Vitamin E-spermine conjugate, synthesis method thereof, and application thereof in nucleic acid drug delivery

Abstract

The present invention discloses a vitamin E-spermine conjugate, a synthesis method thereof, and an application in nucleic acid drug delivery. The present invention discloses a vitamin E-spermine conjugate obtained by coupling vitamin E with spermine, and its general formula is shown in Formula I. The present invention also provides a method for preparing a vitamin E-spermine conjugate. The spermine group in the vitamin E-spermine conjugate provided by the present invention can specifically bind to nucleic acids with double-stranded structures such as siRNA and plasmids, and can further self-assemble, thereby effectively avoiding the problem of lysosomal retention and achieving efficient delivery of nucleic acids such as targeted gene siRNA and plasmids. The present invention verifies that the vitamin E-spermine conjugate / siRNA can achieve RNAi-induced gene silencing of the target gene in multiple aspects such as mRNA levels, cell cycle changes, and changes in nuclear morphology, and also has a very good delivery effect for nucleic acid drugs with larger molecular weights such as DNA plasmids.

Description

Technical Field

[0001] The invention relates to a conjugate used as a nucleic acid drug delivery carrier, in particular to a vitamin E-spermine conjugate and a synthesis method thereof and application in nucleic acid drug delivery, belonging to the field of nucleic acid drug delivery carriers. Background Art

[0002] How to safely and efficiently deliver nucleic acids into cells is one of the core issues of gene therapy. The negatively charged phosphate backbone of nucleic acids repel each other with the negatively charged cell membrane, so nucleic acids must be assisted to enter cells to exert their effects. Although various types of delivery systems such as adenovirus vectors, cationic liposomes, and cationic polymers have been widely reported, safe and efficient nucleic acid delivery is still difficult to achieve. The new nucleic acid delivery vector constructed using receptor molecules that specifically bind to nucleic acids has the characteristics of simplicity, efficiency, and versatility, and shows great advantages in avoiding lysosomal retention and reducing the cytotoxicity of the vector.

[0003] Cationic polymers are also used for the delivery of nucleic acid drugs. Synthetic positively charged polymers such as polyethyleneimine (PEI) and polylysine (PLL) are common positively charged polymer materials used for gene delivery. Positively charged polymers can effectively deliver a variety of nucleic acids including siRNA and plasmid DNA. Their rich positive charges can effectively encapsulate and protect nucleic acids to form nanoparticles that can be taken up by cells. At the same time, their rich amine groups give them a strong proton buffering capacity, allowing them to exert a proton sponge effect and successfully escape from lysosomes. However, polycations also face the problem of cytotoxicity.

[0004] At present, cationic liposomes are the most widely used. The main mechanism of their nucleic acid loading is to form lipid-DNA / RNA complexes through electrostatic interactions between negatively charged nucleic acids and positively charged lipids and be wrapped in vesicle nanoparticles with a diameter of 100nm. These nanoparticles are further taken up and internalized by cells, and then release the encapsulated nucleic acids. However, although the lipid-DNA complexes formed have relatively high stability, in nucleic acid delivery, the positive and negative charge ratios of cationic liposomes relative to nucleic acids are still high, and side effects are difficult to avoid. Therefore, it is urgently needed in the field of nucleic acid drugs to provide a gene delivery vector that takes into account transfection efficiency and cytotoxicity and can efficiently deliver nucleic acid drugs into cells. Summary of the invention

[0005] One of the purposes of the present invention is to provide a class of vitamin E-spermine conjugates or vitamin E-triethylenetetramine conjugates;

[0006] The second object of the present invention is to provide a method for preparing the vitamin E-spermine conjugate or the vitamin E-triethylenetetramine conjugate;

[0007] The third purpose of the present invention is to apply the vitamin E-spermine conjugate or vitamin E-triethylenetetramine conjugate to nucleic acid drug delivery.

[0008] The above object of the present invention is achieved through the following technical solutions:

[0009] A vitamin E-spermine conjugate, the general formula of which is shown in Formula I:

[0010]

[0011] Wherein, R is selected from any one of the following (1)-(4):

[0012] (1)(CH 2 ) n C=O, wherein n is any integer from 1 to 5; (2) C=O(CH 2 ) m C=O, wherein m is any integer from 1 to 6; (3) CH 2 C=ONH(CH 2 CH 2 O) p C=O, wherein p is any integer from 1 to 6; (4) C=O(CH 2 ) q X(CH 2 ) q C=O, wherein q is any integer from 1 to 3, and X is SS or OC(CH 3 ) 2 -O and other cleavable functional groups.

[0013] Preferably, the vitamin E-spermine conjugate is selected from any one of the following formula II to formula V:

[0014]

[0015] The present invention further provides a method for preparing the vitamin E-spermine conjugate shown in formula I, comprising:

[0016] (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine (Sper) with tert-butyloxycarbonyl (Boc), leaving a free primary amine as a coupling site to obtain single-reaction site Sper-t-Boc;

[0017] (2) reacting the product Sper-t-Boc with the N-hydroxysuccinimide activated ester of a vitamin E derivative to obtain a Boc-protected vitamin E-spermine conjugate, and removing the Boc protecting group to obtain the conjugate.

[0018] Furthermore, the present invention provides a method for preparing the vitamin E-spermine conjugates of formula II to formula V.

[0019] A method for preparing the vitamin E-spermine conjugate of formula II, comprising:

[0020] (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine (Sper) with tert-butyloxycarbonyl (Boc), leaving a free primary amine as a coupling site to obtain single-reaction site Sper-t-Boc;

[0021] (2) After vitamin E is reacted with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and the carboxyl group and the amino group are coupled with Sper-t-Boc through a peptide bond condensation reaction, or through an amide ester reaction mediated by N,N'-disuccinimidyl carbonate or N'N-carbonyldiimidazole to obtain the product.

[0022] A method for preparing the vitamin E-spermine conjugate of formula III, comprising:

[0023] (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine (Sper) with tert-butyloxycarbonyl (Boc), leaving a free primary amine as a coupling site to obtain single-reaction site Sper-t-Boc;

[0024] (2) Vitamin E reacts with succinic anhydride, and the reaction product is coupled with Sper-t-Boc to obtain.

[0025] A method for preparing the vitamin E-spermine conjugate of formula IV, comprising:

[0026] (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine (Sper) with tert-butyloxycarbonyl (Boc), leaving a free primary amine as a coupling site to obtain single-reaction site Sper-t-Boc;

[0027] (2) Using dithioglycolic acid as a raw material, dehydration is carried out in trifluoroacetic anhydride to obtain a cyclic anhydride containing a disulfide bond, which is then directly coupled with vitamin E, and finally coupled with Sper-t-Boc to obtain the product.

[0028] A method for preparing the vitamin E-spermine conjugate of formula V, comprising:

[0029] (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc;

[0030] (2) After vitamin E reacts with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and then the carboxyl group reacts with 1-amino-3,6,9-trioxa-11-undecanol, and the reaction product is coupled with Sper-t-Boc through a peptide bond condensation reaction between the carboxyl group and the amino group to obtain the vitamin E-spermine conjugate described in formula V.

[0031] As a preferred specific embodiment of the present invention, the method for preparing Sper-t-Boc with a single reaction site comprises: first, at a low temperature of -78°C, adding an equimolar amount of ethyl trifluoroacetate to react with spermine to obtain spermine trifluoroacetylated with a single primary amine group (ethyl trifluoroacetate is slowly added dropwise to a reaction bottle containing spermine, ensuring that the concentration of spermine in the reaction system is much higher than that of ethyl trifluoroacetate, and the low temperature of -78°C greatly improves the selectivity of the reaction, so that spermine trifluoroacetylated with a single primary amine group can be obtained), then adding an excess of Boc anhydride, protecting the remaining two secondary amines and one primary amine with a Boc protecting group, and finally removing the trifluoroacetyl group under alkaline conditions to obtain Sper-t-Boc with a single reaction site.

[0032] Another aspect of the present invention is to provide a vitamin E-triethylenetetramine conjugate (VE-TETeA) of formula VI:

[0033]

[0034] The present invention further provides a method for preparing the vitamin E-triethylenetetramine conjugate compound of formula VI, comprising:

[0035] (1) Preparation of single-reaction site TETeA-t-Boc: Protect the three nitrogen atoms of triethylenetetramine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain single-reaction site TETeA-t-Boc;

[0036] (2) After vitamin E is reacted with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and the carboxyl group and the amino group are coupled with TETeA-t-Boc through a peptide bond condensation reaction, or the amide ester reaction mediated by N,N'-disuccinimidyl carbonate or N'N-carbonyldiimidazole is coupled with TETeA-t-Boc to obtain the product.

[0037] The present invention uses PAGE gel electrophoresis and microcalorimetry (MST) to study and find that the binding force of four vitamin E-spermine conjugates to siRNA double strands is significantly stronger than that of the analog VE-TETeA (vitamin E-triethylenetetramine conjugate), indicating that the spermine group in the vitamin E-spermine conjugate can specifically bind to the siRNA double strand. Further, molecular dynamics (MD) simulation is used to study the binding mode of the vitamin E-spermine conjugate and siRNA, and it is found that the spermine group in the vitamin E-spermine conjugate acts on the major groove of the siRNA double strand, and the nucleic acid phosphate backbone forms three salt bridges and one hydrogen bond with the spermine part in the major groove. In addition, the height of the VE-Su-Sper / siRNA complex is observed to be significantly increased compared to the height of pure siRNA by atomic force microscopy (AFM), further confirming that the vitamin E-spermine conjugate specifically binds to the siRNA double strand, and the siRNA is non-covalently "labeled" with vitamin E all around to form a hydrophobic coat. At the same time, when the concentration of vitamin E spermine conjugate is further increased, the vitamin E-spermine / siRNA complex is further assembled into nanoparticles with a neutral surface potential and a particle size of about 40nm. Cell uptake experiments show that the vitamin E-spermine conjugate / siRNA complex is different from the VE-TETeA / siRNA liposome. It enters the cell through a caveolin-mediated pathway and the vitamin E-spermine conjugate / siRNA complex after entry is almost not co-localized with the lysosome, thus effectively avoiding the problem of lysosomal retention. At the same time, the delivery of siRNA targeting the EG5 gene was successfully achieved, and the vitamin E-spermine conjugate / siRNA can achieve RNAi-induced gene silencing of the target gene in multiple aspects such as mRNA levels, cell cycle changes, and changes in nuclear morphology. At the same time, it also has a very good delivery effect for nucleic acid drugs with larger molecular weights, such as DNA plasmids and mRNA.

[0038] Therefore, the vitamin E-spermine conjugate or vitamin E-triethylenetetramine conjugate provided by the present invention can be used as a gene delivery vector to efficiently deliver gene drugs into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 There are four different vitamin E-spermine conjugates and the structural formula of VE-TETeA.

[0040] Figure 2 These are the PAGE gel electrophoresis results after different vitamin E-spermine conjugates with different N / Ps acted on siRNA; (a) VE-Sper, (b) VE-TETeA, (c) VE-Su-Sper, (d) VE-SS-Sper, (e) VE-PEG-Sper.

[0041] Figure 3is the MST binding curve; the abscissa is the concentration (mol·L -1 ), the ordinate is the normalized fluorescence intensity; the concentration of FITC-labeled siRNA used in all experiments was 50 nM.

[0042] Figure 4 The morphology (a, b) and particle size distribution (c) of VE-Su-Sper / siRNA complex.

[0043] Figure 5 The cellular uptake amounts of vitamin E-spermine conjugate / siRNA complexes at different molar ratios (the ratio of the molar number of vitamin E-spermine conjugate to the molar number of siRNA); (a) VE-Sper, (b) VE-Su-Sper, (c) VE-SS-Sper, and (d) VE-PEG-Sper.

[0044] Figure 6 The cellular uptake of vitamin E-spermine conjugate / siRNA complex changes with siRNA concentration under the condition of fixed N / P=3.9.

[0045] Figure 7 Real-time cellular entry of the complex; (a) Real-time cellular entry of VE-Su-Sper / siRNA complex (scale bar: 10 μm); (b) Caveolin / lipid raft structure and VE-Su-Sper / siRNA complex co-localize at the cell contraction end (top) and enter the cell together (bottom) (scale bar: 5 μm).

[0046] Figure 8 The subcellular localization of four vitamin E-spermine conjugate / siRNA complexes and VE-TETeA / siRNA complexes 2 hours after uptake (scale bar: 10 μm).

[0047] Fig. 9 Statistical analysis of the EG5 gene silencing results of four vitamin E-spermine conjugate / siRNA complexes and VE-TETeA / siRNA complex (siRNA concentration was 100 nM).

[0048] Fig.10 The changes in mRNA content after silencing the EG5 gene.

[0049] Fig.11 Changes in nuclear morphology; (Hoechst 33342 staining, scale bar 10 μm) (a) negative control group, (b) affected control group, (c) VE-Su-Sper / siRNA complex group.

[0050] Fig.12The figure shows the gene silencing effect of VE-Su-Sper / siRNA complexes with different concentrations; PC is Lipo delivering 100nM siRNA.

[0051] Fig.13 To encapsulate and deliver mCherry plasmid (2.5 μg mL -1 ). Confocal fluorescence imaging shows the expression of mCherry fluorescent protein after 48 hours. a) Blank plasmid, b) Lipo2000, c) VE-Su-Sper, N / P is 5.4. DETAILED DESCRIPTION

[0052] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0053] Example 1 Synthesis and Characterization of Vitamin E-Spermine Conjugate

[0054] In order to synthesize the vitamin E-spermine conjugate, a carboxyl group is introduced from vitamin E and then coupled with spermine through a carboxyl amide reaction. According to the method reported in the literature, the three nitrogen atoms of spermine (Sper) are protected with tert-butyloxycarbonyl (Boc), leaving a free primary amine as a coupling site to obtain the product Sper-t-Boc, which makes the coupling with vitamin E and the subsequent purification steps easy to carry out.

[0055] In order to compare the effects of different linkers between vitamin E and spermine on the delivery of siNRA by vitamin E-spermine conjugates, four different types of linkers were designed: a short linker, a longer hydrophobic linker, a longer hydrophilic linker, and an easily degradable disulfide linker (for VE-Sper, VE-Su-Sper, VE-PEG-Sper, and VE-SS-Sper, respectively). Figure 1 )The specific synthesis route is as follows.

[0056] Synthesis of Sper-t-Boc

[0057] 1g of spermine was added to a 100mL eggplant-shaped bottle, filled with nitrogen for protection, 20mL of methanol was added to dissolve the product, and the temperature was lowered to -78°C in a low-temperature reactor. 703mg of ethyl trifluoroacetate was dissolved in 30mL of methanol and slowly added dropwise to the eggplant-shaped bottle containing spermine for 30min. After the addition was completed, the reaction continued at -78°C for 30min, then the temperature was raised to 0°C, and Boc anhydride was added dropwise for 30min to 60min. After reacting at room temperature for 18h, concentrated ammonia was added until the pH was about 11, and then reacted at room temperature for 15h. After the reaction was completed, methanol was removed by rotary evaporation, and the product 1.66g (yield 67%) was purified by silica gel column. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )3.35-3.09(m,10H,-CONHCH 2 -), 2.94(t, J=6.4Hz, 2H,-NH 2 CH 2 -), 1.89-1.97 (m, 2H, CH 2 ), 1.70-1.62 (m, 2H, CH 2 ), 1.51-1.41 (m, 4H, -CH 2 CH 2 -, 27H, 9×CH 3 ).

[0058] Synthesis of VE-Sper

[0059] 1.666g of sodium hydride was added to an eggplant-shaped bottle containing 60mL of redistilled tetrahydrofuran, filled with nitrogen for protection, and stirred at 0°C for 10min. 8.260g of vitamin E was dissolved in 20mL of redistilled tetrahydrofuran, slowly added to the sodium hydride suspension, and stirred at 0°C for 15min, and then 3.924g of ethyl bromoacetate was added, and the reaction was reacted at room temperature for 3.5h. After the reaction was completed, 30mL of ethyl acetate was added to dilute the reaction solution, and 30mL of water was slowly added to quench the reaction, and then the organic phase was separated with a separatory funnel. The obtained organic phase was washed twice with 30mL of water, and then washed three times with 30mL of saturated brine, followed by drying the organic phase with anhydrous sodium sulfate, filtering under reduced pressure to remove sodium sulfate, and finally removing the organic solvent by rotary evaporation. The obtained crude product has a high purity and does not require further purification, and the product is 8.95g with a yield of 90%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )4.35-4.30(m,4H,-COOCH 2 -,-OCH 2 CO-), 2.59(t, J=6.8Hz, 2H, -PhCH 2 -), 2.21(s, 3H, -PhCH 3), 2.17(s, 3H, -PhCH 3 ), 2.10(s, 3H, -PhCH 3 ), 1.88-1.74(m, 2H), 1.57-1.10(m, 27H, 9×CH 2 , 3×CH, 2×CH 3 ), 0.93-0.86 (m, 12H, 4×CH 3 )

[0060] The above 8.95g compound was dissolved in 70mL tetrahydrofuran, and 20mL sodium hydroxide solution (1.67mol / L) was added. The reaction was allowed to react overnight at room temperature. After the reaction was completed, tetrahydrofuran was removed by rotary evaporation, and 100mL water was added. The pH value was adjusted to acidic using dilute hydrochloric acid, and then 50mL dichloromethane was added for extraction, and the organic phase was collected. The organic phase was washed 3 times with 50mL of water having a pH value of about 3 to 4, and then washed 3 times with 50mL of saturated salt water, followed by drying the organic phase with anhydrous sodium sulfate, filtering under reduced pressure to remove sodium sulfate, and finally rotary evaporation to remove the organic solvent. 8.137g of product was obtained with a yield of 96%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )4.39(s,2H,-PhOCH 2 -), 2.60(t, J=6.8Hz, 2H, -PhCH 2 -), 2.21(s, 3H, -PhCH 3 ), 2.17(s, 3H, -PhCH 3 ), 2.10(s, 3H, -PhCH 3 ), 1.88-1.74(m, 2H), 1.57-1.10(m, 24H, 9×CH 2 , 3×CH,CH 3 ), 0.93-0.86 (m, 12H, 4×CH 3 ).

[0061] The compound (792 mg) from the previous step was dissolved in 6 mL of redistilled tetrahydrofuran, followed by the addition of 450 μL of triethylamine, 400 mg of dicyclohexylcarbodiimide, and 224 mg of N-hydroxysuccinimide, and the reaction was carried out at room temperature for 6 h. The tetrahydrofuran was removed by rotary evaporation, and a mixed solution of redistilled petroleum ether and redistilled ethyl acetate in a volume ratio of 5:1 was added, and the insoluble matter was filtered off under reduced pressure. Finally, 670 mg of the product was obtained by silica gel column separation, with a yield of 71%.

[0062] 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )4.67(s,2H,-PhOCH 2-), 2.90(s, 4H, -COCH 2 CH 2 CO-), 2.60(t, J=6.8Hz, 2H, -PhCH 2 -), 2.22(s, 3H, -PhCH 3 ), 2.18(s, 3H, -PhCH 3 ), 2.11(s, 3H, -PhCH 3 ), 1.88-1.74(m, 2H), 1.57-1.10(m, 24H, 9×CH 2 , 3×CH,CH 3 ), 0.93-0.86 (m, 12H, 4×CH 3 ).

[0063] 461 mg of the compound from the previous step was dissolved in 5 mL of redistilled dichloromethane, and 396 mg of Sper-t-Boc and 130 μL of N,N-diisopropylethylamine were added, and the mixture was reacted at room temperature for 4 h. After the reaction, the product VE-Sper-t-Boc was purified by silica gel column to obtain 654 mg of the product with a yield of 81%.

[0064] 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )4.18(s,2H,-PhOCH 2 -), 3.41-3.10(m, 12H, -CONHCH 2 -), 2.61-2.57(t, J=6.8Hz, 2H, -PhCH 2 -), 2.18-2.10(m, 9H, 3×PhCH 3 ), 1.85-1.75(m, 4H), 1.67(brs, 2H), 1.57-1.09(m, 55H), 0.89-0.85(m, 12H, 4×CH 3 ).

[0065] Dissolve 308 mg of VE-Sper-t-Boc from the previous step in 3 mL of dichloromethane, add 3 mL of trifluoroacetic acid, and react at room temperature for 4 hours. After the reaction is completed, add 20 mL of dichloromethane to dilute the reaction solution, and remove the organic solvent by rotary evaporation. Since trifluoroacetic acid is highly corrosive, an absorption device containing sodium hydroxide needs to be added to the air guide tube of the rotary evaporator. The crude product after removing the organic solvent is dissolved in as little dichloromethane as possible, and a large volume of ice ether is added. A white precipitate is found to precipitate, and the product is placed at -20 ° C overnight. The next day, the insoluble matter in the suspension was collected by centrifugation at 1200 rpm for 15 minutes. The collected white precipitate was washed with ice ether, centrifuged at 1200 rpm for 15 minutes, and the supernatant was discarded. Repeat this step 5 times. After drying, the product VE-Sper 171 mg was obtained with a yield of 53%.

[0066] 1 H NMR (δ / ppm, 400MHz, Pyridine-d5) 4.47 (s, 2H, -PhOCH 2 -), 3.73 (q, J=6.4Hz, 2H, -CONHCH 2 -), 3.55(t,J=7.0Hz, 2H, -NCH 2 -), 3.44 (t, J=7.3Hz, 2H, -NCH 2 -),3.34(t,J=7.3Hz,2H,-NCH 2 -), 3.20-3.15 (m, 4H, -NCH 2 -), 2.60-2.53 (m, 4H, CH 2 , -PhCH 2 -), 2.34-2.27 (m, 2H, CH 2 ), 2.22-2.18(m, 9H, 3×PhCH 3 ), 2.04-2.01 (m, 4H, 2×CH 2 ), 1.82-1.13(m, 26H, 10×CH 2 , 3×CH,CH 3 ), 0.93-0.87 (m, 12H, 4×CH 3 ) 13C NMR (101 MHz, Pyridine-d5) δ 171.62 (TFA), 162.96, 149.98, 149.04, 129.03, 127.29, 121.03, 119.51, 118.09 (TFA), 76.45, 73.45, 48.45, 46.61, 41.61, 40.84, 38.99, 37.56, 34.20, 32.71, 29.46, 28.73, 26.85, 26.01, 25.17, 23.99, 22.64, 22.08, 21.03, 14.03, 13.37, 13.18. ESI-MS: theory: 673 [M+H] + , Experiment: 673[M+H] + Synthesis of VE-TETeA.

[0067] Synthesis of VE-Su-Sper

[0068] 2.15 g vitamin E and 0.75 g succinic anhydride were dissolved in 10 mL redistilled tetrahydrofuran, and then 180 μL triethylamine was added and reacted at room temperature for 24 h. After the reaction was completed, the tetrahydrofuran solvent was removed by rotary evaporation, 20 mL dichloromethane was added to dissolve the residue after rotary evaporation, and 20 mL ddH2O with a pH value of about 1 to 2 was used to dissolve the residue. 2 The mixture was washed with 20 mL of saturated sodium chloride for 3 times, and then washed with 20 mL of saturated sodium chloride for 3 times. The organic phase was then dried with anhydrous sodium sulfate, filtered under reduced pressure to remove the sodium sulfate, and finally the organic solvent was removed by rotary evaporation to obtain a crude product. Purification by silica gel column gave 1.80 g of the product with a yield of 68%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 2.97-2.93 (m, 2H, -COCH 2 -), 2.87-2.83 (m, 2H, -COCH 2 -), 2.60(t,J=6.9Hz, 2H, PhCH 2 ), 2.11(s, 3H, -PhCH 3 ), 2.03(s, 3H, -PhCH 3 ), 1.99(s, 3H, -PhCH 3 ), 1.87-1.75 (m, 2H, CH 2 ),1.57-1.07(m,24H,9×CH 2 , 3×CH,CH 3 ), 0.90-0.86 (m, 12H, 4×CH 3 ).

[0069] Dissolve 317 mg of the compound and 667 mg of HBTU in 5 mL of redistilled tetrahydrofuran and fill with nitrogen. Then use a syringe to add 938 μL of N,N-diisopropylethylamine, and then add a small amount of redistilled dichloromethane containing 300 mg of Sper-t-Boc to the reaction solution, and react at room temperature for 5 hours. After the reaction is completed, filter under reduced pressure to remove insoluble matter, and dilute the obtained organic phase with 15 mL of dichloromethane and 20 mL of ddH 2 O twice, and then washed three times with 20mL saturated brine, then dried the organic phase with anhydrous sodium sulfate, filtered under reduced pressure to remove sodium sulfate, and finally rotary evaporated to remove the organic solvent to obtain a crude product. Purification on a silica gel column gave 482mg of the product VE-Su-Sper-t-Boc with a yield of 79%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 3.35-3.12(m, 12H, -CONHCH 2 -), 3.00 (t, J=6.3Hz, 2H, -COCH 2 -), 2.65-2.58 (m, 4H, -COCH 2 -, PhCH 2 ), 2.10(s, 3H, -PhCH 3 ), 2.02(s, 3H, -PhCH 3 ), 1.98(s, 3H, -PhCH 3 ), 1.83-1.06 (m, 61H), 0.89-0.85 (m, 12H).

[0070] 241 mg VE-Su-Sper-t-Boc was dissolved in 3 mL dichloromethane, followed by the addition of 3 mL trifluoroacetic acid and the reaction was carried out at room temperature for 1 h. After the reaction, the treatment method was the same as that for VE-Sper. After drying, 166 mg VE-Su-Sper was obtained with a yield of 66%. 1 HNMR (δ / ppm, 400MHz, Pyridine-d5): 3.56-3.12(m, 14H, -CONHCH 2 -,-COCH 2 -), 2.93-2.88 (m, 2H, -COCH 2 -), 2.61-2.50(m, 4H, CH2, PhCH 2 ), 2.21-2.14(m, 11H, 3×PhCH 3 , CH 2 ), 2.03-1.98 (m, 4H, 2×CH 2 ), 1.74-1.10(m, 26H, 10×CH 2, 3×CH,CH 3 ), 0.94-0.87 (m, 12H, 4×CH 3 ) 13 C NMR(101MHz,Pyridine-d5)δ173.67(TFA),173.01,162.91,150.21,142.40,128.2 8,126.69,120.64,118.88,117.70(TFA),76.26,48.02,46.33,46.26,40.46,38.43 ,37.39,33.95,33.84,31.66,30.43,29.10,28.20,26.53,25.99,25.66,24.99,24.80,23.72,22.26,21.66,20.82,20.75,14.12,13.28,12.96.ESI-MS: Theoretical: 713 [M+H] + , Experiment: 713[M+H] + .

[0071] Synthesis of VE-SS-Sper

[0072] 1.822 g of dithioglycolic acid was added to 15 mL of trifluoroacetic anhydride and reacted at 30 °C for 2 h. The reaction system was diluted with a large volume of toluene and the organic solvent, excess anhydride and byproduct trifluoroacetic acid were removed by rotary evaporation. This was repeated 3 times. 20 mL of redistilled dichloromethane was added to the residue after rotary evaporation, followed by 862 mg of vitamin E, 49 mg of DMAP and 288 μL of triethylamine. The reaction was allowed to react overnight at room temperature. After the reaction was completed, the reaction system was diluted with 20 mL of dichloromethane and 40 mL of ddH 2 The reaction mixture was washed with 40 mL of saturated sodium chloride for 3 times, and then washed with 40 mL of saturated sodium chloride for 3 times. The organic phase was then dried over anhydrous sodium sulfate, filtered under reduced pressure to remove the sodium sulfate, and finally the organic solvent was removed by rotary evaporation to obtain a crude product. The product was purified by silica gel column to obtain 520 mg of the product, with a total yield of 44% in two steps. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 3.87 (s, 2H, -SCH 2 -), 3.68(s, 2H, -SCH 2 -), 2.59 (t, J=6.8Hz, 2H, -PhCH 2 -), 2.09(s, 3H, -PhCH 3 ), 2.04(s, 3H, -PhCH 3 ), 2.00(s, 3H, -PhCH 3), 1.83-1.71(m, 2H), 1.59-1.02(m, 24H, 9×CH 2 , 3×CH,CH 3 ), 0.87-0.83 (m, 12H, 4×CH 3 ).

[0073] The compound (310 mg) and 965 mg HBTU were dissolved in 5 mL redistilled dichloromethane and filled with nitrogen. Then 790 μL N, N-diisopropylethylamine was added using a syringe, and a small amount of redistilled dichloromethane containing 206 mg Sper-t-Boc was added to the reaction solution, and the reaction was carried out at room temperature for 3.5 h. After the reaction was completed, the insoluble matter was removed by filtration under reduced pressure, and 15 mL of dichloromethane was added to the obtained organic phase for dilution, and 20 mL of ddH 2 The reaction mixture was washed with 20 mL of saturated sodium chloride solution for 2 times, and then washed with 20 mL of saturated sodium chloride solution for 3 times. The organic phase was then dried over anhydrous sodium sulfate, and the sodium sulfate was filtered off under reduced pressure. Finally, the organic solvent was removed by rotary evaporation and purified by silica gel column to obtain 220 mg of the product VE-SS-Sper-t-Boc with a yield of 39%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 3.88 (s, 2H, -SCH 2 -), 3.54(s, 2H, -SCH 2 -), 3.30-3.11(m, 12H, -CONHCH 2 -), 2.61(t,J=6.8Hz,2H,-PhCH 2 -), 2.11(s, 3H, -PhCH 3 ), 2.06(s, 3H, -PhCH 3 ), 2.02(s, 3H, -PhCH 3 ), 1.88-1.10(m, 61H), 0.89-0.86(m, 12H, 4×CH 3 ).

[0074] Dissolve 220 mg of VE-Sper-t-Boc in 3 mL of dichloromethane, then add 3 mL of trifluoroacetic acid and react at room temperature for 1 hour. After the reaction, the treatment method is the same as that of VE-Sper. Dry to obtain 131 mg of the product VE-SS-Sper with a yield of 57%. 1 HNMR (δ / ppm, 400MHz, Pyridine-d5): 4.38 (s, 2H, -SCH 2 -), 3.97(s, 2H, -SCH 2 -), 3.61-3.15(m, 12H, -CONHCH 2-), 2.63-2.53 (m, 4H, CH 2 , PhCH 2 ), 2.23-2.13(m, 11H, 3×PhCH 3 , CH 2 ), 2.05-2.20 (m, 4H, 2×CH 2 ), 1.76-1.10(m, 26H, 10×CH 2 , 3×CH,CH 3 ), 0.95-0.87 (m, 12H, 4×CH 3 ) 13 CNMR(101MHz,Pyridine-d5)δ169.44(TFA),168.51,161.45,149.08,141.12,126.96,125.40 ,119.44,117.86,116.50(TFA),75.20,46.88,45.28,44.99,43.06,40.84,39.32,37.49,37.4 0,37.29,37.21,36.74,32.81,32.70,27.95,26.85,25.28,25.24,24.87,24.52,23.67,23.59,22.58,22.49,21.14,20.50,19.68,19.62,19.52,12.99,12.16,11.81.ESI-MS: Theoretical: 779 [M+H] + , Experiment: 779[M+H] + .

[0075] Synthesis of VE-PEG-Sper

[0076] 1.692g of vitamin acetic acid derivative VE-CO-NHS was dissolved in 10mL redistilled dichloromethane, and 558mg of 1-amino-3,6,9-trioxa-11-undecyl alcohol and 502μL of N,N-diisopropylethylamine were added and reacted at room temperature for 4h. After the reaction, 15mL of dichloromethane was added to dilute the system and 20mL of ddH 2 The organic phase was dried over anhydrous sodium sulfate, filtered under reduced pressure to remove the sodium sulfate, and finally the organic solvent was removed by rotary evaporation. The product was purified by silica gel column to obtain 670 mg of the product with a yield of 35%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 4.20 (s, 2H, -PhOCH 2 -), 3.86-3.35 (m, 16H, -OCH 2-), 2.59 (t, J=6.8Hz, 2H, -PhCH 2 -), 2.18-2.10(m, 9H, 9×PhCH 3 ), 1.84-1.07(m, 26H, 10×CH 2 , 3×CH,CH 3 ), 0.90-0.86 (m, 12H, 4×CH 3 ).

[0077] The compound (278 mg) from the previous step was dissolved in 10 mL of redistilled dichloromethane, and then 654 μL of N,N-diisopropylethylamine and 428 mg of N,N'-disuccinimidyl carbonate were added and reacted at room temperature for 24 h. After the reaction was completed, the insoluble matter was filtered out and 20 mL of ddH 2 O for 2 times, and then washed 3 times with 20mL saturated brine, then dried the organic phase with anhydrous sodium sulfate, and filtered under reduced pressure to remove sodium sulfate. After the solvent was removed by rotary evaporation, the second step of the reaction was directly carried out. 5mL of redistilled dichloromethane was added to dissolve the obtained product, and 210mg of Sper-t-Boc and 100μL of N,N-diisopropylethylamine were added to react at room temperature for 4h. After the reaction was completed, as before, after washing, drying and filtering, the obtained product was purified by silica gel column to obtain 142mg of product VE-PEG-Sper-t-Boc with a yield of 31%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 ): 4.21-4.19 (m, 4H, -PhOCH 2 -,-COOCH 2 -), 3.71-3.63 (m, 14H, -OCH 2 -), 3.33-3.11(m, 12H, -CONHCH 2 -), 2.59(t,J=6.9Hz,2H,-PhCH 2 -), 2.15-2.10(m, 9H, 9×PhCH 3 ), 1.86-1.10(m, 61H), 0.90-0.86(m, 12H, 4×CH 3 ).

[0078] 142 mg VE-PEG-Sper-t-Boc was dissolved in 3 mL dichloromethane, followed by the addition of 3 mL trifluoroacetic acid and the reaction was carried out at room temperature for 4 h. After the reaction, the treatment method was the same as that for VE-Sper. Finally, 52 mg VE-PEG-Sper was obtained with a yield of 33%.

[0079] 1H NMR (δ / ppm, 400MHz, Pyridine-d5): 4.46 (s, 2H, -PhOCH 2 -), 4.38(t, J=4.9Hz, 2H, -COOCH 2 -), 3.83-3.64 (m, 14H, -OCH 2 -), 3.57-3.17(m, 12H, -CONHCH 2 -), 2.61-2.55 (m, 4H, CH 2 , PhCH 2 ), 2.23-2.18(m, 11H, 3×PhCH 3 , CH 2 ), 2.03-2.02 (m, 4H, 2×CH 2 ), 1.80-1.13(m, 26H, 10×CH 2 , 3×CH,CH 3 ), 0.94-0.87 (m, 12H, 4×CH 3 ) 13 C NMR(101MHz,Pyridine-d5)δ170.46(TFA),162.98,149.95,149.09,129.02,127.29,120.94,119. 51,118.00(TFA),76.44,73.62,72.08,72.04,71.87,71.37,71.10,65.44,48.39,46.88,46.52,41 .50,40.83,40.59,39.55,38.90,38.75,34.31,34.19,32.71,29.46,28.75,26.80,26.35,26.01,25.15,24.09,23.99,22.62,22.09,21.18,21.12,21.01,14.05,13.39,13.19.ESI-MS: Theoretical: 892 [M+H] + , Experimental: 892[M+H] + .

[0080] Synthesis of VE-TETeA (same as synthesis of VE-Sper)

[0081] 723 mg of triethylenetetramine was added to a 100 mL eggplant-shaped bottle, nitrogen was filled for protection, 20 mL of methanol was added to dissolve the product, and the temperature was cooled to -78 ° C in a low-temperature reactor. 703 mg of ethyl trifluoroacetate was dissolved in 30 mL of methanol and slowly added dropwise to the eggplant-shaped bottle containing triethylenetetramine for 30 min. After the addition was completed, the reaction continued at -78 ° C for 30 min, then the temperature was raised to 0 ° C, and Boc anhydride was added dropwise for 30 to 60 min. After reacting at room temperature for 18 h, concentrated ammonia was added until the pH was about 11, and then reacted at room temperature for 15 h. After the reaction was completed, the methanol was removed by rotary evaporation, and the product 402 mg TETeA-t-Boc was purified by silica gel column with a yield of 18%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )3.60-3.28(m,12H,-CONHCH 2 -, -NH 2 CH 2 -), 1.47-1.42(m, 27H, 9×CH 3 ).

[0082] 315 mg VE-CO-NHS was dissolved in 5 mL redistilled dichloromethane, and 200 mg TETeA-t-Boc and 78 μL N,N-diisopropylethylamine were added, and the mixture was reacted at room temperature for 4 h. After the reaction, the product was purified by silica gel column to obtain 200 mg VE-TETeA-t-Boc with a yield of 41%. 1 H NMR (δ / ppm, 400 MHz, CDCl 3 )4.17(s,2H,-PhOCH 2 -), 3.35-3.28(m, 12H, -CONHCH 2 -), 2.58(t, J=6.8Hz, 2H, -PhCH 2 -), 2.13-2.07 (m, 9H, -PhCH 3 ), 1.87-1.04(m, 57H), 0.89-0.85(m, 12H, 4×CH 3 ).

[0083] 200 mg VE-TETeA-t-Boc was dissolved in 3 mL dichloromethane, and then 3 mL trifluoroacetic acid was added and reacted at room temperature for 24 h. After the reaction, the treatment method was similar to that of VE-Sper, but the solvent used for precipitation and washing was replaced with petroleum ether. After drying, 115.6 mg VE-TETeA was obtained with a yield of 55%. 1 H NMR (δ / ppm, 400MHz, DMSO) 4.08 (s, 2H, -PhOCH 2-), 3.35-3.28(m, 12H, -CONHCH 2 -), 2.54(t, J=6.8Hz, 2H, -PhCH 2 -), 2.10(s, 3H, -PhCH 3 ), 2.08(s, 3H, -PhCH 3 ), 2.00(s, 3H, -PhCH 3 ), 1.74(t,J=7.2Hz,2H), 1.54-1.07(m, 24H, 9×CH 2 , 3×CH,CH 3 ), 0.88-0.82 (m, 12H, 4×CH 3 )13C NMR (101 MHz, DMSO) δ 169.52 (TFA), 158.84, 158.52, 148.06, 147.24, 127.41, 125.85, 118.95, 117.98, 115.98 (TFA), 75.04, 71.75, 37.19, 37.05, 35.43, 32.45, 27.83, 24.59, 24.13, 23.98, 23.01, 22.92, 20.08, 20.03, 12.91, 12.12, 12.05.ESI-MS: theory: 617 [M+H] + , Experiment: 617[M+H] + .

[0084] Experimental Example 1: Interaction experiment between vitamin E-spermine conjugate and siRNA

[0085] Four vitamin E-spermine conjugates were co-incubated with siRNA in N / P format, and the interaction between the two was studied by non-denaturing PAGE gel electrophoresis. The experimental results are as follows: Figure 2 As shown, free siRNA will form obvious bands after electrophoresis, but when the N / P of the vitamin E-spermine conjugate incubated with it reaches 7.2, the siRNA band can no longer be detected. This shows that the siRNA has been completely encapsulated by the vitamin E-spermine conjugate at this time, and there is no free siRNA. In contrast, the analog of the vitamin E-spermine conjugate VE-TETeA cannot completely encapsulate nucleic acids at the same ratio, and even when the N / P reaches 14.4, a light gray band can still be seen. This difference indicates that the specific binding between spermine and nucleic acids plays a key role in the process of nucleic acid encapsulation by vitamin E spermine-conjugate.

[0086] MST technology can measure the dissociation equilibrium constant of molecular binding through the change of molecular MST signal. Figure 3As shown in Figure 2, the dissociation equilibrium constants of VE-Su-Sper, VE-PEG-Sper, VE-SS-Sper and VE-Sper for binding to siRNA were 1.49±0.08μM, 1.14±0.07μM, 3.4±0.3μM and 3.4±0.9μM, respectively, which are consistent with the binding affinity of 1-naphthylacetyl-spermine reported in the literature. However, the K of the vitamin E-modified spermine analog VE-TETeA was D The value was 16±2 μM, which was one order of magnitude lower than that of the four vitamin E-spermine conjugates, VE-Su-Sper, VE-PEG-Sper, VE-SS-Sper and VE-Sper.

[0087] In this experiment, an atomic force microscope (AFM) was used to observe the morphology of the VE-Su-Sper / siRNA complex formed under the condition of N / P=0.45. Prior to this, the state of siRNA under AFM was observed first. In order to easily find siRNA, a high-concentration siRNA sample was used. Since the structure of siRNA is very small (about 5-6nm) and the horizontal resolution of AFM is very limited, the precise structure of siRNA cannot be observed, and only a large number of circular to elliptical white dots can be observed. However, the z-axis resolution of AFM is high and it is sensitive to sample height information. The height of these small white dots measured by AFM fluctuates within the range of 1.0-1.5nm, confirming that the white dot structure observed by AFM is a double-stranded siRNA. Under low-concentration siRNA conditions, small elliptical white dots with a height of 1.0-1.5nm were also observed under AFM ( Figure 4 (a)). Under the same concentration conditions, compared with siRNA, the average height of the VE-Su-Sper / siRNA complex (N / P=0.45) observed under AFM increased from 1.0-1.5nm to about 2.0nm. Further increasing the N / P of VE-Su-Sper and siRNA to 3.6 will induce further assembly of the VE-Su-Sper / siRNA complex. The morphology of the vitamin E-spermine conjugate / siRNA complex was observed using STEM (scanning transmission electron microscopy). As shown, the morphology of the vitamin E-spermine conjugate / siRNA complex is spherical, and its particle size is about 40nm. Carefully observe the internal structure of the sphere ( Figure 4 (b)), it was found that it is composed of many smaller units stacked together, presenting a structural pattern of multiple "leaves" arranged in a radial manner. This "leaves" is similar to the vitamin E "labeled" siRNA observed by AFM.

[0088] Experimental Example 2 Cellular uptake experiment of vitamin E-spermine conjugate / siRNA complex

[0089] Vitamin E-spermine conjugate was incubated with Cy3-labeled siRNA (Cy3-siRNA) at different N / P ratios to obtain vitamin E-spermine conjugate / siRNA complexes, and flow cytometry was used to detect the uptake of the complexes by cells. Figure 5 As shown, with the increase in the proportion of the added compound, the cellular uptake of the complexes formed by the four vitamin E-spermine conjugates and siRNA all showed a trend of first rising and then falling. For the compounds VE-Sper and VE-PEG-Sper, the cellular uptake of the complex reached the maximum when the N / P was 3.6, while for VE-Su-Sper and VE-SS-Sper, the uptake of the complex was the maximum when the N / P was 7.2. The formation of the multi-"leaf" spoke-axis structure of the vitamin E-spermine conjugate / siRNA complex is affected by the ratio of the vitamin E-spermine conjugate to the siRNA. Too little vitamin E-spermine conjugate will result in too little vitamin E "labeled" on the double-stranded surface of the siRNA, and there will be a large amount of negative charge remaining, which is not conducive to the assembly of siRNA into nanostructures with the assistance of the vitamin E-spermine complex. Too much vitamin E-spermine conjugate may also interfere with the self-assembly of the nanostructure. At the same time, excessive vitamin E derivatives in the solution will affect the interaction between vitamin E on the surface of the nanoparticles and the cell membrane. This phenomenon was also found in the previous work of the inventor's research group. Unlike the four vitamin E-spermine conjugates, VE-TETeA has a weaker binding affinity with siRNA. At the same N / P ratio, its ability to deliver siRNA is very low. At high ratios, the nonspecific effects of positive and negative charges begin to play an important role. Therefore, only at high ratios can VE-TETeA encapsulate siRNA and achieve its delivery.

[0090] Under the condition that the N / P ratio of vitamin E-spermine conjugate to siRNA is 3.6, the concentration dependence of cellular uptake of vitamin E-spermine conjugate / siRNA complex is shown in the following experimental results: Figure 6As shown, the cellular uptake of vitamin E-spermine conjugate / siRNA complexes increases with the increase of siRNA concentration. Compounds VE-Sper, VE-Su-Sper, and VE-PEG-Sper can all efficiently deliver siRNA into cells. When the siRNA concentration reaches 100nM, the uptake of these three vitamin E-spermine conjugate / siRNA complexes is higher than the cellular uptake of siRNA delivered by Lipo. When the siRNA concentration is 200nM, the uptake of VE-Su-Sper is even about twice that of the Lipo group. These results all illustrate the great potential of vitamin E-spermine conjugates for siRNA delivery, among which VE-Su-Sper performs best.

[0091] Experimental Example 3 Vitamin E-spermine conjugate / siRNA complex uptake mechanism experiment

[0092] Based on the structure of flow cytometry analysis, VE-Su-Sper showed the highest siRNA delivery efficiency, so VE-Su-Sper was selected as an example. First, the real-time cell entry of VE-Su-Sper / siRNA complex was observed using scanning laser confocal microscopy (LSCM). Figure 7 As shown in (a), it was found that the VE-Su-Sper / siRNA complex is not randomly taken up from all directions of the cell, but is always adsorbed on the leading edge and retracting tail of the cell (the active part of the cell membrane during movement and migration, yellow arrow in the figure), and then taken up into the cell. The cell contraction end is the area where the caveolin / lipid raft structure is enriched, and the VE-Su-Sper / siRNA complex is always taken up through the contraction end of the cell. There is a strong correlation between the two. Further, the caveolin / lipid raft structure was labeled with fluorescently labeled CTB, and it was found that it co-localized with the VE-Su-Sper / siRNA complex at the leading edge of the cell ( Figure 7 (b), upper), this evidence points to the fact that the VE-Su-Sper / siRNA complex enters the cell via the caveolin-mediated pathway. At the same time, the VE-Su-Sper / siRNA complex that enters the cell co-localizes with the caveolin / lipid raft structure in the cell, indicating that the VE-Su-Sper / siRNA complex binds to the caveolin / lipid raft and then enters the cell together with it ( Figure 7 (b)), below).

[0093] Laser scanning confocal microscopy was further used to observe the subcellular localization of the vitamin E-spermine conjugate / siRNA complex that entered the cells. Figure 8As shown, it was observed that the intracellular distribution of Cy3-labeled siRNA delivered by vitamin E-spermine conjugate, VE-TETeA and Lipo was different. After the Lipo / siRNA complex enters the cell, it colocalizes strongly with the lysosome (represented by the red signal in the figure), which is manifested by the high orange-yellow signal in the figure. The distribution of the VE-TETeA / siRNA complex after entering the cell is similar to that of the Lipo / siRNA complex, and it has strong colocalization with the lysosome, but its transfection efficiency is lower than that of Lipo. The vitamin E-spermine conjugate / siRNA complex exhibits different properties from the aforementioned Lipo / siRNA complex and VE-TETeA / siRNA complex. It was further determined that the vitamin E-spermine conjugate / siRNA complex can effectively avoid lysosomes, which is a major advantage in gene delivery.

[0094] Experimental Example 4: Experiment on silencing EG5 gene by vitamin E-spermine conjugate / siRNA complex

[0095] Vitamin E-spermine conjugates can specifically bind to siRNA double strands and further self-assemble into nanoparticles, which enter cells through caveolin-mediated pathways and avoid lysosomal escape. Taking the EG5 gene target as an example, vitamin E-spermine conjugates were used to deliver siRNA for gene silencing experiments.

[0096] The experimental results are as follows Fig. 9 As shown in the figure, VE-Su-Sper can effectively deliver siRNA and mediate gene silencing, and the proportion of cells in the G2 / M phase increased 5 times compared with the control group, reaching 30%. VE-Su-Sper and VE-Sper can also deliver siRNA to achieve gene silencing, but the proportion of cells in the G2 / M phase only increased from 5% to 20%. Under the same transfection conditions, VE-TETeA cannot effectively deliver siRNA into cells, and naturally cannot achieve effective gene silencing in gene silencing experiments. Therefore, VE-Su-Sper is the most effective carrier among several vitamin E-spermine conjugates. The delivery strategy of non-covalently "labeling" vitamin E to the siRNA surface through the action of spermine and siRNA is feasible and can achieve effective gene silencing.

[0097] At the same time, q-PCR technology was used to determine the changes in the mRNA level of EG5 gene knockout. The VE-Su-Sper / siRNA complex can significantly reduce the mRNA level ( Fig.10 ). Further, the cell nucleus was stained with Hoechst 33342 dye to observe the changes in the cell nuclear morphology after EG5 gene silencing. Fig.11As shown, the nuclei of the negative control group were normal in shape and regular in shape, while after silencing the EG5 gene by using Lipo to deliver siRNA, the nuclei showed irregular multinuclear morphology. Using VE-Su-Sper to deliver siRNA can also change the nuclei from regular spheres to multinuclear morphology with blocked mitosis. The knockdown of mRNA and the change in nuclear morphology confirmed that the change in the ratio of cells in the G2 / M phase was the result of the target-specific knockdown of the EG5 gene.

[0098] Finally, the concentration dependence of gene silencing by VE-Su-Sper / siRNA complex was studied. Fig.12 ). It was found that with the increase of siRNA concentration, the silencing effect of VE-Su-Sper / siRNA complex increased linearly. When the concentration reached 100nM, the silencing effect reached a level equivalent to that of the positive control group, and the proportion of cells in the G2 / M phase increased to 30%. At a concentration of 100nM, the silencing effect of VE-Su-Sper / siRNA complex reached a platform, and when the concentration continued to rise, its gene silencing efficiency no longer changed significantly. When studying the changes in the silencing efficiency of VE-Su-Sper / siRNA complex gene silencing complex over time, it was found that it showed different characteristics from the Lipo group.

[0099] In addition to delivering siRNA, related vectors can also bind to DNA, mRNA and other double-stranded nucleic acids and achieve efficient delivery. Taking DNA plasmid as an example, VE-Su-Sper vector and mCherry fluorescent protein plasmid were packaged and added to HepG2 cells. After 48 hours, the cells were imaged with selected fluorescence, and the transfection efficiency was observed to be comparable to that of commercial reagents ( Fig.13 ).

Claims

1. A vitamin E-spermine conjugate, It is characterized in that Its general formula is shown in Formula I: Wherein, R is selected from any one of the following (1)-(4): (1)(CH 2 ) n C=O, wherein n is any integer from 1 to 5; (2) C=O(CH 2 ) m C=O, wherein m is any integer from 1 to 6; (3) CH 2 C=ONH(CH 2 CH 2 O) p C=O, wherein p is any integer from 1 to 6; (4) C=O(CH 2 ) q X(CH 2 ) q C=O, wherein q is any integer from 1 to 3, and X is SS or OC(CH 3 ) 2 -O.

2. The method for preparing the vitamin E-spermine conjugate according to claim 1, It is characterized in that include: (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc; (2) reacting Sper-t-Boc with N-hydroxysuccinimide activated ester of vitamin E derivative to obtain Boc-protected vitamin E-spermine conjugate, and removing the Boc protecting group to obtain.

3. The vitamin E-spermine conjugate according to claim 1, It is characterized in that The vitamin E-spermine conjugate is selected from any one of the following formulas II to V:

4. The method for preparing the vitamin E-spermine conjugate according to claim 3, It is characterized in that include: The method for preparing the vitamin E-spermine conjugate of formula II comprises: (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc; (2) After vitamin E is reacted with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and the carboxyl group and the amino group are coupled with Sper-t-Boc through a peptide bond condensation reaction, or through an amide ester reaction mediated by N,N'-disuccinimidyl carbonate or N'N-carbonyldiimidazole, to obtain; The method for preparing the vitamin E-spermine conjugate of formula III comprises: (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc; (2) Vitamin E reacts with succinic anhydride, and the reaction product is coupled with Sper-t-Boc to obtain; The method for preparing the vitamin E-spermine conjugate of formula IV comprises: (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc; (2) using dithioglycolic acid as a raw material, dehydrating it in trifluoroacetic anhydride to obtain a cyclic anhydride containing a disulfide bond, directly coupling it with vitamin E, and finally coupling it with Sper-t-Boc to obtain the vitamin E-spermine conjugate of formula IV; The method for preparing the vitamin E-spermine conjugate of formula V comprises: (1) Preparation of single-reaction site Sper-t-Boc: Protect the three nitrogen atoms of spermine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain a single-reaction site Sper-t-Boc; (2) After vitamin E reacts with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and then the carboxyl group reacts with 1-amino-3,6,9-trioxa-11-undecanol, and the reaction product is coupled with Sper-t-Boc through a peptide bond condensation reaction between the carboxyl group and the amino group to obtain the vitamin E-spermine conjugate described in formula V.

5. The preparation method according to any one of claims 2 or 4, It is characterized in that The method for preparing a single-reaction-site Sper-t-Boc comprises: firstly, at a low temperature of -78°C, adding an equimolar amount of ethyl trifluoroacetate to react with spermine to obtain spermine trifluoroacetylated with a single primary amine group, then adding an excess of Boc anhydride, protecting the remaining two secondary amines and one primary amine with a Boc protecting group, and finally removing the trifluoroacetyl group under alkaline conditions to obtain a single-reaction-site Sper-t-Boc.

6. A vitamin E-triethylenetetramine conjugate, It is characterized in that Its structural formula is shown in VI:

7. The method for preparing the vitamin E-triethylenetetramine conjugate according to claim 6, It is characterized in that include: (1) Preparation of single-reaction site TETeA-t-Boc: Protect the three nitrogen atoms of triethylenetetramine with tert-butyloxycarbonyl, leaving a free primary amine as a coupling site to obtain single-reaction site TETeA-t-Boc; (2) After vitamin E is reacted with ethyl bromoacetate, the ester bond is hydrolyzed into carboxylic acid, and the carboxyl group and the amino group are coupled with TETeA-t-Boc through a peptide bond condensation reaction, or the amide ester reaction mediated by N,N'-disuccinimidyl carbonate or N'N-carbonyldiimidazole is coupled with TETeA-t-Boc to obtain the product.

8. Use of the vitamin E-spermine conjugate according to claim 1 or 3 in the preparation of delivery functional nucleic acids or gene drugs.

9. Use of the vitamin E-triethylenetetramine conjugate according to claim 6 in the preparation of functional nucleic acid or gene medicine for delivery.

10. A gene therapy drug comprising a target gene and a gene delivery vector, It is characterized in that The gene delivery vector is the vitamin E-spermine conjugate according to claim 1 or the vitamin E-triethylenetetramine conjugate according to claim 6.

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