Acid response gene vector as well as construction method and application thereof

Abstract

The invention relates to the technical field of biochemical engineering, in particular to an acid response gene vector as well as a construction method and application thereof. The invention provides acid-responsive supramolecular nanoparticles SNs based on the subject-object interaction of a star-shaped cationic polymer beta-CD-PDMAEMA and adamantane modified polyasparagine Pasp-Benzoic imine-Ad, experimental results show that the mixing ratio (Ad / CD) of the Pasp-Benzoic imine-Ad and the beta-CD-PDMAEMA and the mixing ratio (N / P) of the SNs and DNA can influence the cytotoxicity, the transfection efficiency and the phagocytosis efficiency, the N / P ratio is 20, and the specific surface area of the SNs and the specific surface area of the beta-CD-PDMAEMA and the specific surface area of the beta-CD-PDMAEMA and the specific surface area of the beta-CD-PDMAEMA are different. When the Ad / CD is 2 or 4, the compound has high cell transfection set endocytosis efficiency while having stability; on the basis, the 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 technical field of biochemical engineering, and in particular to an acid-responsive gene vector, a construction method and an application thereof. Background Art

[0002] Tobacco is an important model organism and one of the most valuable scientific research materials. Many pioneering studies in botany and biotechnology are derived from tobacco. On the one hand, tobacco, as a bioreactor, is widely used in many gene expressions, including anti-cancer and anti-AIDS genes related to human health, as well as structural and functional analysis of genes from various other sources. The genes are introduced into tobacco by means of gene transformation, transfection or transduction to obtain the corresponding products. On the other hand, tobacco, as an important economic crop, has important significance for the improvement of plants through multi-purpose development in making full use of tobacco resources, ensuring the stability of tobacco farmers' income, and improving economic and social value.

[0003] The improvement of tobacco plants and the introduction of external genes both require the help of genetic engineering methods; currently, non-viral vectors, as one of the genetic engineering methods, have attracted much attention in recent decades due to their weak immunogenicity, easy construction and relative safety. For polycationic carriers, high molecular weight and sufficient positive charge density are prerequisites for their effective gene encapsulation, but these can lead to high toxicity and hinder the intracellular release of genes. Therefore, people have developed many new strategies, such as cationization of some biocompatible polymers to construct new structural carriers. Among them, α-amino acid-based polypeptides have attracted people's attention because they are easily degraded into non-toxic small molecules and excreted from the body. Biodegradable polyasparagine derivatives can be easily derived from polysuccinimide (PSI), and its side chain can be further modified to introduce reducing or pH-sensitive groups. Therefore, a series of smart materials based on polyasparagine have been widely studied in the field of gene carriers. However, gene vectors need to have stability, specificity, low cytotoxicity and transformation efficiency. Therefore, developing gene vectors with high stability, specificity, low cytotoxicity and high transformation efficiency and expanding the gene vector library have important research value and application prospects. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an acid-responsive gene vector, a construction method and application thereof.

[0005] The present invention provides a composition comprising an adamantane-terminated polyasparagine derivative (Pasp-benzoicimine-Ad) and a star-shaped cationic polymer (β-CD-PDMAEMA);

[0006] The adamantane-terminated polyasparagine derivative is prepared by the following steps:

[0007] Step 1, ethylenediamine with Boc protection at one end is subjected to a ring-opening reaction with polysuccinimide (PSI) as shown in formula I and the Boc protecting group is removed to obtain Pasp-EDA;

[0008] Step 2, Pasp-EDA reacts with adamantane acid benzaldehyde ester (ACB) shown in formula II to obtain an adamantane-modified polyasparagine derivative;

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

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

[0011] Formula I ;

[0012] Formula II ;

[0013] Formula III .

[0014] Furthermore,

[0015] The synthesis of star-shaped cationic polymers includes the following steps:

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

[0017] Step 2, using DMAEMA monomer and brominated β-cyclodextrin represented by formula III as initiator and CuBr / PMDETA as catalyst system to synthesize the star-shaped cationic polymer;

[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, it is 0.23: 13.87: 0.55: 0.46; more specifically, in the present invention, the addition amount is 0.23mmol: 13.87mmol: 0.55mmol: 0.46mmol in sequence;

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

[0020] The number average molecular weight of the star-shaped cationic polymer is 5.38 KDa to 13.27 KDa, specifically 8.35 kDa.

[0021] In a specific embodiment of the present invention, size exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS) and 1 H NMR determination of the number average molecular weight (M) of adamantane-modified polyasparagine derivatives and star-shaped cationic polymers n ); Specifically, the relative molecular weight of the adamantane-modified polyasparagine derivative was 43 KDa as determined by SEC-MALLS; the relative molecular weight of the star-shaped cationic polymer was 8.45 kDa; 1 The molecular weight of the star-shaped cationic polymer was determined to be 8.78 kDa by H NMR.

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

[0023] Furthermore, when the molar ratio of the adamantane-modified polyasparagine derivative to the star-shaped cationic polymer is 4, the polymer particle size is small while having high stability and anti-protein adsorption ability as well as the highest transfection efficiency and endocytosis efficiency.

[0024] In a specific embodiment of the present invention, the molar ratio of β-CD-PDMAEMA and Pasp-benzoic imine-Ad as gene vectors was optimized; the particle size of the mixture of β-CD-PDMAEMA and DNA was very large (about 500 nm); after adding Pasp-benzoic imine-Ad, the particle size of the particles was significantly reduced (below 200 nm), especially when the Ad / CD (molar ratio of the adamantane end group in the polyasparagine derivative with adamantane end group and the β-cyclodextrin in the star-shaped cationic polymer) ratio was 4 and 8, the particle size of SNs was reduced to about 100 nm, and the morphology and size were more uniform, and the particle size was the smallest when Ad / CD was 8; the stability of Pasp-benzoic imine-Ad / β-CD-PDMAEMA in saline solution first increased and then decreased, and the stability was the highest when Ad / CD was 4; Pasp-benzoic imine-Ad / β-CD-PDMAEMA in saline solution increased first and then decreased, and the stability was the highest when Ad / CD was 4; The anti-protein adsorption ability of imine-Ad / β-CD-PDMAEMA increased with the increase of Ad / CD ratio, and the anti-protein adsorption ability was the best when Ad / CD was 8, but there was no significant difference with Ad / CD of 4; the β-CD-PDMAEMA / pDNA complex showed low cytotoxicity. After adding the polymer Pasp-benzoic imine-Ad, the cytotoxicity of SNs increased at the same N / P ratio, and the cytotoxicity was the highest when Ad / CD was 8. When Ad / CD was 4 or 2, the cytotoxicity was lower at the same N / P (molar ratio of nitrogen atoms in the carrier to phosphorus atoms in the nucleic acid to the molar ratio of the carrier) ratio; when Pasp-benzoic imine-Ad / β-CD-PDMAEMA had the same N / P ratio, the transfection efficiency and endocytosis efficiency of SNs were the highest when the Ad / CD molar ratio was 4.

[0025] The present invention provides the use of the composition as a carrier in carrying and / or carrying substances.

[0026] Furthermore, the substance includes nucleic acid.

[0027] Furthermore,

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

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

[0030] In the present invention, a polyasparagine derivative modified with adamantane (Pasp-benzoic imine-Ad) and a star-shaped cationic polymer (β-CD-PDMAEMA) are mixed and used as a nucleic acid carrier for nucleic acid expression and silencing;

[0031] In a specific embodiment of the present invention, the molar ratio (N / P) of the complex formed by the polymer formed by β-CD-PDMAEMA and Pasp-benzoic imine-Ad and the pDNA was optimized. The experimental results showed that the transfection efficiency and endocytosis efficiency were the highest when the molar ratio of the complex formed by the polymer formed by β-CD-PDMAEMA and Pasp-benzoic imine-Ad and the pDNA was 20.

[0032] In the present invention, the nucleic acid and the vector form a complex for expression and / or silencing of the nucleic acid;

[0033] 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;

[0034] 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;

[0035] 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.

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

[0037] 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.

[0038] 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;

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The present invention provides application of the composition in preparing products for carrying and / or carrying substances.

[0043] The present invention provides a product for carrying and / or carrying a substance, which comprises auxiliary materials and the composition of the present invention as claimed in claim 1.

[0044] 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.

[0045] 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;

[0046] 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;

[0047] 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.

[0048] 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.

[0049] The present invention provides the use of the product in the expression and / or silencing of nucleic acid.

[0050] The present invention provides a method for carrying and / or carrying a substance, which comprises using the composition of the present invention to carry out carrying and / or carrying of the substance.

[0051] 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;

[0052] 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.

[0053] The present invention relates to the field of biochemical engineering technology, and in particular to an acid-responsive gene vector, a construction method and an application thereof. The present invention provides acid-responsive supramolecular nanoparticles SNs based on the host-guest interaction of Pasp-benzoic imine-Ad of star-shaped cationic polymer β-CD-PDMAEMA and adamantane-modified polyasparagine. Experimental results show that the mixing ratio of Pasp-benzoic imine-Ad and β-CD-PDMAEMA (Ad / CD) and the mixing ratio of SNs and DNA (N / P) will affect cytotoxicity, transfection efficiency and phagocytosis efficiency. Among them, the complex with an N / P ratio of 20 and an Ad / CD ratio of 2 or 4 has high cell transfection and endocytosis efficiency while being stable. Based on this, the vector of the present invention can be used for the expression, gene silencing and / or cell 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

[0054] Figure 1 Schematic diagram showing the formation of supramolecular polymer / pDNA nanocomplex and the degradation process stimulated by intracellular pH;

[0055] Figure 2 The synthesis process of Pasp-benzoic imine-Ad and β-CD-PDMAEM is shown, wherein A is the synthesis of Pasp-benzoic imine-Ad; B is the synthesis of β-CD-PDMAEMA;

[0056] Figure 3 Show 1H NMR spectrum, where A is Boc-EDA (solvent is CDCl3); 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 Pasp-benzoic imine-Ad and β-CD-PDMAEMA 1 H NMR spectrum, where A is Pasp-benzoic imine-Ad; B is β-CD-PDMAEMA;

[0058] Figure 5 The 2D1H NOESY NMR spectra of Pasp-benzoic imine-Ad / β-CD-PDMAEMA (solvent: D2O) are shown. The concentration of Pasp-benzoic imine-Ad / β-CD-PDMAEMA is 10 mg / mL, and the molar ratio of Ad / CD is 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 blocking test of SNs with different Ad / CD ratios under different N / P ratios, where a~d are cultured under pH 7.4 PBS conditions, and c' is cultured in pH 5.0 acetate buffer for 4 h when Ad / CD=4; B is the particle size and potential of SNs with different Ad / CD ratios under different N / P conditions, where a is the particle size and b is the potential; C is the TEM image of SNs with different Ad / CD ratios at an N / P ratio of 20, where a~d are cultured in pH 7.4 PBS, and b'~d' are cultured in pH 5.0 acetate buffer for 4 h; where the scale bars are 500 nm for a~d, 100 nm for inset, and 2 μm for b'~d');

[0060] Figure 7 Figure 2 shows the Pasp-benzoic imine-Ad (solvent: D2O) before and after incubation in pH 5.0 acetate buffer. 1 H NMR and FTIR spectra, where A is 1 H NMR spectrum; B is infrared spectrum (FTIRspectra);

[0061] Figure 8Figure 4 shows the stability and anti-protein adsorption ability test of SNs, where A is the particle size of nanocomposites with different Ad / CD ratios (N / P is 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 adsorption by nanocomposites with different Ad / CD ratios at N / P ratio of 20; where the relative adsorption ratio of BSA is defined as the amount of adsorbed BSA / mg, *** p < 0.001, t test, data are expressed as mean ± SD, n = 3;

[0062] Fig. 9 The cell viability of polycationic / pDNA nanocomplexes with different Ad / CD ratios under different N / P conditions is shown, where A is 293T cells; B is B16-F10 cells; C is CHO-K1 cells; data are expressed as mean ± SD, n = 3;

[0063] Fig.10 Figure 2 shows the in vitro expression of luciferase protein mediated by PEI / pDNA nanocomplexes and the comparison of polycationic / pDNA nanocomplexes with different Ad / CD ratios under different N / P conditions in serum culture medium, where A is 293T cells; B is B16-F10 cell line; C is CHO-K1 cell line; data are expressed as mean ± SD, n = 3;

[0064] Fig.11 The in vitro EGFP expression of 293T cells treated with nanocomplexes with different Ad / CD ratios is shown; a1 is a picture of EGFP-expressing cells, with a scale bar of 100 nm; a2 is the ratio of EGFP-positive cells; a3 is the mean fluorescence intensity (MFI);

[0065] Fig.12 The in vitro EGFP expression of B16-F10 cells treated with nanocomplexes with different Ad / CD ratios is shown; b1 is a picture of EGFP-expressing cells, with a scale bar of 100 nm; bb2 is the ratio of EGFP-positive cells; bb3 is the mean fluorescence intensity (MFI);

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

[0067] Fig.14The EGFP fluorescence images and relative average fluorescence intensity of HeLa-GFP cells treated with nanocomplexes with different Ad / CD ratios (N / P is 20) are shown, where A is the EGFP fluorescence image; B is the relative average fluorescence intensity, scale bar 100 nm;

[0068] Fig.15 The laser confocal scanning images and cell counting diagrams of 293T cells co-incubated with complexes with different Ad / CD ratios for 4 hours are shown, where A is the laser confocal scanning image, B is the cell endocytosis efficiency; C is the mean relative fluorescence intensity (MFI); plasmids, cell nuclei and cell membranes were stained with TOTO-3 (red), Hoechst33342 was used to stain the cell nuclei in blue, and WGA-488 was used to stain the cell membrane in green. The scale bar is 20μm. DETAILED DESCRIPTION

[0069] The present invention provides acid responsive gene vector, its construction method and application, those skilled in the art can learn from this article content, appropriately improve process parameters and realize.It is particularly important to point out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present invention.The method and application of the present invention have been described by preferred embodiments, and the relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention, to realize and apply the technology of the present invention.

[0070] 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:

[0071] Example 1 Construction and application of acid-responsive gene vector

[0072] The present invention provides acid-responsive supramolecular nanoparticles (SNs) constructed by host-guest interaction based on β-CD-PDMAEMA and adamantane-modified polyasparagine (adamantane units are connected to the polymer backbone via acid-sensitive phenylimide bonds), such as Figure 1 The present invention regulates the properties of particles by adjusting the mixing ratio of Ad and CD, studies in detail the effects of Ad / CD on the physicochemical properties, cytotoxicity, transfection efficiency, phagocytosis efficiency, etc. of SNs, and optimizes the Ad / CD molar ratio in the hope of obtaining a SNs gene vector with excellent biocompatibility and high transfection efficiency.

[0073] 1. Experimental steps

[0074] 1.1 Reagents Polysuccinimide (PSI, Mn = 17.8 kDa, PDI = 1.46) and adamantane benzaldehyde ester (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%), and N, N'-carbonyldiimidazole (CDI) were purchased from China National Pharmaceutical Group. Bromoisobutyryl bromide (BIBB, 98%) and trifluoroacetic acid were purchased from Aladdin. Branched polyethyleneimine (PEI, 25 kDa), thiazolyl blue (MTT), and Lipofectamine® RNAiMAX transfection reagent were purchased from Sigma-Aldrich. TOTO-3, wheat germ AlexaFluor488 (WGA488), and Hoechst33342 were purchased from Life Technologies, Inc., USA. Plasmid pcDNA3-Luc, pEGFP, anti-GFP siRNA, and negative control siRNA) were purchased from Sigma.

[0075] 1.2 Synthesis

[0076] 1.2.1 The synthesis of polyaspartamide derivatives containing benzoimide bonds and adamantane modification (Pasp-benzoicimine-Ad) was based on the previous literature report of our laboratory (Yu H, Sun J, Zhang Y, et al. 2015. pH-and β-cyclodextrin-responsive micelles based on polyaspartamidederivatives as drug carrier[J]. Journal of Polymer Science Part A: PolymerChemistry, 53(11): 1387-1395.), which is mainly divided into the following steps.

[0077] 1.2.1.1 Synthesis of Boc-EDA

[0078] Take 10mL of ethylenediamine (EDA) and dissolve it in 80mL of dichloromethane. Take another 50mL of dichloromethane to dissolve 8.1g of Boc anhydride and place it in a constant pressure dropping funnel. Add Boc anhydride dropwise to ethylenediamine under ice bath conditions and continue the reaction at room temperature for 12h. After the reaction is completed, spin dry the solvent and add an appropriate amount of secondary water, stir and filter. Collect the filtrate and add NaCl to it until saturated, then extract with ethyl acetate (80mL×3), spin dry the organic phase, dissolve it with 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 3.2g Boc-EDA and dissolve it in 10mL DMF, add it to 10mL DMF solution containing 1.0g PSI, react in 70°C oil bath for 24h, the reaction product precipitates in ether, filter to get the product, pass N2 to remove ether, vacuum dry at room temperature for 24h to get white solid Pasp-EDA-Boc. Dissolve the white solid in 15mL trifluoroacetic acid (TFA) at room temperature for 3h, precipitate in ether, vacuum dry the precipitate at room temperature to get white solid, and characterize it by hydrogen nuclear magnetic resonance spectrum.

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

[0082] 0.8 g Pasp-EDA and 0.43 g ACB were dissolved in 10 mL DMF, and a small amount of NaOH was added as a catalyst. The mixture was reacted in an oil bath at 50 °C for 24 h, precipitated with ether and centrifuged, and dried in vacuum at room temperature for 24 h to obtain a light yellow product, which was characterized by hydrogen nuclear magnetic resonance spectroscopy.

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

[0084] The synthesis was based on previous literature reports (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 is mainly divided into 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, stirred and dissolved under nitrogen protection, then cooled to 0°C in an ice bath, and a DMF solution containing 1 mL BIBB was added dropwise over 1 hour, and the mixture was stirred for 2 hours under ice bath conditions, then returned to room temperature and stirred for 24 hours. After the reaction was completed, the solvent was concentrated under reduced pressure, precipitated in excess ether, then washed with acetone and distilled water, and vacuum dried at 40°C for 12 hours to obtain a white solid, which was characterized by hydrogen nuclear magnetic resonance spectrum.

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

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

[0089] 1.2.3 Structural characterization

[0090] The H NMR spectrum was tested using a Varian INOVA-400MHz NMR instrument, with deuterated chloroform, deuterated dimethyl sulfoxide and deuterated water as solvents. The H NMR spectrum was used to characterize the changes in the structure of the polymer Pasp-benzoicimine-Ad before and after degradation. Fourier transform infrared spectroscopy (FT-IR) was tested using a Spectrumone spectrometer (Perkin-Elmer), and FT-IR was used to characterize the infrared absorption difference of Pasp-benzoicimine-Ad before and after degradation. The interaction between Pasp-benzoicimine-Ad and β-CD-PDMAEMA was characterized by two-dimensional nuclear magnetic NOESY spectra, which was tested using a Varian INOVA-600MHz NMR instrument, with deuterated water (D2O) as solvent. The concentration of all polymers was 5 mg / mL, and the polymer solution was left to stand at room temperature for 30 minutes before testing. The molecular weight and distribution of the polymer were measured by size exclusion chromatography-multi-angle laser light scattering analyzer (SEC-MALLS). The instrument used a dual detector system, including a multi-angle laser light scattering signal detector (DAWNEOS, Wyatt Technology, laser wavelength of 690nm) and a differential refractive index detector (OptilabDSP, Wyatt Technology). The chromatographic separation column consisted of Styragel HR4 WAT 045900 and Styragel HR3 WAT045885. DMF (containing 10mM LiBr) was used as the mobile phase with a flow rate of 0.3mL / min, and 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 that stably express green fluorescent protein were cultured in DMEM medium containing 10% FBS. Chinese hamster ovary cells CHO-K1 were cultured in DMEM medium containing 2mM L-glutamyl and 10% FBS. All the above cells were cultured in an incubator at 37°C and 5% CO2 concentration.

[0093] 1.2.5 Polymer / pDNA

[0094] Pasp-benzoicimine-Ad and β-CD-PDMAEMA were dissolved in Hepes (20mM, pH 7.4), HBS (20mM, 130mM NaCl, pH 7.4) or HBG (20mM, 5% glucose, pH 7.4) buffer solution (Pasp-benzoicimine-Ad and β-CD-PDMAEMA, each polymer was dissolved in 3 solvents, and solutions containing different polymers in different solvents were obtained for subsequent different experiments) to prepare 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 seconds and incubated at room temperature for 30 minutes (the incubation solution included pH 7.4 PBS buffer or pH 5.0 acetate buffer) 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 of complex electrophoresis samples (each sample contains 100 ng DNA) were prepared. While incubating, a 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 well, and added to the gel wells. The gel was run at 90 V for 90 min in Tris-acetate buffer (TAE), and then imaged and photographed under UV (254 nm) illumination using a gel imager (Bio-Rad, USA).

[0097] The pH responsiveness of the complex was also analyzed by agarose gel electrophoresis. The complexes with different mass ratios were incubated in pH 5.0 acetate buffer for 4 h, then electrophoresed at 90 V for 90 min in an electrophoresis instrument and observed by a UV imager.

[0098] 1.2.7 Determination of composite particle size and potential

[0099] 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 1.2.5. Then they were placed in a four-sided square glass cuvette for particle size testing. 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 ζ potential measurement. The stability of the Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complex was characterized by testing the particle size change within six days.

[0100] 1.2.8 Transmission electron microscopy (TEM) observation The morphology of Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complexes (N / P20) with different Ad / CD molar ratios and their reaction in pH 5.0 acetate buffer for 4 h was observed by transmission electron microscopy (Tecnai Spirit, 120 kV).

[0101] 1.2.9 Characterization of the anti-protein adsorption of the complex The anti-protein adsorption ability of the Pasp-benzoicimine-Ad / β-CD-PDMAEMA / pDNA complex (N / P20) with different Ad / CD molar ratios was tested as follows:

[0102] 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 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).

[0103] 1.2.10 In vitro cytotoxicity analysis

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

[0105] The complexes were incubated in Hepes solution containing 10 mM DTT or without DTT. The changes in the particle size of the above complexes over time were tested by dynamic light scattering (DLS), and their morphological changes were characterized by TEM.

[0106] 1.2.11 Study on the in vitro transfection efficiency of the complex

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

[0108] The cells were seeded at 8000 per well in a 96-well plate and cultured for 24 hours. Then, 75 μL of fresh culture medium was replaced, and the complex was added to the well (25 μL per well, 0.25 μg pDNA) and allowed to react with the cells for 4 hours. Subsequently, the culture medium containing the complex was aspirated, 100 μL of fresh culture medium was replaced, and the culture was continued for 44 hours. After transfection, luciferase expression was analyzed using the Steady-Glo® Luciferase Assay System kit. The relative light intensity RLU of luciferase was measured by the Infinite M200 plate reader, and the protein concentration was detected using the BCA protein assay kit (Pierce). Luciferase activity was expressed as RLU / mg Protein.

[0109] 1.2.11.2 Analysis of green fluorescent protein expression

[0110] Select pEGFP as the reporter gene, select 293T, B16-F10 and CHO-K1 cells as transfected cells, and 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] The cells were seeded in a 24-well plate at 20,000 cells per well and cultured for 24 hours. Then, 400 μL of fresh culture medium was replaced, and the complex was added to the wells (100 μL per well, 1 μg pDNA) and allowed to react with the cells for 4 hours. Subsequently, the culture medium containing the complex was aspirated, 500 μL of fresh culture medium was replaced, and the culture was continued for 44 hours. After transfection, the cells were directly observed and photographed using an inverted fluorescence 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 in vitro siRNA silencing efficiency The silencing efficiency of anti-GFP siRNA complex on green fluorescent protein expression at different Ad / CD molar ratios and different N / P ratios was studied using human cervical cancer cells HeLa-GFP that can stably express green fluorescent protein. The specific method is as follows:

[0113] 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 added to each well (0.1 mL of siRNA complex solution (25 pmol siRNA) was added to each well and allowed to react with the cells for 4 hours. Subsequently, the complex-containing culture medium was aspirated, replaced with fresh culture medium, and cultured for another 44 hours. After transfection, the cells were directly observed and photographed using an inverted fluorescence microscope (Olympus IX 71), 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 transfection results were expressed as relative fluorescence intensity (Relative MFI). The relative fluorescence intensity (Relative MFI) is defined as follows:

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

[0115] Among them, MFI sample The mean fluorescence intensity (MFI) of cells interacting with the complex control Represents the mean fluorescence intensity of blank cells, MFI negative The mean fluorescence intensity of cells treated with negative control is shown.

[0116] 11.2.12 Study on cell endocytosis efficiency

[0117] 293T cells were used as research cells, and laser confocal microscopy and flow cytometry were used to characterize the cell endocytosis efficiency. The specific methods are as follows:

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

[0119] For CLSM observation and photography, after the complex and cells interacted for 4 hours, the medium containing the complex was removed and washed three times with PBS. Then, 200 μL of 4% paraformaldehyde solution was added to each well to fix the cells for 10 min at room temperature. Then, they were washed three times with PBS, 200 μL of WGA 488 (5 μg / mL) was added to each well, stained for 5 min at 4 °C, and then the WGA 488 solution was removed and washed three times with PBS. Then, 200 μL of Hoechst 33342 (0.1 mg / mL) was added to each well and stained for 10 min at room temperature. Finally, the Hoechst 33342 solution was removed and washed three times with PBS. A Nikon A1R+ (Nikon Corporation, Japan) laser confocal microscope was used for observation and photography.

[0120] For flow cytometry, after the complexes interacted with cells for 4 h, the cells were washed three times with PBS, and then harvested by trypsin digestion and tested in a flow cytometer (Apogee A50-Micro).

[0121] 1.2.13 Statistical analysis

[0122] Statistical differences were analyzed using t-test. *P<0.05 indicates that the results have significant statistical differences; **P<0.01 indicates that the results have very significant statistical differences; ***P<0.001 indicates that the results have extremely high significant statistical differences; NS indicates that the results have no significant statistical differences. All data results were performed at least three times in parallel and are expressed as mean ± variance.

[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. The side chain of this polymer was connected to the adamantyl group via an acid-sensitive imine bond. The specific synthesis route is shown in Figure 2 ( Figure 2In the above, A is the synthesis of Pasp-benzoic imine-Ad; B is the synthesis of β-CD-PDMAEMA; wherein n in A represents the number of monomer units in 1 mol of Pasp polymer, all n in A are 159, and x represents the number of moles of adamantane introduced in 1 mol of polymer. According to NMR calculation, the degree of adamantane substitution is about 11%, that is, there are about 17 moles of adamantane groups in 1 mol of polymer, that is, x=17; in B, an average of 4 ATRP reaction sites are introduced on each cyclodextrin, and β-CD-PDMAEMA is obtained by ATRP reaction, and an average of 48 DMAEMA units are introduced on each CD. According to calculation, an average of 12 DMAEMA units are introduced on each reaction site, so n is 12). First, ethylenediamine (Boc-EDA, 1HNMR as Figure 3 The PSI was ring-opened and the Boc group was removed using trifluoroacetic acid to obtain the amino-functionalized polyasparagine derivative Pasp-EDA ( 1 H NMR Figure 3 The aldehyde group on adamantane benzaldehyde ester (ACB) and the amino group on the polymer side chain react to form an acid-sensitive imine bond, thereby introducing an adamantane group on the polymer side chain.

[0125] The structure of Pasp-benzoicimine-Ad is shown in Figure 2. 1 H NMR confirmed that Figure 4 As shown in A in the figure. The signal peak at 8.1ppm is attributed to hydrogen e on the imine bond, the signal peaks at 6.8 and 7.5ppm are attributed to hydrogen f and g on the benzene ring, and the signal peaks between 1.4 and 1.9ppm are hydrogen h and i on the adamantane group. The NMR spectrum shows that the target polymer Pasp-benzoicimine-Ad was successfully synthesized, and it not only provides the structural characteristics of the polymer, but also provides a basis for our quantitative analysis. The access ratio of the adamantane group 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 main chain. The degree of substitution of adamantane is about 11%. At the same time, the molecular weight of the polymer Pasp-benzoicimine-Ad was measured by SEC-MALLS, as shown in Table 1.

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

[0127]

[0128] a Depend on 1 H NMR determination;

[0129] bis the result of SEC-MALLS measurement; PDI = weight average molecular weight / number average molecular weight or M w / M n .

[0130] The cationic polymer β-CD-PDMAEMA is mainly synthesized in two steps, such as Figure 2 First, the ATRP initiator CD-Br was synthesized and characterized by 1HNMR, as shown in Figure 3 As shown in D, based on the integrated areas at 1.92ppm (a, methyl proton peak in isobutyryl bromide) and 4.88ppm (c, 1-position methyl proton peak in cyclodextrin cavity), it can be calculated that an average of 4 ATRP reaction sites are introduced on each cyclodextrin. Then, β-CD-PDMAEMA is obtained by ATRP reaction, and its structure is characterized by 1HNMR. Figure 4 As shown in Figure B, 0.81~0.10ppm (1) and 2.2ppm (3) are attributed to the methyl proton peaks on the β-CD-PDMAEMA arm. 1.86ppm and 2.65ppm (2, 4) are attributed to the methylene proton peaks on the β-CD-PDMAEMA arm, respectively. 4.07ppm (5) is attributed to the methylene proton peak connected to the ester bond oxygen. By comparing the integral area ratio of 5 to H2~H6, it can be calculated that an average of 48 DMAEMA units are introduced on each CD, from which the molecular weight of β-CD-PDMAEMA can be calculated to be approximately 8.78kDa. In addition, the molecular weight and distribution of β-CD-PDMAEMA were tested by SEC-MALLS as shown in Table 1. The test results (8.45kDa) are also close to the NMR calculation results. The results of NMR and SEC-MALLS show that we have successfully synthesized β-CD-PDMAEMA.

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

[0132] The host-guest interaction between the polymers Pasp-benzoicimine-Ad and β-CD-PDMAEMA was characterized by two-dimensional NMR NOESY spectra, such as Figure 5 As shown in the figure, the proton peak of the cyclodextrin cavity at 3.0-4.0 ppm and the proton peak of the adamantane at 1.5-2.0 ppm produced a NOE correlation signal, indicating that Pasp-benzoicimine-Ad and β-CD-PDMAEMA can indeed undergo a host-guest intercalation. 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 ability of SNs to DNA was studied by agarose gel electrophoresis. Figure 6 As shown in Figure A, with the increase of the Ad / CD molar ratio, the binding ability of SNs to DNA is greatly improved. Especially when the Ad / CD ratio is 4, SNs can bind DNA at a very low N / P ratio of 0.5. In order to detect how the ratio of Ad and CD affects the particle size of SNs, dynamic light scattering analysis was used to analyze the prepared SNs. Figure 6 As shown in B (a) in Figure 1, when Pasp-benzoicimine-Ad was not added, β-CD-PDMAEMA was directly mixed with DNA, and even at a high N / P ratio of 20, large particles (about 500 nm) were still obtained. Subsequently, the particle size of SNs with different Ad / CD molar ratios was tested, as shown in Figure 1. Figure 6 As shown in B (a) in Figure 1, after adding Pasp-benzoicimine-Ad, the particle size of the particles was significantly reduced (below 200nm), especially when the Ad / CD ratio was 4 and 8, the particle size of SNs was reduced to about 100nm, which is very suitable for cell endocytosis. The morphology of SNs was then observed using a transmission electron microscope, as shown in Figure 1. Figure 6 As shown in Figure C, it can be seen from the TEM image that SNs are relatively uniform spherical particles, especially after the addition of Pasp-benzoicimine-Ad, the morphology and size are more uniform, which is consistent with the DLS results.

[0134] 2.3 pH responsiveness test of SNs

[0135] Since the adamantane group is introduced into the polyasparagine chain through an acid-sensitive imine bond, the polymer Pasp-benzoicimine-Ad should be acid-responsive. Pasp-benzoicimine-Ad was degraded in a pH 5.0 buffer for 4 days, and then the polymer structure before and after degradation was characterized by nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. Figure 7 As shown in A 1HNMR, the proton peaks a, b belonging to the phenyl group and the proton peak c belonging to the imine bond are clearly visible in D2O before degradation, and after degradation, these characteristic proton peaks disappear, indicating that the acid sensitivity of the imine bond can cause the degradation of Pasp-benzoicimine-Ad. Figure 7 In B, the out-of-plane bending vibration peak of the benzene ring (798 cm -1 and 835cm -1 ) and the in-plane bending vibration peak (1126cm -1 and 1174cm -1) disappeared after the polymer degradation, which further illustrates the acid degradability of Pasp-benzoicimine-Ad. The degradation of the polymer Pasp-benzoicimine-Ad will affect the binding ability of the complex to pDNA, thereby changing the morphology and size of SNs. The experiment found that after incubation in pH 5.0 acetate buffer for a period of time, the complex could not compress DNA as effectively as at pH 7.4. 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 in Figure 6 As shown in Figure C, before degradation, SNs are compact spherical nanoparticles ( Figure 6 C in (b, c, d)), while after degradation under acidic conditions, SNs become loose and irregular ( Figure 6 C (b', c', d') in Figure 2), the particle size changes from nanometers to micrometers as the morphology changes. Therefore, the pH responsiveness of SNs is confirmed by the above results, which will facilitate 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 carrier, it will encounter inorganic salts and serum proteins in body fluids in a complex physiological environment. Therefore, it is very important to maintain stability in the blood circulation. In order to characterize the dynamic stability of SNs, DLS was used to evaluate the changes in the particle size of SNs (N / P20) at different Ad / CD molar ratios. Here, a 130mM NaCl solution was prepared to simulate the salt concentration in the physiological environment. Figure 8 As shown in A, the addition of Pasp-benzoicimine--Ad helps to improve the salt stability of the complex. When Ad / CD=4, SNs can remain stable and not aggregate for up to 5 days. However, when Pasp-benzoicimine-Ad was continued to be added to Ad / CD=8, the complex did not show better stability. On the contrary, it was worse than when Ad / CD=4, indicating that the more Pasp-benzoicimine-Ad is not necessarily better. This may be because after the addition of excessive Pasp-benzoicimine-Ad, the chimerism of Ad and CD has been saturated, resulting in more free polymers in the complex solution, causing entanglement and aggregation between the complexes, thereby reducing the stability of the complex. In summary, SNs can remain stable in salt solution at a suitable Ad / CD (Ad / CD=4). Bovine serum albumin BSA was selected as a model protein to simulate the adsorption of the complex on non-specific proteins. As shown Figure 8As shown in Figure B, when Pasp-benzoicimine-Ad was not added, the anti-protein adsorption ability of SNs was similar to that of the control 25kDaPEI, both of which were very weak. After adding Pasp-benzoicimine-Ad, the anti-protein adsorption ability of SNs was significantly improved, which shows that the carrier system formed by Pasp-benzoicimine-Ad / β-CD-PDMAEMA through the host-guest interaction has good anti-protein adsorption ability.

[0138] 2.5 In vitro cytotoxicity analysis of SNs

[0139] Cytotoxicity is an important consideration when designing gene vectors. In this chapter, the MTT method was used to evaluate the cytotoxicity of SNs with different Ad / CD molar ratios at different N / P. Fig. 9 As shown in the figure, the cytotoxicity of SNs is related to the molar ratio of Ad / CD and the N / P ratio, and increases with the increase of the Ad / CD ratio and the N / P ratio. Compared with the control 25kDaPEI, the β-CD-PDMAEMA / pDNA complex showed lower cytotoxicity (for example, at an N / P of 20, the survival rate of B16-F10 cells was higher than 75%). 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 Figure 2, after the addition of Pasp-benzoicimine-Ad, SNs showed a higher ζ potential and an increase in relative molecular weight, thereby increasing cytotoxicity. However, in the acidic environment inside the cell, due to the pH responsiveness of SNs, β-CD-PDMAEMA can fall off from the Pasp-benzoicimine-Ad chain, causing SNs to disassemble into low-toxic poly-α-amino acid chains and β-CD-PDMAEMA. Therefore, compared with 25kDa PEI, SNs still show lower cytotoxicity.

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

[0141] 293T, B16-F10 and CHO-K1 cells were selected as transfected cells, and plasmids expressing luciferase and green fluorescent protein were selected as reporter genes to explore the in vitro transfection efficiency of SNs. Fig.10 As shown in the figure, compared with β-CD-PDMAEMA / pDNA complex, SNs showed 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 have better DNA compression ability, smaller particle size and higher surface potential ( Figure 6A and B in the figure), which are conducive to higher transfection efficiency. On the other hand, SNs are pH-responsive. In the acidic environment inside the cell, SNs become unstable ( Figure 1 ), which is conducive to the release of DNA and improves transfection efficiency. Interestingly, among the three cells, at the same N / P ratio, the transfection efficiency of SNs was the highest when the Ad / CD molar ratio was 4, which is consistent with the previous agarose gel electrophoresis and DLS results (SNs had the best DNA binding ability and the smallest particle size at an Ad / CD molar ratio of 4).

[0142] In addition, pEGFP was selected as the reporter gene, and the expression of green fluorescent protein in three cell lines, 293T, B16-F10 and CHO-K1, was directly observed using a fluorescence microscope. Fig.11 A (a1) in the figure is a picture of EGFP expression in 293T cells of 25kDaPEI / pDNA complex, β-CD-PDMAEMA / pDNA complex and SNs (different Ad / CD). It can be seen from the figure that compared with β-CD-PDMAEMA / pDNA complex, under the same N / P conditions, SNs showed more protein expression and stronger fluorescence intensity ( Fig.11 A (a2, a3) in ), especially when Ad / CD=4, which is consistent with the previous luciferase results ( Fig.10 We also obtained similar results in B16-F10 and CHO-K1 cells ( Fig.12 and Fig.13 ).

[0143] In addition to plasmid DNA, the possibility of using SNs to deliver siRNA was also explored. Anti-GFP siRNA was selected as the silencing gene, HeLa-GFP was used as the silencing cell, and the silencing efficiency of Lipofectamine2000, 25kDa PEI, β-CD-PDMAEMA / pDNA complex and SNs (N / P20, different Ad / CD) was measured. When Ad / CD=4, SNs can reduce the fluorescence intensity of HeLa-GFP cells by 40%, such as Fig.14 As shown in the figure, this result 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 plasmid DNA and siRNA transfection results show that the addition of polymer Pasp-benzoicimine-Ad can improve the transfection ability of SNs in specific Ad / CD.

[0144] 2.7 Study on SNs cell endocytosis efficiency

[0145] The efficiency of cellular endocytosis is very important for an effective gene vector system. The present invention uses laser confocal microscopy CLSM and flow cytometry to evaluate the cellular endocytosis efficiency of SNs with different Ad / CDs, using TOTO-3 red fluorescent dye to label DNA, green fluorescent dye WGA488 to stain cell membranes, and blue fluorescent dye Hoechst 33342 to label cell nuclei. CLSM is used to directly observe and take pictures to study the differences in the cellular endocytosis of the complex. Compared with the β-CD-PDMAEMA / pDNA complex, the cells acting on SNs showed a large amount of red fluorescence ( Fig.15 A in the figure). It can be observed that when Ad / CD=4, the cell endocytosis efficiency of SNs is the highest, which further explains the result that the transfection efficiency is the highest when Ad / CD=4 ( Fig.10 , Fig.11 , Fig.12 and Fig.13 ). Tested by flow cytometry ( Fig.15 Figure 2 (B, C) shows the endocytosis 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). Compared with 25 kDa PEI / pDNA and β-CD-PDMAEMA / pDNA complexes, the endocytosis efficiency of SNs is higher, which is due to the higher surface charge of SNs ( Figure 6 B (b) in the figure is more conducive to the binding of nanoparticles to the negatively charged cell membrane surface, thereby improving the endocytosis efficiency.

[0146] 3. Conclusion

[0147] In the invention, a new type of supramolecular nanoparticle SNs was constructed, which was formed by the host-guest interaction of pH-sensitive adamantane-modified polyasparagine and cationic polymer β-CD-PDMAEMA. This nanoparticle has good DNA compression ability and stability, and has excellent biocompatibility and high transfection efficiency. All the results in this chapter show that under the appropriate Ad / CD molar ratio conditions, SNs can become a very potential gene carrier

[0148] 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 composition, characterized in that including adamantane-terminated polyasparagine derivatives and star-shaped cationic polymers; The star-shaped cationic polymer is prepared from dimethylaminoethyl methacrylate monomer using brominated β-cyclodextrin shown in formula III as an initiator; Formula III .

2. The composition according to claim 1, characterized in that The number average molecular weight of the adamantane-terminated polyasparagine derivative is 30Kda to 50Kda; The number average molecular weight of the star-shaped cationic polymer is 5.38KDa ~ 13.27kDa.

3. The composition according to claim 1 or 2, characterized in that The molar ratio of the adamantane end group in the adamantane end group polyasparagine derivative to the beta-cyclodextrin in the star-shaped cationic polymer is (2-8):

1.

4. Use of the composition according to any one of claims 1 to 3 as a carrier in carrying and / or carrying a substance.

5. The use according to claim 4, characterized in that: The substances include nucleic acids.

6. The use according to claim 4 or 5, characterized in that: The molar ratio of nitrogen atoms in the carrier to phosphorus atoms in the nucleic acid to the carrier is (5-20):

1.

7. Use of the composition according to any one of claims 1 to 3 in the preparation of products for carrying and / or carrying substances.

8. Products for carrying and / or carrying substances, characterized in that: The invention comprises auxiliary materials and the composition according to any one of claims 1 to 3.

9. Use of the product according to claim 8 in the expression and / or silencing of nucleic acids.

10. A method for carrying and / or transporting a substance, characterized in that: The method comprises using the composition according to any one of claims 1 to 3.

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