HR4 WAT 045900 and Styragel HR3 WAT 045885. DMF (containing 10 mM LiBr) was used as the mobile phase, and the flow rate was 0.3 mL / min. The experimental data were processed using Astra software (Wyatt Technology).

[0091] 1.2.4 Cell culture

[0092] Human cervical cancer cells HeLa, mouse melanoma cells B16-F10 and human cervical cancer cells HeLa-GFP stably expressing green fluorescent protein were cultured in DMEM medium containing 10% FBS. Chinese hamster ovary cells CHO-K1 were cultured in DMEM medium containing 2 mM L-glutamine and 10% FBS. All the above cells were cultured in an incubator at 37 °C and a CO2concentration of 5%.

[0093] 1.2.5 Polymer / pDNA

[0094] Pasp-benzoicimine-Ad and β-CD-PDMAEMA were dissolved in Hepes (20 mM, pH 7.4), HBS (20 mM, 130 mM NaCl, pH 7.4) or HBG (20 mM, 5% glucose, pH 7.4) buffer solution (Pasp-benzoicimine-Ad and β-CD-PDMAEMA, each polymer was dissolved in 3 kinds of solvents, respectively, to obtain different polymer solutions with different solvents for subsequent different tests), and prepared into a 1 mg / mL solution. Different volumes of Pasp-benzoicimine-Ad and β-CD-PDMAEMA were added to 40 μL pcDNA3-Luc solution (50 μg / mL, 20 mM Hepes), vortexed for 5 s, and incubated at room temperature for 30 min (incubated solution includes PBS buffer at pH 7.4 or acetic acid buffer at pH 5.0), to prepare Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes with different Ad / CD molar ratios and different N / P ratios.

[0095] 1.2.6 Agarose gel electrophoresis analysis

[0096] The pH responsiveness of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes was also analyzed by agarose gel electrophoresis. According to different N / P ratios, 25 μL electrophoresis samples of complexes were prepared (each sample contained 100 ng DNA), and 1% (w / v) agarose gel containing 5 μL SYBR® Safe DNA gel stain dye was prepared in Tris-acetate solution during incubation. 5 μL Loading Buffer was added to the electrophoresis sample of the complex, mixed well, and added to the gel hole. In Tris-acetate buffer (TAE), run at 90V for 90 min, then use gel imager (Bio-Rad, USA) to image and take pictures under ultraviolet (254 nm) irradiation.

[0097] The pH responsiveness of the complexes was also analyzed by agarose gel electrophoresis. Different mass ratios of complexes were incubated in acetic acid buffer at pH 5.0 for 4 h, then electrophoresed in an electrophoresis apparatus at 90V for 90 min, and observed by ultraviolet imaging instrument.

[0098] 1.2.7 Determination of complex particle size and potential

[0099] Particle size and zeta potential of the complexes were measured by Nano-ZS ZEN3600 (Malvern Instruments). A series of complex HBS solutions were prepared according to the method in 1.2.5 with different N / P ratios. Then they were placed in a four-side square glass cuvette for particle size test. Similarly, a series of complex HBG solutions were prepared according to the method in 1.2.5 and placed in a potential sample cell for zeta potential determination. The stability of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes was characterized by testing the change of particle size within six days.

[0100] 1.2.8 Transmission electron microscopy (TEM) observation of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes (N / P 20) with different Ad / CD molar ratios and their morphologies after 4 h incubation in pH 5.0 acetic acid buffer were observed by transmission electron microscopy (Tecnai Spirit, 120 kV).

[0101] 1.2.9 Protein adsorption resistance characterization of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes (N / P 20) with different Ad / CD molar ratios were tested for their protein adsorption resistance as follows:

[0102] The carrier material (1 mg / mL) and the complex of the carrier material / pDNA under the optimal N / P ratio condition were dissolved in pure water. Bovine serum albumin (BSA) was used as a model protein and prepared into a 2 mg / mL aqueous solution. 1 mL of the BSA solution was mixed with 1 mL of the solution of the carrier or the complex, and incubated at 37°C for 30 min. Then, the sample was centrifuged at 8000 rpm, and the supernatant was tested for ultraviolet absorption at 280 nm. The mass of BSA adsorbed by unit carrier material or complex was calculated by the standard curve of the BSA aqueous solution, and was defined as: adsorbed = ([BSA]0- [BSA] s ) / W polymer , wherein [BSA]0represents the total amount of BSA (mg) in the initial mixed solution, [BSA] s represents the content of BSA (mg) in the supernatant, and W polymer is the content of the carrier material (mg) in the mixed solution.

[0103] 1.2.10 In vitro cytotoxicity analysis

[0104] 293T, B16-F10 and CHO-K1 cells were selected as test cells, and MTT method was used to evaluate the cytotoxicity of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complex at different Ad / CD molar ratios and at different N / P ratios. The specific method is as follows:

[0105] The complexes were incubated in Hepes solution containing 10 mM DTT and without DTT, respectively. The change of the above complex particle size with time was tested by dynamic light scattering (DLS), and the change of their morphology was characterized by TEM.

[0106] 1.2.11.1 Luciferase expression analysis

[0107] 11.2.11.1 Luciferase expression analysis pcDNA3-Luc was selected as a reporter gene, and 293T, B16-F10 and CHO-K1 cells were selected as transfection cells to test the luciferase expression efficiency of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complex at different Ad / CD molar ratios and different N / P ratios. The specific method is as follows:

[0108] The cells were seeded in 96-well plates at 8000 per well and cultured for 24 h. Then, 75 μL of fresh culture medium was replaced, and then the complex was added to the well (25 μL per well, 0.25 μg of pDNA), and reacted with the cells for 4 h. Subsequently, the culture medium containing the complex was aspirated, and 100 μL of fresh culture medium was replaced, and the culture was continued for 44 h. After transfection, luciferase expression analysis was performed using Steady-Glo® Luciferase Assay System kit, the relative light intensity RLU of luciferase was measured by InfiniteM200 plate reader, and the protein concentration was detected using BCA protein assay kit (Pierce), and the luciferase activity was expressed as RLU / mg Protein.

[0109] 1.2.11.2 Green fluorescent protein expression analysis

[0110] pEGFP was selected as a reporter gene, and 293T, B16-F10 and CHO-K1 cells were selected as transfection cells to test the green fluorescent protein expression efficiency of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complex at different Ad / CD molar ratios and different N / P ratios. The specific method is as follows:

[0111] Cells were seeded at 20000 per well in 24-well plates and incubated for 24 h. Then, 400 μL fresh medium was replaced, and the complexes were added to the wells (100 μL, 1 μg pDNA per well) and allowed to interact with the cells for 4 h. Subsequently, the medium containing the complexes was aspirated, and 500 μL fresh medium was added, and the incubation was continued for 44 h. After the end of the transfection, the cells were directly observed and photographed using a fluorescence inverted microscope (Olympus IX 71). The number of cells expressing green fluorescent protein and the mean fluorescence intensity (MFI) were measured by flow cytometry (Apogee A50-Micro).

[0112] 1.2.11.3 Analysis of the efficiency of siRNA silencing in vitro The anti-GFP siRNA complexes were studied for their efficiency in silencing the expression of green fluorescent protein at different Ad / CD molar ratios and different N / P ratios using human cervical cancer cells HeLa-GFP stably expressing green fluorescent protein. The method was as follows:

[0113] Cells were seeded at 40,000 per well in 24-well plates and incubated for 24 h. Then, 0.4 mL fresh medium was added to each well (0.1 mL siRNA complex solution (25 pmol siRNA) was added to each well, and the cells were allowed to interact with the complexes for 4 h. Subsequently, the medium containing the complexes was aspirated, and fresh medium was added, and the incubation was continued for 44 h. After the end of the transfection, the cells were directly observed and photographed using a fluorescence inverted microscope (Olympus IX 71), and then the cells were digested and collected, and the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (Apogee A50-Micro), and the transfection results were expressed as relative fluorescence intensity (Relative MFI). The relative fluorescence intensity (Relative MFI) was defined as follows:

[0114] Relative MFI = (MFI sample -MFI negative ) / (MFI control -MFI negative ) x 100%

[0115] where MFI sample represents the mean fluorescence intensity of the cells interacting with the complexes, MFI control represents the mean fluorescence intensity of the blank cells, and MFI negative represents the mean fluorescence intensity of the cells interacting with the negative control.

[0116] 11.2.12 Study of cellular endocytosis efficiency

[0117] The 293T cells were used as the research cells, and the laser confocal microscope and flow cytometry were used to characterize the cellular endocytosis efficiency. The method was as follows:

[0118] Cells were seeded at 20,000 per well in 8-well Lab-Tek (Thermo Fisher Scientific, USA) and incubated for 24 h. Then, 0.4 mL fresh medium was added to each well and 0.1 mL complex solution (mass ratio 20, 1 pg TOTO-3 labeled pcDNA3-Luc per well) was added to each well.

[0119] For CLSM observation, after 4 h incubation of the complex with cells, the medium containing the complex was removed and the cells were washed with PBS three times. Then, 200 pL 4% paraformaldehyde solution was added to each well to fix the cells at room temperature for 10 min. Then, the cells were washed with PBS three times, 200 pL WGA 488 (5 pg / mL) was added to each well and the cells were stained at 4 °C for 5 min. Then, the WGA 488 solution was removed and the cells were washed with PBS three times. Then, 200 pL Hoechst 33342 (0.1 mg / mL) was added to each well and the cells were stained at room temperature for 10 min. Finally, the Hoechst 33342 solution was removed and the cells were washed with PBS three times. The cells were observed and photographed using a Nikon A1R+ (Nikon Corporation, Japan) laser confocal microscope.

[0120] For flow cytometry test, after 4 h incubation of the complex with cells, the cells were washed with PBS three times and then collected by trypsin digestion for flow cytometry test (Apogee A50-Micro).

[0121] 1.2.13 Statistical analysis

[0122] Statistical difference analysis was performed using t-test. *P < 0.05 indicates that the results have a significant statistical difference; **P < 0.01 indicates that the results have a very significant statistical difference; ***P < 0.001 indicates that the results have a very high significant statistical difference; N.S. indicates that the results have no significant statistical difference. All data results were obtained from at least three parallel experiments and are expressed as mean ± standard deviation.

[0123] 2. Results and discussion

[0124] 2.1 Synthesis and characterization of polymers In this study, an acid-degradable polymer Pasp-benzoicimine-Ad was synthesized, which contains adamantane groups on the side chain through acid-sensitive imine bonds. The specific synthesis route is shown in Figure 2 Figure 2 ​In this paper, A represents the synthesis of Pasp-benzoic imine-Ad; B represents the synthesis of β-CD-PDMAEMA. In A, n represents the number of monomer units in 1 mole of Pasp polymer, and all n values ​​in A are 159. x represents the number of moles of adamantane introduced into 1 mole of polymer. NMR calculations show that the degree of adamantane substitution is approximately 11%, meaning there are approximately 17 adamantane groups in 1 mole of polymer, i.e., x = 17. In B, an average of 4 ATRP reaction sites are introduced per cyclodextrin. β-CD-PDMAEMA is obtained via the ATRP reaction, with an average of 48 DMAEMA units introduced per CD. Calculations show an average of 12 DMAEMA units introduced per reaction site, so n is 12. First, ethylenediamine with one end protected by Boc (Boc-EDA, 1H NMR as shown) is used. Figure 3 (As shown in A) Ring-opening PSI, and using trifluoroacetic acid to remove the Boc group to obtain the amino-functionalized polyasparagine derivative Pasp-EDA ( 1 H NMR such as Figure 3 (As shown in C). An acid-sensitive imine bond is generated through the aldehyde reaction between the aldehyde group on adamantane benzaldehyde ester (ACB) and the amino group on the polymer side chain, introducing an adamantyl group into the polymer side chain.

[0125] The structure of Pasp-benzoicimine-Ad is obtained through 1 H NMR determination, such as Figure 4 As shown in Figure A, the signal peak at 8.1 ppm is attributed to the hydrogen (e) on the imine bond, the signal peaks at 6.8 and 7.5 ppm are attributed to the hydrogens (f and g) on ​​the benzene ring, and the signal peaks between 1.4 and 1.9 ppm are the hydrogens (h and i) on the adamantyl group. The NMR spectrum indicates the successful synthesis of the target polymer Pasp-benzoicimine-Ad, providing not only structural characteristics but also a basis for quantitative analysis. The proportion of adamantyl groups in the polymer can be calculated based on the proton peaks (h and i) of the adamantane group and the proton peaks (a and b) of the polyasparagine backbone, with an adamantane substitution degree of approximately 11%. The molecular weight of the polymer Pasp-benzoicimine-Ad was determined by SEC-MALLS, as shown in Table 1.

[0126] Table 1. Molecular weight and dispersibility of polymers

[0127]

[0128] a Depend on 1 H NMR determination;

[0129] bDetermined by SEC-MALLS; PDI = weight average molecular weight / number average molecular weight or Mw / Mn w / M n .

[0130] Cationic polymer β-CD-PDMAEMA was synthesized mainly through two steps, as shown in Figure 2 . First, ATRP initiator CD-Br was synthesized and characterized by 1H NMR, as shown in D of Figure 3 . According to the integral area at 1.92 ppm (a, methyl proton peak in isobutyryl bromide) and 4.88 ppm (c, 1 -substituted methylene proton peak in the cavity of CD), it can be calculated that an average of 4 ATRP reaction sites were introduced on each CD. Then, β-CD-PDMAEMA was obtained by ATRP reaction, and its structure was characterized by 1H NMR. As shown in B of Figure 4 , 0.81-0.10 ppm (1) and 2.2 ppm (3) belong to the methyl proton peaks on the arm of β-CD-PDMAEMA. 1.86 ppm and 2.65 ppm (2, 4) belong to the methylene proton peaks on the arm of β-CD-PDMAEMA. 4.07 ppm (5) belongs to the methylene proton peak connected to the oxygen of the ester bond. By the ratio of the integral area of 5 to H2-H6, it can be calculated that an average of 48 DMAEMA units were introduced on each CD, and thus the molecular weight of β-CD-PDMAEMA was calculated to be about 8.78 kDa. In addition, the molecular weight and distribution of β-CD-PDMAEMA were also tested by SEC-MALLS, as shown in Table 1, and the test results (8.45 kDa) were also close to the nuclear magnetic calculation results. The results of nuclear magnetic resonance and SEC-MALLS showed that we successfully synthesized β-CD-PDMAEMA.

[0131] 2.2 Study on the construction of nanoparticles by host-guest interaction between polymer Pasp-benzoicimine-Ad and β-CD-PDMAEMA

[0132] The host-guest interaction between polymer Pasp-benzoicimine-Ad and β-CD-PDMAEMA was characterized by two-dimensional nuclear magnetic NOESY spectrum, as shown in Figure 5 . The NOE correlation signal was generated between the cavity proton peak at 3.0-4.0 ppm and the adamantane proton peak at 1.5-2.0 ppm, indicating that Pasp-benzoicimine-Ad and β-CD-PDMAEMA can indeed undergo host-guest inclusion interaction. By adjusting the mixing ratio of the two modules, supramolecular nanoparticles (SNs) with different Ad / CD molar ratios can be constructed.

[0133] The binding and compression capabilities of SNs to DNA were studied using agarose gel electrophoresis, such as... Figure 6 As shown in Figure A, the binding ability of SNs to DNA increases significantly with increasing Ad / CD molar ratio. Especially at an Ad / CD ratio of 4, SNs can bind DNA at a very low N / P ratio of 0.5. Dynamic light scattering analysis was used to analyze the prepared SNs to determine how the Ad / CD ratio affects the particle size of the SNs. Figure 6 As shown in B(a), when Pasp-benzoicimine-Ad was not added, direct mixing of β-CD-PDMAEMA with DNA still yielded large particles (approximately 500 nm), even at a high N / P ratio of 20. Subsequently, the particle size of SNs with different Ad / CD molar ratios was tested, as shown in Figure B(a). Figure 6 As shown in B(a), the particle size of the SNs was significantly reduced (below 200 nm) after the addition of Pasp-benzoicimine-Ad, especially at Ad / CD ratios of 4 and 8, where the particle size of the SNs decreased to around 100 nm, a size very suitable for cellular endocytosis. The morphology of the SNs was then observed using transmission electron microscopy, as shown in Figure B(a). Figure 6 As shown in C in the figure, the TEM image shows that the SNs are relatively uniform spherical particles, especially after the addition of Pasp-benzoicimine-Ad, the morphology and size are even more uniform, which is consistent with the DLS results.

[0134] 2.3 pH responsiveness test of SNs

[0135] Since the adamantyl group is introduced onto the polyasparagine chain via an acid-sensitive imine bond, the polymer Pasp-benzoicimine-Ad should exhibit acid responsiveness. Pasp-benzoicimine-Ad was degraded in a pH 5.0 buffer solution for 4 days, and the polymer structure before and after degradation was characterized by 1H NMR spectroscopy and Fourier transform infrared spectroscopy. Figure 7 As shown in the ¹H NMR, proton peaks a and b attributed to the phenyl group and proton peak c attributed to the imine bond were clearly visible in D₂O before degradation, but these characteristic proton peaks disappeared after degradation. This indicates that the acid sensitivity of the imine bond can induce the degradation of Pasp-benzoicimine-Ad. Figure 7 In B, the out-of-plane bending vibration peak of the benzene ring (798 cm⁻¹) -1 and 835cm -1 ) and in-plane bending vibration peak (1126cm) -1 and 1174cm -1) disappeared after the degradation of the polymer, which further demonstrated the acid-degradability of Pasp-benzoicimine-Ad. The degradation of polymer Pasp-benzoicimine-Ad would affect the binding ability of the complex to pDNA, thus changing the morphology and size of SNs. It was found that after incubation in pH 5.0 acetate buffer for a period of time, the complex could not compress DNA as efficiently as at pH 7.4, as shown by A (c') in Figure 6 . This indicates that as Pasp-benzoicimine-Ad degrades under acidic conditions, the complex becomes unstable, which is more conducive to the release of DNA from the complex. The pH responsiveness of SNs was further confirmed by TEM, as shown by C in Figure 6 . Before degradation, SNs were compact spherical nanoparticles (C (b, c, d) in Figure 6 ), while after degradation under acidic conditions, SNs became very loose and irregular (C (b', c', d') in Figure 6 ), with the particle size changing from nanoscale to microscale. Therefore, the pH responsiveness of SNs was confirmed by the above results, which would be conducive to the release of DNA in tumor cells and promote the improvement of transfection efficiency.

[0136] 2.4 Study on the stability and anti-protein adsorption ability of SNs

[0137] As a gene vector, it will encounter inorganic salts and serum proteins in body fluids in a complex physiological environment, so it is very important to remain stable in the blood circulation. In order to characterize the dynamic stability of SNs, DLS was used to evaluate the change of particle size of SNs (N / P 20) at different Ad / CD molar ratios. Here, 130 mM NaCl solution was prepared to simulate the salt concentration in the physiological environment. As shown by A in Figure 8 , the addition of Pasp-benzoicimine-Ad helps to improve the salt stability of the complex. At Ad / CD = 4, SNs can remain stable and not aggregate for up to 5 days. However, when Pasp-benzoicimine-Ad is continued to be added to Ad / CD = 8, the complex does not show better stability, but is less than that at Ad / CD = 4, indicating that Pasp-benzoicimine-Ad is not the more the better. This may be due to the fact that after adding too much Pasp-benzoicimine-Ad, the chelation of Ad and CD has been saturated, resulting in more free polymers in the complex solution, causing the entanglement and aggregation of the complexes, thereby reducing the stability of the complex. In summary, SNs can remain stable in a suitable Ad / CD (Ad / CD = 4) in a salt solution. Bovine serum albumin BSA was selected as a model protein to simulate the adsorption of the complex to non-specific proteins. As shown by Figure 8As shown in Figure B, without the addition of Pasp-benzoicimine-Ad, the anti-protein adsorption capacity of SNs was similar to that of the control 25kDa PEI, both being very weak. After the addition of Pasp-benzoicimine-Ad, the anti-protein adsorption capacity of SNs significantly improved. This demonstrates that the carrier system formed by Pasp-benzoicimine-Ad / β-CD-PDMAEMA through host-guest interaction possesses excellent anti-protein adsorption capacity.

[0138] 2.5 In vitro cytotoxicity analysis of SNs

[0139] Cytotoxicity is an important consideration when designing gene vectors. This chapter uses the MTT assay to evaluate the cytotoxicity of gene vectors (SNs) with different Ad / CD molar ratios at different N / P ratios. Figure 9 As shown, the cytotoxicity of SNs was correlated with the Ad / CD molar ratio and the N / P ratio, increasing with increasing Ad / CD and N / P ratios. Compared to the control 25 kDa PEI, the β-CD-PDMAEMA / pDNA complex exhibited lower cytotoxicity (e.g., B16-F10 cell survival was above 75% at an N / P ratio of 20). After the addition of the polymer Pasp-benzoicimine-Ad, the cytotoxicity of SNs increased at the same N / P ratio. This is consistent with the previous results ( Figure 6 Consistent with B(b) in the above, the addition of Pasp-benzoicimine-Ad resulted in higher zeta potentials and increased relative molecular weight in the SNs, thereby increasing cytotoxicity. However, in the acidic intracellular environment, due to the pH-responsiveness of the SNs, β-CD-PDMAEMA can detach from the Pasp-benzoicimine-Ad chain, leading to the disassembly of the SNs into the less toxic poly-α-amino acid chain and β-CD-PDMAEMA. Therefore, compared to the 25 kDa PEI, the SNs still exhibited lower cytotoxicity.

[0140] 2.6 Analysis of in vitro transfection efficiency of SNs

[0141] Three cell lines—293T, B16-F10, and CHO-K1—were selected as transfection cells. Plasmids expressing luciferase and green fluorescent protein were chosen as reporter genes to investigate the in vitro transfection efficiency of SNs. Figure 10 As shown, compared with the β-CD-PDMAEMA / pDNA complex, SNs exhibited higher transfection efficiency (2-10 times), especially at a high N / P ratio of 20. On the one hand, compared with β-CD-PDMAEMA, SNs possessed better DNA compression ability, smaller particle size, and higher surface potential (…). Figure 6A and B in Figure 6), which are all beneficial to higher transfection efficiency. On the other hand, SNs are pH-responsive, in the acidic environment of cells, SNs become unstable ( Figure 1 ), which is beneficial to the release of DNA and the improvement of transfection efficiency. Interestingly, in all three cell lines, SNs all have the highest transfection efficiency at Ad / CD molar ratio of 4 at the same N / P ratio, which is consistent with the results of agarose gel electrophoresis and DLS (SNs have the best DNA binding ability and the smallest particle size at Ad / CD molar ratio of 4).

[0142] In addition, pEGFP was also chosen as the reporter gene to directly observe the expression of green fluorescent protein in 293T, B16-F10 and CHO-K1 cell lines by fluorescence microscopy. Figure 11 Figure 6 (a1) is the picture of EGFP expression in 293T cells of 25kDa PEI / pDNA complex, β-CD-PDMAEMA / pDNA complex and SNs (different Ad / CD), from which it can be seen that, compared with β-CD-PDMAEMA / pDNA complex, SNs show more protein expression and stronger fluorescence intensity (green color) at the same N / P ratio. Figure 11 Figure 6 (a2, a3), especially at Ad / CD = 4, which is consistent with the previous luciferase results ( Figure 10 ). In B16-F10 and CHO-K1 cells, we also obtained similar results ( Figure 12 and Figure 13 ).

[0143] In addition to plasmid DNA, the possibility of using SNs to deliver siRNA was also explored. Anti-GFP siRNA was chosen as the silenced gene, and HeLa-GFP was chosen as the silenced cell to determine the silencing efficiency of Lipofectamine 2000, 25kDa PEI, β-CD-PDMAEMA / pDNA complex and SNs (N / P 20, different Ad / CD). At Ad / CD = 4, SNs can reduce the fluorescence intensity of HeLa-GFP cells by 40%, as shown in Figure 14 , which is very close to the commercial reagent Lipofectamine 2000 (50% silencing efficiency) and better than 25kDa PEI (35% silencing efficiency). This is consistent with the transfection results of plasmid DNA, which shows that SNs can effectively deliver siRNA. The above transfection results of plasmid DNA and siRNA show that, at a specific Ad / CD, the addition of polymer Pasp-benzoicimine-Ad can improve the transfection ability of SNs.

[0144] 2.7 Study on the endocytosis efficiency of SNs

[0145] Cellular uptake efficiency is very important for an effective gene vector system. In the present invention, the cellular uptake efficiency of SNs at different Ad / CD was evaluated by laser confocal microscope CLSM and flow cytometry. TOTO-3 red fluorescent dye was used to label DNA, green fluorescent dye WGA488 was used to label cell membrane, and blue fluorescent dye Hoechst 33342 was used to label cell nucleus. The differences of cellular uptake of complexes were studied by direct observation and photographing with CLSM. Compared with β-CD-PDMAEMA / pDNA complexes, a large number of red fluorescence appeared in cells treated with SNs (A in Figure 15 ). It can be seen from the observation that the cellular uptake efficiency of SNs was the highest at Ad / CD = 4, which further explained the result that the transfection efficiency was the highest at Ad / CD = 4 (B in Figure 10 , Figure 11 、 Figure 12 and Figure 13 ). The cellular uptake efficiency and fluorescence intensity of 25 kDa PEI / pDNA, β-CD-PDMAEMA / pDNA, SNs (Ad / CD = 2), SNs (Ad / CD = 4) and SNs (Ad / CD = 8) were tested by flow cytometry (B, C in Figure 15 ). Compared with 25 kDa PEI / pDNA and β-CD-PDMAEMA / pDNA complexes, the cellular uptake efficiency of SNs was higher, which was due to the higher surface charge of SNs (B (b) in Figure 6 ), which was more conducive to the combination of nanoparticles with the negatively charged cell membrane surface and thus improved the cellular uptake efficiency.

[0146] 3. Conclusion

[0147] In the present invention, a new type of supramolecular nanoparticles SNs was constructed, which was formed by host-guest interaction between pH-sensitive adamantane-modified polyaspartamide and cationic polymer β-CD-PDMAEMA. The nanoparticles had good DNA compression capacity and stability, and also had excellent biocompatibility and higher transfection efficiency. All the results in this chapter showed that under the condition of suitable Ad / CD molar ratio, SNs could become a very potential gene vector

[0148] The above is only the preferred embodiment of the present invention, and it should be pointed out that for those skilled in the art, without departing from the principles of the present invention, some improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Supramolecular nanoparticles with acid response, characterized in that, The adamantane end group polyasparagine derivative and the star cationic polymer are obtained by host-guest interaction; The adamantane end group polyasparagine derivative is obtained by connecting adamantane units to the polymer main chain through acid-sensitive benzamide bonds; The star cationic polymer is prepared from dimethylaminoethyl methacrylate monomers using brominated β-cyclodextrin shown in formula III as an initiator; Formula III ; The molar ratio of adamantane end groups in the adamantane end group polyasparagine derivative to β-cyclodextrin in the star cationic polymer is (2-8):

1.

2. The supramolecular nanoparticle of claim 1, wherein, The number average molecular weight of the adamantane end group polyasparagine derivative is 30Kda-50Kda; The number average molecular weight of the star cationic polymer is 5.38Kda-13.27kDa.

3. Use of the supramolecular nanoparticle of claim 1 or 2 as a carrier for nucleic acid loading and / or carrying for non-disease diagnosis and treatment purposes. The molar ratio of nitrogen atoms in the supramolecular nanoparticle to phosphorus atoms in the nucleic acid is (10-20):

1.

4. Use of the supramolecular nanoparticle of claim 1 or 2 in the preparation of a nucleic acid loading and / or carrying product.

5. A nucleic acid supported and / or carried product, characterized in that, The supramolecular nanoparticle of claim 1 or 2 and an auxiliary material.

6. Use of the product of claim 5 in the expression and / or silencing of nucleic acids for non-disease diagnosis and treatment purposes.

7. A method for nucleic acid carrying and / or bearing for diagnostic and therapeutic purposes other than disease, characterized in that, The supramolecular nanoparticle of claim 1 or 2 is used.

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