CN108478807B - Nucleic acid drug delivery system and application thereof - Google Patents

Abstract

The invention discloses a nucleic acid drug delivery system and application thereof. The system consists of a neutral base lipid carrier and a metal salt, whereinThe structure of the neutral base lipid carrier is shown as a formula I. In addition, the delivery system also comprises metal salt, and experiments prove that the existence of the metal ions can effectively improve the silencing activity of the nucleic acid medicament and realize the effective transportation of the nucleic acid medicament in cells and in vivo. The invention uses chemical modification methods such as D, L-isonucleoside modification, deoxyinosine modification, peptide conjugation modification and phosphorylation modification and the like in the research of a nucleic acid delivery system, fully discovers the advantages and rules of a composite modification mode on nucleic acid delivery, and uses the modification strategy in the research of nucleic acid drugs. Researches show that the product obtained by the chemical modification is more suitable for the system and has the advantages of stable physicochemical property, good bioactivity, good membrane permeability and the like. The nucleic acid drug delivery system has wide application prospect in the field of gene therapy.

Description

Nucleic acid drug delivery system and application thereof

Technical Field

The invention relates to a nucleic acid delivery system and also relates to application of the nucleic acid delivery system in nucleic acid molecule delivery. The invention belongs to the field of biological medicine.

Background

Gene therapy provides a new treatment mode for various acquired and hereditary diseases, only used for treating single-gene hereditary diseases at early stage, and the treatment range is expanded to polygenic diseases seriously threatening human health, including cardiovascular diseases, hereditary diseases, malignant tumors, metabolic diseases, infectious diseases (such as hepatitis b and AIDS) and the like. Gene therapy can be classified into in vitro gene therapy and in vivo gene therapy depending on the introduction mode. In vitro gene therapy refers to the process of obtaining certain cells from a patient body for culture, screening and amplifying in vitro, and then inputting the cells into the patient body again. The in vivo therapeutic method is to introduce exogenous target gene directly into organism via specific vector, and the used gene medicine may be gene segment (including RNA or DNA) or complete gene for large scale production. Gene (nucleic acid) drugs for in vivo gene therapy have to meet the following requirements: (1) high specificity and affinity to the target sequence; (2) has high stability in vivo, and can resist degradation of various nucleases; (3) has the ability to penetrate the cell membrane to reach the target site. Since natural genes (nucleic acids) cannot meet the requirements, structural modification needs to be carried out on the natural genes by certain technology, and the natural genes (nucleic acids) are introduced into biological cells by using a nucleic acid delivery vector, and the biocompatibility and high efficiency of a nucleic acid drug delivery system are often key factors for success and failure of treatment. The requirement is difficult to be met by single-technology modified nucleic acid drugs, and the synergistic effect of multiple modes such as chemical modification, carrier entrapment and the like is an effective solution.

In the field of biological research, plasmid DNA is an important tool for modern biological research, plays an increasingly important role in the research of genomes and functions thereof, and is also a tool molecule in gene therapy research. Achieving efficient delivery of plasmid DNA is critical to its function.

Nucleic acid vectors can be divided into viral vectors and non-viral vectors. The viral vector method has high transfection efficiency, but has the defects of difficult preparation, cytotoxicity, immunogenicity, variability, lack of target cell positioning and the like, and limits the wide application of the viral vector method. The non-viral delivery method mostly uses artificially synthesized vectors, has the advantages of simple preparation, controllable performance, low toxicity, low immunity and the like, and has great application potential in the field of gene therapy.

The cationic liposome is a non-viral gene vector which is most widely applied, generally comprises a cationic head, an aliphatic tail and a connecting arm, and can effectively entrap nucleic acid by combining the cationic head, the aliphatic tail and the connecting arm with the nucleic acid through the coulomb force action between positive charges and negative charges. However, the cationic liposome has many limiting factors to be solved, such as strong cytotoxicity and serum protein binding capacity, strong immunogenicity and liver accumulation; in addition, the stronger electric property makes the combination with nucleic acid more compact, and the effective release after transmembrane is difficult (Biomaterials 2008,29,3477-3496), and these defects greatly limit the further application of cationic liposome in clinical research. The present inventors previously designed and synthesized Cationic Liposomes (CLD) which can stably transfect and exert effective biological activity at a proper ratio of liposome and nucleic acid, but have a certain cytotoxicity at a high concentration.

The invention constructs basic acetamide glyceryl ether molecules, and at the early stage, basic acetic acid glyceryl ether molecules (Chinese patent 201310006506.9) which have basic property heads and can realize effective entrapment of nucleic acid through hydrogen bond action and electron cloud pi-pi accumulation action, and in addition, the entrapment method is optimized, certain metal ions are added, and a chemical modification method is combined, so that a high-efficiency and low-toxicity neutral nucleic acid drug entrapment delivery system is obtained.

Disclosure of Invention

The invention aims to provide a high-efficiency and low-toxicity nucleic acid drug delivery system and application thereof in delivering nucleic acid drugs.

In order to achieve the purpose, the invention adopts the following technical means:

the invention relates to a nucleic acid delivery system, which consists of a neutral base lipid carrier and a metal salt, wherein the structural formula of the neutral base lipid carrier is shown as a formula I:

wherein X is an oxygen atom or a nitrogen atom, and B is a natural purine or pyrimidine base, i.e., adenine, guanine, hypoxanthine, cytosine, thymine and uracil, preferably a cytosine-1-yl group or a thymine-1-yl group.

Among them, preferred is the aliphatic long chain-C contained in formula I₁₈H₃₅Is structured as

Preferably, the neutral base lipid carrier has a structure shown in the following formula:

wherein, preferably, the metal salt is calcium salt or manganese salt, preferably CaCl₂。

Furthermore, the invention also provides the application of the nucleic acid delivery system in preparing the nucleic acid carrying reagent.

Wherein, preferably, the nucleic acid comprises oligonucleotide, aptamer, siRNA and plasmid DNA.

Preferably, the nucleic acid is a nucleic acid analogue modified by at least one of D, L-isonucleoside modification, deoxyinosine modification, peptide conjugation modification and phosphorylation modification.

Compared with the prior art, the invention has the beneficial effects that:

1. the nucleic acid delivery system consists of a neutral base lipid carrier and metal salt, wherein the neutral base lipid carrier has a base property head, and forms supermolecule combination and liposome by utilizing hydrogen bond and pi-pi action between a nucleoside base and a nucleic acid base of the carrier, so that the surface of a nanoparticle is prevented from generating cations, and the transfection of nucleic acid into cells is realized. In addition, metal ions are added into the delivery system, and experiments prove that the existence of the metal ions can effectively improve the silencing activity of the nucleic acid medicament and realize the effective intracellular transport and in vivo transport of the nucleic acid medicament.

2. The invention uses the chemical modification methods of nucleic acid such as D, L-isonucleoside modification, deoxyinosine modification, peptide conjugation modification, phosphorylation modification and the like in the research of a nucleic acid delivery system, fully discovers the advantages and rules of a composite modification mode for nucleic acid delivery, and uses the modification strategy in the research of nucleic acid drugs. The product obtained by the chemical modification method has the advantages of stable physicochemical property, good biological activity, good membrane permeability and the like, and can be widely used in research of nucleic acid drugs. The delivery system can also be used for plasmid DNA, thereby having wide application prospect in the field of gene research.

Drawings

FIG. 1 is a scheme of the synthesis of the base acetamide glycerol ether molecule DNTA;

FIG. 2 is a scheme of the synthesis of the base acetamide glycerol ether molecule DNCA;

FIG. 3 shows scanning electron microscope observations of DNTA (c, d) and DNCA (a, b) liposomes;

FIG. 4 shows CD spectra before and after annealing of Cenersen (20bp, 4. mu.M), a mixture of DOCA and Cenersen (base ratio 1: 1);

FIG. 5 shows CD spectra before and after annealing of Cenersen (22bp, 4. mu.M), DNCA and a mixture of Cenersen (base ratio 5: 1);

FIG. 6 shows the examination of antiproliferative activity of G3139 encapsulated in a basic lipid carrier;

(A) a549 cells; (B) A549/TXL cells (DNXA: 7.5. mu.M); (C) effect of NC and DNCA-loaded G3139 on cell viability of A549/TXL cells (AONs: 400 nM);

FIG. 7 shows the efficiency of DNCA on ss/ds-miR-122 entrapment by acrylamide gel electrophoresis (20 pmol/well for FAM-RNA samples; DNCA 0.44nmol (N/P1: 1));

FIG. 8 is a graph showing the intracellular stability of DNCA-entrapped conjugated RNA through acrylamide gel electrophoresis;

RNA samples (20pmol) were added to freshly prepared cell lysates (300 cells/. mu.L, 1. mu.L) and incubated at 37 ℃ for various times; N/P is 5: 1;

FIG. 9 shows the cellular uptake capacity of DNCA, lipo encapsulating peptide conjugated ss/dsRNA;

(A) the entry of unmodified RNAs into the cell; (B) peptide-conjugated modified RNAs entry;

FIG. 10 shows that cck-8 detects the cell proliferation inhibition effect of DNCA and lipo encapsulating peptide conjugated ss/dsRNA;

(A) cytotoxicity of nanoparticles to HEK 293A; (B) proliferation inhibitory activity of nanoparticles on HepG 2.

FIG. 11 is a gel electrophoresis investigation of the encapsulation efficiency of DNCA-entrapped single-stranded DNA;

N/P-0 represents pure nucleic acid sequence, no DNCA vector;

fig. 12 shows Tm values after annealing AS1411(26bp,4 μ M), DNTA, and AS1411 mixture (base ratio 3: 1);

FIG. 13A is a cell viability assay of DNTA/DOTA/DNCA/DOCA on MCF-7 cells for 72 hours;

FIG. 13B is the antiproliferative activity of AS1411 (entrapped by DNTA/DOTA/DNCA/DOCA) on A549, MCF-7 and K562 cells;

cell viability was determined 48 hours after addition of AS1411 using CCK-8 assay: a set of three different experiments (each replicated three times) was performed and each value was expressed AS the mean. + -. standard deviation (1: control; 2: 26. mu.M DNCA; 3: 200nM AS 1411; 4: 200nM AS1411+ 26. mu.M DNTA; 5: 200nM AS1411+ 26. mu.M DNOTA; 6: 200nM AS1411+ 26. mu.M DNCA; 7: 200nM AS1411+ 26. mu.M DOCA);

fig. 13C is the uptake of FAM-labeled AS1411 in a549 cells;

FIG. 14 shows the effect of ion and amino acid components in GenOpti on inhibiting the proliferation of A549 and A549/TXL cells;

solvent: PBS; the components are as follows: 50 mu M; AS1411 (a): 200 nM; dnca (d): 15 mu M; 0.2g/L of Ca-2 (C); mn-2:9.9 x 10^-8g/L；

FIG. 15 shows CaCl₂The transfection concentration and the annealing temperature have the inhibition effect on the proliferation of A549/TXL cells;

FIG. 16 shows the transfection efficiency (N ═ 3) of the base lipid vector transfected pEGFP-N1 plasmid (+ indicating serum-containing transfection for 24h and-indicating serum supplementation after 4h of serum-free transfection);

figure 17 is a toxicity study of the base lipid vector for transfection of the pMB3 plasmid (48h, n-2).

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Synthesis of lipid Carriers with first partial bases

Example 1 Synthesis of base acetic acid glyceryl Ether ester molecule DOTA and DOCA

The basic lipid carriers DOTA and DOCA are synthesized according to the method described in Chinese patent application with the patent application number of CN201310006506.9 and the invention name of 'a basic glycerol ether acetate molecule, a chemical synthesis method and the application thereof in the field of gene therapy'.

Example 2 Synthesis of base acetamide Glycerol Ether ester molecule DNTA and DNCA

(1) Synthesis of oleyl alcohol methylsulfonyl ester

Oleyl alcohol (50g, 85% purity,158mmol), Et₃N (40mL,286mmol) was added to a 1L round bottom flask, DCM (500mL) was added, and the mixture was stirred well on an ice bath to bring the temperature down to 0 ℃. Methanesulfonyl chloride (16mL,206mmol) was added slowly via syringe and the solution became cloudy. The ice bath was then removed and the reaction was allowed to slowly return to room temperature and stirring was continued for 12 h. Water (250mL) was added to quench the reaction, and the organic phase was separated by a separatory funnel. The aqueous phase was back-extracted with DCM (250 mL. times.2) and the organic phases were combined. The combined organic phases were treated successively with 1N hydrochloric acid (250mL), 10% NaHCO₃Washed with aqueous solution (250mL) and saturated brine (250mL) anhydrous Na₂SO₄And (5) drying. The organic phase is evaporated to dryness under reduced pressure and the residue is separated by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 20/1, R)_f0.3) to yield 44.3g of a pale yellow oily liquid in 81% yield.¹H NMR(400MHz,CDCl₃):δ＝5.30-5.43(m,2H),4.22(t,J＝6.6Hz,2H),3.00(s,3H),1.90-2.10(m,4H),1.70-1.80(m,2H),1.20-1.40(m,22H),0.88(t,J＝6.8Hz,3H)；¹³C NMR(100MHz,CDCl₃):δ＝130.2,129.9,70.3,37.5,32.0,29.90,29.83,29.66,29.46,29.29,29.26,29.15,27.36,27.30,25.6,22.8,14.3；IR(neat)ν＝2925.5,2854.5,1463.6,1355.9,1175.4,974.8,947.8,831.7,721.6,528.8；MS(ESI-TOF⁺)for C₁₉H₃₈O₃SNa[M+Na]⁺found369.2315,calcd369.2434；Anal.calcdfor C₁₉H₃₈O₃S:C 65.85,H 11.05,Found:C 65.63,H 10.98.

(2) Synthesis of 1-triphenylmethoxy glycerol

Glycerol (40g,435mmol), triphenylchloromethane (30g,107mmol), DMAP (300mg,2.46mmol) were placed in a dry 500mL round bottom flask, THF (80mL) and Et were added₃N (18mL), stirred at room temperature for 12 h. Water (100mL) was added to the reaction solution to quench the reaction, which was then diluted with ethyl acetate (150 mL). After sufficient shaking, the mixture was transferred to a separatory funnel and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (100 mL. times.2), and then the organic phases were combined. The combined organic phases were successively saturated with NaHCO₃Washed with aqueous solution (200mL), water (200mL) and saturated brine (200mL) and anhydrous Na₂SO₄And (5) drying. After filtration, the solvent was evaporated to dryness to give a yellow oil. This was dissolved in toluene/n-hexane (200mL, v/v. 1/1), and left at room temperature for 24h to crystallize 29g of a white solid with a yield of 85%. 1H NMR (400MHz, CDCl)₃):δ＝7.38-7.48(m,6H),7.20-7.35(m,9H),3.84(s,1H),3.63-3.71(m,1H),3.53-3.63(m,1H),3.20-3.28(m,2H),2.74(brs,1H),2.35(brs,1H)；13C NMR(100MHz,CDCl₃):δ＝143·8，128·7，128·0，127·3，87·1，71·3，65·1，64·4；IR(film，KBr)＝3380·8，3058·1，2920.0,2866.8,1490.0,1447.8,1081.5,1028.5,699.8；MS(EI)for C₂₂H₂₂O₃[M]⁺found 334.5,calcd 334.2；Anal.calcd for C₂₂H₂₂O₃:C 79.02,H 6.63,Found:C 79.26,H 6.49.

(3) Synthesis of 1-triphenylmethyl-2, 3-dioleyl ether-glycerol

1-Triphenylmethoxy-glycerol-2, 3-diol (8g,23.1mmol), KOH (3.3g,58.9mmol) and oleyl p-methylsulfonyl ester (19.2g,55.42mmol) were mixed and dissolved in a dry solution of benzene (150mL), equipped with a water separator, heated to 80 ℃ and refluxed for 32 hours. Thereafter, 100mL of ethyl acetate and 150mL of water were added thereto, followed by extraction and separation of the organic phase. Aqueous phase with ethyl acetate(150 mL. times.3) and the combined organic phases were extracted with anhydrous Na₂SO₄Drying, evaporating the solvent, and separating by reduced pressure silica gel column chromatography to obtain target product 6.1g with a yield of 31%. In addition, 3.7g of 1-triphenylmethoxy-3-oleyl alcohol ether-glycerol-2-ol was obtained, the yield was 27%. The target product was a light yellow liquid.¹H NMR(400MHz,CDCl₃):δ＝7.40-7.50(m,6H),7.18-7.32(m,9H),5.26-5.43(m,4H),3.50-3.60(m,5H),3.35-3.45(m,2H),3.12-3.20(m,2H),1.92-2.08(m,8H),1.50-1.58(m,4H),1.26(brs,44H),0.88(t,J＝6.6Hz,6H)；¹³C NMR(100MHz,CDCl₃):δ＝144.31,130.07,130.00,128.90,127.85,127.02,86.64,78.45,71.76,71.33,70.84,63.73,32.77,32.06,30.28,29.94,29.93,29.85,29.82,29.72,29.68,29.65,29.47,27.37,27.06,26.31,26.25,22.84,14.27；IR(film,KBr)ν＝3004.4,2925.3,2854.1,1742.6,1597.7,1490.7,1450.0,1220.6,1118.7,763.9,745.0,704.1,632.8cm^-1；MS(ESI-TOF+)for C₅₈H₉₀O₃Na[M+Na]⁺found 857.9059,calcd 857.6782；Anal.calcd for C₅₈H₉₀O₃:C 83.39,H 10.86,Found:C 83.10,H 10.62.

(4) Synthesis of 1, 2-dioleoyl ether-glycerol-3-ol

1-triphenylmethyl-2, 3-dioleyl ether-glycerol (8.34g,10mmol) was suspended in a methanol-tetrahydrofuran (100mL, v/v ═ 1/1) mixed solution, and concentrated hydrochloric acid (2mL,12M) was added thereto, and the mixture was stirred at room temperature for 2 hours. TLC detection found that the starting material had reacted completely. The solvent was evaporated under reduced pressure, ethyl acetate (50mL) and water (100mL) were added to the residue, and the organic phase was separated after extraction. The aqueous phase was extracted with ethyl acetate (3X 100mL), and the organic phases were combined, anhydrous Na₂SO₄And (5) drying. Filtering to remove desiccant, evaporating solvent under reduced pressure, and separating with silica gel column chromatography (eluent: petroleum ether/ethyl acetate 20/1, R)_f0.2), the objective product was obtained in 3.7g with a yield of 62%. Light yellow oily liquid.¹H NMR(400MHz,CDCl₃):δ＝5.30-5.45(m,4H),3.40-3.75(m,9H),2.18(s,1H),1.90-2.10(m,8H),1.55-1.65(m,4H),1.25-1.40(brs,44H),0.88(t,J＝6.4Hz,6H)；¹³C NMR(100MHz,CDCl₃):δ＝130.10,129.97,78.39,72.00,71.07,70.54,63.27,32.06,30.23,29.92,29.85,29.81,29.77,29.67,29.65,29.60,29.47,29.41,27.36,26.25,22.83,14.25；IR(film,KBr)ν＝3470.1,3004.4,2925.4,2854.0,1651.2,1463.2,1376.2,1117.5,1041.3,968.0,721.9cm^-1；MS(ESI-TOF+)for C₃₉H₇₆O₃Na[M+Na]⁺found 615.7213,calcd 615.5687；Anal.calcd for C₃₉H₇₆O₃:C 78.99,H 12.92,Found:C 78.72,H 12.68.

(5)1, 2-dioleoyl alcohol ether-3-glycerol methanesulfonate

Triethylamine (0.54mL, 3.91mmol) and 1, 2-dioleoyl ether-glycerol-3-ol (1.932g, 3.26mmol) were taken in a 25mL single-neck flask, dried dichloromethane (5mL) was added, the mixture was dissolved by magnetic stirring, the reaction system was cooled to 0 ℃ by an ice-water bath, methanesulfonyl chloride (0.3mL,3.91mmol) was added dropwise, and the reaction was continued at 0 ℃ for 2 hours. The reaction was added to saturated NaHCO₃(50mL) of the solution, the organic phase was separated and the aqueous phase was treated with CH₂Cl₂(2X 50mL), and the combined organic phases are dried over anhydrous Na₂SO₄Dry overnight. The drying agent was removed by filtration, the solvent was evaporated under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: petroleum ether/methanol: 120/1) to obtain the objective product 2.02mg (yield 92.6%).¹H NMR(400MHz,CDCl₃)δ5.37(s,4H),4.40(d,J＝10.9Hz,1H),4.27(d,J＝10.7,5.8Hz,1H),3.54(d,J＝29.7,27.4,20.6Hz,8H),3.06(s,3H),2.03(d,J＝5.2Hz,7H),1.58(s,4H),1.29(d,J＝10.6Hz,44H),0.90(t,J＝6.1Hz,6H).¹³C NMR(101MHz,CDCl₃)δ127.93,127.27,77.34,77.22,77.02,76.70,76.38,71.90,70.83,69.08,37.40,32.62,31.92,29.95,29.78,29.71,29.59,29.54,29.52,29.33,29.28,27.23,26.08,26.02,22.70,14.13.MS(EI)for C₄₀H₇₈O₅S[M+Na]⁺found 693.36,calcd 693.55；

(6)1, 2-dioleoyl ether-glycerol-3-azido

Taking dry compound 1, 2-dioleyl ether-glycerol-3-methanesulfonate (970mg, 1.44mmol) in a 25mL single-neck flask, adding dry DMF (8mL) solution, magnetically stirring to dissolve, adding NaN₃(188mg, 2.88mmol) under argon and the reaction was warmed to 70 ℃ for 15 hours. Cooling the reaction solution to the chamberAfter diluting with acetone (30mL), the reaction mixture was filtered through celite, the filtrate was evaporated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 200/1) to obtain 345.5mg (yield 56%) of the objective product as a colorless oil.¹H NMR(400MHz,CDCl₃)δ5.37(s,4H),3.60–3.36(m,9H),2.31(s,1H),2.07–1.95(m,8H),1.59(d,J＝8.3Hz,4H),1.30(d,J＝10.8Hz,44H),0.90(s,6H).¹³C NMR(101MHz,CDCl₃)δ129.89(d,J＝10.6Hz),77.90(s),77.45–76.95(m),76.95–76.86(m),76.70(s),71.79(s),70.66(s),70.11(s),52.08(s),32.62(s),31.92(s),30.38–29.54(m),29.37(t,J＝11.7Hz),27.22(s),26.07(d,J＝7.5Hz),22.70(s),14.13(s).MS(EI)for C₃₉H₇₅N₃O₂[M+Na]⁺found 640.49,calcd 640.58.

(7)1, 2-dioleoyl ether-glycerol-3-amine

Adding 1, 2-dioleoylether-glycerol-3-azide (309mg,0.5mmol) into dry THF (10ml), magnetically stirring to dissolve, cooling the solution to 0 deg.C, adding LiAlH₄(95mg,2.5mmol) under the protection of argon, reacting for 45min, heating the solution to room temperature, continuing to react for 2.5h, adding saturated Na₂SO₄The reaction was quenched with a solution (0.7mL), the reaction mixture was filtered through celite, the filtrate was evaporated to dryness under reduced pressure, and the residue was subjected to column chromatography on a silica gel column (eluent: dichloromethane/methanol-50/1) to obtain 233.5mg of the objective product (yield 79%). A light yellow oil.

¹H NMR(400MHz,CDCl₃):δ＝5.30-5.45(m,4H),3.40-3.75(m,9H),2.18(s,1H),1.90-2.10(m,8H),1.55-1.65(m,4H),1.25-1.40(brs,44H),0.88(t,J＝6.4Hz,6H)；¹³C NMR(100MHz,CDCl₃):δ＝130.10,129.97,78.39,72.00,71.07,70.54,63.27,32.06,30.23,29.92,29.85,29.81,29.77,29.67,29.65,29.60,29.47,29.41,27.36,26.25,22.83,14.25；MS(ESI-TOF+)for C₃₉H₇₇NO₂[M-H]^-found 590.75,calcd 591.60；

(8) Synthesis of (thymin-1-yl) -acetic acid

Thymidine (10.0g,79.3mmol) was suspended in H₂O (150mL), to which was added an aqueous KOH solution (50 m)L, 3.6M). After the mixture was stirred at room temperature for 10min, the solution gradually became clear. Chloroacetic acid (15.0g,159mmol) was then added and the reaction heated to reflux for 90 min. After cooling the reaction mixture to room temperature, it was acidified with concentrated hydrochloric acid to pH 3 and then left overnight at 4 ℃ to precipitate a white crystalline precipitate. Filtration gave the white crystalline precipitate, P₂O₅Drying in vacuum gave 4.5g of the desired product (31% yield).¹H NMR(400MHz,DMSO-d₆):δ＝13.11(s,1H),11.34(s,1H),7.50(s,1H),4.37(s,2H),1.75(s,3H)；¹³C NMR(100MHz,DMSO-d₆):δ＝169.6,164.4,151.0,141.8,108.4,48.4,11.9；IR(film,KBr)ν＝3180.2,3076.2,3027.0,2962.3,2835.8,1737.7,1708.4,1664.8,1631.9,1418.3,1356.3,1201.7,1147.0,829.8,566.9cm^-1；MS(EI):m/z(％):184.1(39)[M⁺],95.9(100)；Anal.Calcd for C₇H₈N₂O₄:C 45.66,H 4.38,N 15.21,Found:C 45.59,H 4.40,N 15.25.

(9) Synthesis of (thymin-1-yl) -acetyl- (N-hydroxysuccinimide) -ester

To a dry 25mL eggplant-shaped bottle were added (thymin-1-yl) -acetic acid (3g,16.3mmol) and dry DMF (30mL), and the mixture was stirred to completely dissolve the compound. N-hydroxysuccinimide (2.38g,21mmol) and N, N' -dicyclohexylcarbodiimide (DCC,3.36g,16.3mmol) were then added thereto. Stirring overnight at room temperature precipitated a large amount of white precipitate. The precipitate was removed by filtration, the filtrate was distilled under reduced pressure, and the residue was redissolved in DMF (5 mL). Anhydrous ether (30mL) was added to the reaction solution to precipitate a white solid. The solid was obtained by filtration and dried in vacuo to obtain 4.6g (yield 61%) of the objective product. A white solid.¹H NMR(400MHz,DMSO-d₆):δ＝11.52(s,1H),7.63(s,1H),4.96(s,2H),2.83(brs,4H),1.77(s,3H)；¹³C NMR(100MHz,DMSO-d₆):δ＝169.8,165.0,164.2,150.7,140.8,109.3,46.4,25.5,11.9；IR(film,KBr)ν＝3154.4,3003.4,2830.5,1827.4,1785.4,1739.8,1697.2,1467.8,1422.8,1382.8,1358.6,1213.7,1106.8,1065.1,793.9,651.3cm^-1；MS(ESI-TOF+)for C₁₁H₁₁N₃O₆Na[M+Na]⁺found 304.0489,calcd 304.0540；Anal.Calcd for C₁₁H₁₁N₃O₆:C 46.98,H 3.94,N 14.94,Found:C 46.75,H 3.96,N 14.95.

(10) Synthesis of 1, 2-di (oleyl) -glycero-3-amine- (thymin-1-yl) -acetyl ester (DNTA)

The synthetic route for DNTA is shown in figure 1.

(thymin-1-yl) -acetyl- (N-hydroxysuccinimide) -ester (280mg,1.0mmol), 1,2, -di (oleyl) -glycero-3-amine (709mg,1.2mmol), DMPA (14.6mg,0.1mmol), pyridine (0.4mL) and anhydrous DMF (20mL) were mixed under argon and stirred at room temperature overnight. Ethyl acetate (200mL) was added to dilute, transferred to a separatory funnel, and diluted with dilute hydrochloric acid (0.1M), saturated NaHCO in that order₃The aqueous solution, water and saturated brine were washed with water, and the organic phase was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, the filtrate was evaporated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: dichloromethane/methanol-50/1) to obtain 537.5mg of the objective product (yield 71%). A light yellow oil.¹H NMR(400MHz,CDCl₃)δ＝8.12(s,1H),7.09(s,1H),6.43(s,1H),5.38(d,J＝18.8Hz,4H),4.31(s,2H),3.58(s,1H),3.52-3.42(m,6H),2.05-1.94(m,10H),1.60(s,4H),1.29(d,J＝11.7Hz,45H),0.89(d,J＝6.7Hz,6H).(ESI-MS)for C₄₆H₈₃N₃O₅[M-H]^-found756.61,calcd.757.23.¹³C NMR(100MHz,CDCl₃):δ＝167.54,163.69,150.61,140.18,130.13,129.98,111.38,76.35,72.03,70.89,69.97,65.77,48.60,32.06,29.93,29.78,29.67,29.61,29.48,27.37,26.19,14.25,12.45；(ESI-MS)for C₄₆H₈₃N₃O₅[M-H]^-found 756.61,calcd.757.23.

(11) Synthesis of (4-N- (benzhydryloxycarbonyl) -cytosine) -1-acetic acid

Cytosine (10.3g,90mmol,1.0eq) was dissolved in DMF (90mL), potassium tert-butoxide (11.6g,103.5mmol,1.15eq) was added, and the reaction was heated to 100 ℃ for 2 hours. The reaction system was cooled to 10 ℃ and benzyl 2-bromoacetate (16.05mL,101mmol,1.12eq) was added dropwise over 30 minutes, after the addition was complete, the reaction system was warmed to room temperature and allowed to continue reacting for 12 hours, and acetic acid (5.9mL,103.5mmol, 1) was added.2eq) quench the reaction and spin dry the reaction. The residue is resuspended in H₂O (100mL), stirred for 4 hours and filtered, H₂O (4X 150mL) and dried to obtain 20.6g of cytosine-1-benzylacetic acid. Cytosine-1-benzylacetic acid (20.6g,82mmol,1.0eq) was dissolved in DMF (160mL) and N, N' -carbonyldiimidazole (21.25g,131.25mmol,1.6eq) was added. After completion of the TLC detection reaction, methanol was added, stirring was continued for 1.5 hours, and benzhydrol (19.65g,106.5mmol,1.3eq) was added. The reaction was heated to 60 ℃ and diphenylmethanol (2X1.825g,9.9mmol,0.12eq) was added in two batches at 1-hour intervals and the reaction was continued for 6 hours. The heating was stopped for 12 hours and the reaction was quenched by addition of methanol (4.65g,115mmol,1.4 eq). The reaction was spun dry and the residue was further recrystallized from ethanol, followed by methanol (100mL) to give 29.35g of (4-N- (benzhydryloxycarbonyl) -cytosine) -1-acetate. (4-N- (Benzyloxycarbonyl) -cytosine) -1-acetate (29.35g,62.5mmol,1.0eq) was dissolved in acetonitrile MeOH H₂EtOH (2:2:1, 350mL) was added to the mixed solution system, the mixture was heated to dissolve the compound, and then the temperature was reduced to 0 ℃ to add an aqueous solution (196.8mL) of LiOH.H2O (25.5g,0.61mol,9.7 eq). After completion of the reaction by TLC, the reaction was quenched by addition of an aqueous solution (290mL) of citric acid (58.5g,303.5,4.9 eq). Thus, 22.1g of (4-N- (benzhydryloxycarbonyl) -cytosine) -1-acetic acid was obtained.¹H NMR(400MHz,D6-DMSO,25℃)δ8.03-8.01(d,J＝7.5,1H,C₆),7.46(d,J＝7.5,4H,Ph),7.38(t,J＝7.5,4H,Ph),7.3(d,J＝7.5,2H,Ph),6.96(d,J＝7.5,1H,C₅),6.82(s,1H,CH-(C₆H₅)₂),4.50(s,2H,N-CH₂-CO)；¹³C NMR(100MHz,D₆-DMSO,25℃)δ169.9,163.5,155.5,152.8,151.1,140.8,129.0,128.3,126.9,94.2,71.9,51.3；HRMS(ESI)calculated for C₂₀H₁₇N₃O₅:(M+Na⁺):402.1060,found:402.1010.

(12) Synthesis of 1, 2-di (oleyl) -glycero-3-amine- (cytosin-1-yl) -acetyl ester (DNCA)

The synthetic route for DNCA is shown in fig. 2.

To a dry 25mL eggplant-shaped bottle was added (4-N- (benzhydryloxycarbonyl) -cytosine) -1-acetic acid (3.29g,10mmol) and dry DMF (30mL)And stirring to completely dissolve the mixture. N-hydroxysuccinimide (14.73g,13mmol) and N, N' -dicyclohexylcarbodiimide (DCC,2.06g,10mmol) were then added thereto. Stirring overnight at room temperature precipitated a large amount of white precipitate. The precipitate was removed by filtration, the filtrate was distilled under reduced pressure, and the residue was redissolved in DMF (5 mL). Anhydrous ether (30mL) was added to the reaction solution to precipitate a white solid. The solid was filtered and dried in vacuo to give the desired product 2.62g (55% yield). A white solid. (4-N- (Benzyloxycarbonyl) -cytosine) - (N-hydroxysuccinimide) -ester (476mg,1.0mmol), 1,2, -di (oleyl) -glycero-3-amine (910mg,1.2mmol), DMPA (14.6mg,0.1mmol), pyridine (0.4mL) and anhydrous DMF (20mL) were mixed under argon and stirred at room temperature overnight. Ethyl acetate (200mL) was added to dilute, transferred to a separatory funnel, and diluted with dilute hydrochloric acid (0.1M), saturated NaHCO in that order₃The aqueous solution, water and saturated brine were washed with water, and the organic phase was dried over anhydrous sodium sulfate. The drying agent was removed by filtration, the filtrate was evaporated to dryness under reduced pressure, and the residue was separated by silica gel column chromatography (eluent: dichloromethane/methanol: 50/1) to obtain 485.5mg of the objective product (yield 51%). 1, 2-bis (oleyl) -glycero-3-amine- (4-N- (benzhydryloxycarbonyl) -cytosine) -acetyl ester (476mg,0.5mmol) was dissolved in CH with 5% TFA₂Cl₂(20mL), the mixture was stirred at room temperature for 0.5 hour. DCM (30mL) and water (30mL) were added to extract the layers, the aqueous phase was back-extracted with DCM (2 × 30mL) and the organic phases were combined. The organic phase was successively treated with 10% NaHCO₃(200mL), water (200mL) and saturated brine (200mL), and dried over anhydrous sodium sulfate overnight. The organic solvent was evaporated under reduced pressure and separated by silica gel chromatography (eluent: dichloromethane/methanol-50/1) to give 345mg (yield 93%) of the desired product DNCA:¹H NMR(400MHz,CDCl₃)δ＝7.44-7.29(m,4H),5.80(d,J＝3.8Hz,1H),5.37(dd,J＝12.0,7.4Hz,4H),4.45(s,2H),3.59-3.40(m,7H),2.11-1.91(m,8H),1.55(s,4H),1.44-1.10(m,45H),0.90(t,J＝6.4Hz,6H).(ESI-MS)for C₄₅H₈₂N₄O₄[M+H]⁺found 743.50,[M+Na]⁺found 756.44.calcd.742.21.

example 3 Liposome preparation of the base acetamide Glycerol Ether

The basic group acetamide glycerol ether molecule has an amphiphilic structure and can be prepared into supermolecular structures such as liposome and the like. Taking DNTA as an example, the preparation method of the liposome comprises the following steps: DNTA (1.18mg, 1.56. mu. mol) was dissolved in methanol (1mL) and vortexed to dissolve it thoroughly, after centrifugation, DNTA in methanol (12.8. mu.L) was added to a 200. mu.L centrifuge tube, PBS (100. mu.L) was added and vortexed (10s) to mix thoroughly. It was then subjected to sequence annealing in PCR. The procedure is as follows: heating at 95 deg.C for 10min, cooling to 5 deg.C every 5min until 15 deg.C, and storing at 4 deg.C. After annealing, incubating at 40 deg.C for 30min until methanol is completely volatilized, and obtaining liposome solution.

Particle size and morphology:

under a projection electron microscope (TEM), it can be seen that neutral base lipid carrier molecules DNCA and DNTA can form stable and uniform nanoparticles in an aqueous solution, the particle size of liposomes formed by the self-assembly of DNTA molecules in water is about 150nm, and the particle size of liposomes formed by the self-assembly of DNCA carrier molecules in water is about 180nm (FIG. 3).

Example 4 Liposome preparation of the bases Glycerol acetate Ether

The molecular molecule of acetic acid glycerol ether ester, the chemical synthesis method and the application thereof in the field of gene therapy are prepared according to the method described in Chinese patent application with the patent application number of CN201310006506.9 and the invention name of the molecular molecule.

Application of second part base lipid carrier in nucleic acid drug delivery

Example 5 CD Spectroscopy study of binding of base lipid Carriers to nucleic acids

The present invention investigated the binding of nucleobase lipid vectors DOCA, DNCA to antisense nucleic acid Cenersen (22bp,4 μ M) which is an antisense nucleic acid sequence containing 22 bases targeting p53mRNA exon 10 using Circular Dichroism (CD) spectroscopy. Respectively mixing base lipid carriers DOCA and DNCA with Cenersn, and annealing to obtain the compound. The annealing was performed by using a PCR instrument, and the procedure was the same as that for amplifying the nucleic acid double strand. FIGS. 4 and 5 show the CD spectra of the oligonucleotides in aqueous solution. From the results, it can be seen that the CD spectrum did not change significantly when the base lipid carrier was mixed with it. But there is some change in the CD spectrum after annealing.

EXAMPLE 6 transfection efficiency examination of oligonucleotide G3139 Encapsulated with a base lipid Carrier

The invention considers the activity effect of G3139 carried by synthetic neutral base lipid carriers DNCA, DOCA, DNTA and DOTA, wherein G3139 is a full-thio modified antisense nucleic acid containing 18 bases, which is complementary with the first six codons of Bcl-2mRNA, can effectively target the drug-resistant gene Bcl-2 and inhibit the growth of tumors.

1. Method of producing a composite material

Using DNTA and DNCA as examples and FAM-labeled G3139 as a template, the nucleic acid transfection ability of the thymine base acetamidoglyceryl ether was investigated. The specific coating and transfection processes are as follows:

DNTA or DNCA liposome and FAM-G3139 are prepared into a mixed solution according to a certain base ratio. Heating to 96 deg.C, gradually decreasing to 4 deg.C (annealing), and standing at 4 deg.C for 2 days. The cells used in the transfection experiment are A549 and a drug-resistant strain A549/TXL cell line thereof.

(1) Plate paving: adding 30 ten thousand cells into a six-hole plate, wherein the pore volume is 1.8 mL;

(2) after 18-24h, the sample was diluted in the appropriate amount of opti-MEM, pipetted well and 20. mu.L of this mixed solution was added to each well to give a final concentration of 100nM FAM-G3139. The wells without DNA and with DNA only serve as two negative controls, and a transfection reagent Lipofectamine 2000-DNA complex (the transfection reagent and the DNA group are added according to protocol) is added as a positive control;

(3) washed three times with 500 μ L PBS;

(4) after 4h, sucking off the liquid in the culture hole, adding 250 mu L of 5% trypsin for digestion, placing in a 1.5mLEP tube, and adding fixed cells containing 4% paraformaldehyde for 15 min;

(5) centrifuging, removing supernatant, and adding 400 μ LPBS;

(6) the fluorescence intensity was measured by flow cytometry.

2. Results

The results are shown in FIG. 6, and indicate that the delivery capacity of DNCA in A549 and its resistant strain A549/TXL cell line is better than that of other three-base lipid carriers, and about 35% of tumor cell proliferation inhibition effect is shown under the condition of DNCA (7.5. mu.M)/G3139 (400 nM). Furthermore, increasing the DNCA concentration from 7.5 μ M to 15 μ M stepwise under the condition of G3139 — 400nM, there was no significant increase in tumor suppressive activity.

Example 7 efficiency examination of DNCA-entrapped ssRNA

In the experiment, 20% non-denaturing gel electrophoresis is adopted to investigate the entrapment efficiency of DNCA on ss/ds-miR-122. N/P settings were from 1:2 to 10:1, and RNA samples were 6-FAM labeled at the 5' -end.

miR-122 is highly expressed in normal liver tissues and is involved in regulating physiological processes related to various cell metabolisms, but the expression level of miR-122 in liver cancer tissues is obviously reduced. Relevant researches prove that the miR-122 is over-expressed in the liver cancer cells to inhibit the proliferation, metastasis and infiltration of the cancer cells and increase the sensitivity of the cancer cells to drugs. In addition, miR-122 can also target bladder cancer VEGFC gene, and has obvious influence on downstream signal pathways AKT and mTOR, and finally has the effects of efficiently inhibiting tumor growth and angiogenesis. The natural miR-122 has an incompletely matched double-chain structure, leaves along with a chain after being combined with Ago2 protein, forms active RISC and plays a physiological function. Due to the existence of mismatch sites in miR-122, the sequence is extremely easy to recognize and degrade by ribozyme; in addition, the thermodynamic difference between miR-122 ends is not obvious, so that the follower chain has a high possibility to enter RISC, and the off-target effect is generated. Therefore, in the experiment, miR-122 is designed into three forms, namely double-chain incomplete matching, double-chain complete matching and single-chain, in a structural modification mode. In order to reduce the possibility of the random strand entering RISC, L-isonucleoside (L-isoA) modification is carried out at the 5' -end according to the method described in Chinese patent application with the patent application number CN201210020028.2 and the invention name of "a method for chemically modifying siRNA sequence by using isonucleoside, and the product and application thereof", thereby eliminating the activity of the strand. In addition, in order to improve the stability of miR-122, the double-stranded 3' -end is modified by peptide conjugation (3 ' -KALLAL-5 ') to reduce the possibility that RNA is degraded by 3' -exonuclease, and the modification method is as per the patent application number WO2016197264A1, and the invention name is ' a small stem combined with heteronucleoside modification, end peptide conjugation and cationic liposomeThe method of modifying interfering RNA and the preparation are described in the patent application. (Table 1: miR-122(122) represents microRNA of interest; miR-122-mic represents a mimic of miR-122, the follower chain of which is completely matched with the leading chain; m represents a mimic; P represents peptide conjugation modification (3' -KALLAL); 1A_LRepresents that the 1 st position of the 5' end is modified by L-isoA; pi represents a 5' -phosphorylation modification; as represents a main guide chain; s represents a follower chain. Such as: "PP-122 m-S1A_L", indicates that the dipeptide conjugation is associated with a miR-122 mimetic modified with L-isoA at position 1 of the chain. )

TABLE 1 miR122mimics sequences

The results are shown in fig. 7, which shows that the electrophoretic bands of ssRNA and its peptide conjugate products essentially disappeared under N/P5: 1 conditions; however, even under the condition that the N/P is 10:1, the electrophoresis band of the double-stranded RNA is not obviously reduced. The above results indicate that the DNCA carrier can selectively recognize and bind to a single-stranded RNA structure, and has poor double-stranded RNA entrapment capability.

Example 8 intracellular stability Studies of DNCA-Encapsulated ssRNA

Effective vector delivery and chemical modification strategies can play a role in protecting RNAs and reduce the recognition and degradation of ribozymes. Therefore, this experiment examined the change in intracellular stability before and after encapsulation of peptide-conjugated modified ssrnas (exemplified by miR-122) by DNCA.

The results of the experiment show (fig. 8) that single-stranded RNA can be completely entrapped by DNCA under N/P5: 1 conditions and released from the nanoparticles after addition of cell lysate. In addition, the unencapsulated sequence 122-SS (single-chain miR-122) and the Pep-122-SS (terminal peptide LALLAK conjugated modified single-chain miR-122) are completely degraded within 1h, which indicates that the 3' -peptide conjugation modification can not improve the stability of the ssRNA. The stability of the DNCA/122-SS nano-particles is not obviously improved, which indicates that the DNCA and RNA are combined relatively loosely and cannot play an effective ribozyme protection role. However, the serum stability of the DNCA/Pep-122-SS nanoparticles is obviously improved, which indicates that the peptide fragment (LALLAK) and the DNCA carrier interact with each other, so that the assembly mode between DNCA and RNA conjugate is changed, and the compactness of the nanoparticles is improved.

Example 9 examination of transmembrane Capacity of DNCA-entrapped ssRNA

This experiment examined the transmembrane capacity of DNCA entrapped peptide conjugated modified RNA (taking miR-122 as an example, the modification method is the same as that of example 8). And the commercial transfection reagent lipofectamine 2000(lipo) was selected as a control, further illustrating the advantage of the DNCA + peptide conjugation modification strategy in delivering ssRNA.

The results are shown in FIG. 9, which shows that the vector-free RNAs are unable to enter the cell through the membrane. Furthermore, DNCA has higher transfection efficiency relative to lipo under serum-free transfection conditions. Furthermore, DNCA is suitable for transfecting ssRNA in serum conditions with transfection efficiency superior to serum-free conditions. Consistent with the entrapment efficiency results, the efficiency of DNCA transfection of ssRNA was significantly higher than dsRNA, further demonstrating that DNCA can selectively recognize bound ssRNA.

Example 10 DNCA Encapsulated ssRNA safety and anti-tumor cell proliferation Effect

This experiment examined the safety of DNCA-entrapped ssRNA (exemplified by miR-122) against the human embryonic kidney cell (HEK293A) cell line.

DNCA/pi-AS: main chain mimetics representing DNCA-entrapped 5' -phosphorylation modified miR-122

DNCA/Pep-pi-AS: main chain mimetics representing DNCA-entrapped miR-122 that bind to 5 '-phosphorylation modifications and 3' peptide conjugation modifications

The transfection procedure was as in example 6.

The results are shown in FIG. 10 and indicate that neither DNCA/pi-AS nor DNCA/Pep-pi-AS is significantly cytotoxic at high dose concentrations. In contrast, lipo2000 entrapped the same sequence exhibited a significant dose-dependent effect, with HEK293A cells dying more than half at RNA concentrations up to 100 nM. The above results fully demonstrate the better safety and lower cytotoxicity of DNCA compared to lipo 2000. In addition, the activity result aiming at the HepG2 cell line shows that DNCA/pi-AS has no obvious activity effect relative to an NC sequence, but DNCA/Pep-pi-AS shows better tumor cell proliferation inhibition effect under the condition of 200nM, and further illustrates the advantages of the strategy.

Example 11 efficiency of DNCA Loading Single-stranded DNA

To verify whether DNCA can effectively entrap single-stranded DNA, the experimental system examined the entrapment efficiency of DNCA on AS1411((5'-GGT GGT GGT GGT TGT GGTGGT GGT GG-3', 26-mer)), G3139 and N-G3139 under different N/P conditions. The results are shown in fig. 11 and indicate that DNCA can efficiently entrap the corresponding nucleic acid sequence when N/P is 4-5. Where G3139 entrapment was slightly less efficient, this may be because the full-site PS modification affected the interaction between the DNCA-nucleic acids.

Example 12 determination of Tm value of DNCA/AS1411 Complex

The present invention utilizes Circular Dichroism (CD) spectroscopy to study the stability of the complex formed by DNCA and AS1411(20 bp).

As a result, AS shown in FIG. 12, the Tm values of AS1411 after annealing in PBS buffer (41 ℃ for AS 1411; 53 ℃ for AS1411/DNCA complex) are shown. The DNCA is mixed with the DNCA, and after an annealing process, the Tm value of the DNCA/AS1411 compound is obviously changed, and is increased from 42 ℃ to 52 ℃, so that the DNCA and the AS1411 form a new secondary structure which is more stable than the secondary structure formed by the AS 1411.

Example 13 biocompatibility examination of base lipid Carriers

The basic acetamide glyceryl ether molecule is required to be used as a novel biological material and to realize the application in gene therapy, and the basic acetamide glyceryl ether molecule has low cytotoxicity. We examined the cytotoxicity of the four nucleoside base acetamide/glycerol acetate compounds, and the results showed that none of the four compounds had significant cytotoxicity. At the concentration of 65 mu M, after 72 hours of adding the nucleoside base lipid molecular compound, the cell survival rate is over 90 percent, and the good biocompatibility is shown. The results are shown in FIG. 13A.

Example 14 base lipid Carrier-entrapped AS1411 Activity for anti-cck-8 proliferation

In the experiment, the cck-8 proliferation inhibition experiment is used for evaluating the concentration gradient of the nanoparticles of the AS1411 encapsulated by the basic group lipid carrier-the proliferation rate of tumor cells. The experimental result shows that neither AS1411 nor DNCA has obvious tumor inhibition effect; the AS1411/DNCA system shows higher anti-proliferative activity at low concentration, and gradually enhances the killing capacity to A549cells along with the increase of the concentration, which indicates that DNCA effectively delivers AS1411 to enter the cells and play the active role. In addition, the DNCA/AS1411 has obviously enhanced inhibition effect on A549-TXL cells and has the effect of reversing multidrug resistance of tumor cells. The results are shown in FIG. 13B.

Example 15 DNCA-entrapped AS1411 cell uptake assay

Experiments show that the cell uptake of DNCA/AS1411 is about 3 times that of the cell uptake of the unencapsulated AS1411, which indicates that DNCA has good transfection efficiency for the G-4 structure. The results are shown in FIG. 13C.

Example 16 Effect of salt/amino acid on the transfection Effect of DNCA/AS1411

In the experiment, a certain component in the GenOpti solution is found to promote the transfection effect of DNCA on AS1411, in order to solve the influence of each component of GenOpti on the transfection effect of DNCA/AS1411, the concentration of DNCA and AS1411 is respectively increased to 15 mu M and 200nM in the experiment, and the influence of a salt component and a salt/amino acid (50 mu M) component on the transfection effect of AS1411 is respectively examined.

The results of the experiment are shown in FIG. 14, which show that CaCl₂And MnCl₂Can improve the inhibition effect of DNCA/AS1411 on the proliferation of tumor cells, and the inhibition effect is not further improved after the amino acid is added. The metal ion concentration was then reduced to GenOpti solution concentration, CaCl₂MnCl with high activity₂The activity was substantially lost. Elucidation of CaCl in GenOpti₂The composition is an important reason for improving the efficiency of nucleic acid transfection by liposomes.

Example 17CaCl₂Investigation of the Effect of AS1411 transfection efficiency

This experiment was performed on a series of AS1411 and its modified sequence (6)_L12_D24_I) Oligonucleotide G3139, optimized formulationsProtocol (addition of CaCl)₂) A system investigation was performed (table 2).

The results are shown in FIG. 15, and the experimental results show that CaCl₂The nanoparticles in the DNCA-entrapped AS1411 system have ideal value-added inhibitory activity; the DNCA-entrapped G3139 activity effect was lower than that of AS1411, with the sequences 4, 11, 30 and 36 having significantly improved activity (6)_L12_D24_I: 6-position L-heteroside modification, 12-position D-heteroside modification, 24-position deoxyinosine modification of AS 1411).

TABLE 2 scheme

Example 18 neutral lipid vector for transfection of pEGFP-N1 plasmid

pEGFP-N1 plasmid expressing green fluorescent protein was selected, and the transfection efficiencies of neutral lipid vectors DNCA, DNTA and DNXA (DNCA and DNTA mixed at 1:1) in the presence and absence of serum were examined under the same preparation conditions, and the transfection method was the same as in example 6. And compared to the commercial transfection reagent LIPO.

As shown in fig. 16, the results of the experiment showed that neutral lipid vectors DNCA, DNTA and DNXA (DNCA: DNTA ═ 1:1) had better transfection efficiency to plasmids in the presence of serum, and that cells were starved in the absence of serum, resulting in a significant decrease in transfection efficiency. While the cationic transfection reagent LIPO has high transfection efficiency in serum-free state and greatly reduced transfection efficiency in serum-free state. The results show that although the transfection efficiency of the neutral lipid carrier to the plasmid is still to be improved compared with LIPO, the complex formed by the lipid carrier and the plasmid has better stability in serum, the transfection under serum condition can be realized, and the in vivo transfection may have advantages in the future.

EXAMPLE 19 toxicity Studies of neutral lipid vectors for plasmid DNA transfection

The plasmids used in the experiment are nontoxic, and in order to examine the toxicity of the neutral lipid carrier/DNA complex, the experiment is designed, under the condition of the dosage of common lipid (1nmol of lipid carrier is used for loading 10ng of plasmid), the toxicity of DNCA, DNTA and DNXA (DNCA and DNTA are mixed according to the ratio of 1:1) to cells (HEK293A) is simultaneously compared in the process of transfecting the plasmid, and a group of experiment groups with 5 times of dosage of DNCA (5nmol of lipid carrier is used for loading 10ng of plasmid) to transfect the same amount of plasmid is added. The plasmid pMB 310 ng was loaded in 96-well plates with 1nmol DNCA per well at a DNCA effect concentration of 10. mu.M and transfected for 48h under serum conditions. The control group is LIPO, each well is 0.1 μ L, serum-free effect is achieved for 4h, blood is enriched and cleared, then culture is continued for 44h, and cell viability is detected by CCK-8 cell viability detection reagent and neutral lipid carrier group at the same time. The transfection procedure was as in example 6.

The results are shown in FIG. 17, which shows that LIPO showed significant toxicity, DNCA and DNXA were substantially non-toxic at the usual level (10. mu.M), DNTA had little effect on cytotoxicity, and 5-fold toxic effect of DNCA level (50. mu.M) was also significant. The result shows that the neutral lipid carriers DNCA and DNTA as plasmid transfection reagents have low toxicity and wide application prospect. A small amount of DNTA has toxicity, possibly related to the solubility of DNTA, the complex solution of DNTA and plasmid is relatively turbid, a few floating particles can be observed after the DNTA and the plasmid complex solution is added into a culture medium, insoluble large particles easily stimulate cell membranes to damage cells, and acute toxicity is generated, and accumulated toxicity can be caused if the complex particles cannot be degraded in time after entering the cells. Neutral lipid carriers can be transfected under serum conditions, are more favorable for normal cell proliferation compared with a serum-free environment, and have certain advantages in vivo transfection compared with a cationic preparation LIPO.

The information shown and described in detail herein is sufficient to achieve the above-mentioned objects of the present invention, and therefore the preferred embodiments of the present invention represent the subject matter of the present invention, which is broadly encompassed by the present invention. The scope of the present invention fully encompasses other embodiments that may become obvious to those skilled in the art, and that the scope of the present invention is therefore not limited by anything other than the appended claims, in which the singular form of an element used herein does not mean "one and only" one "unless explicitly so stated, but rather" one or more ". All structural, compositional, and functional equivalents to the elements of the above-described preferred embodiments and additional embodiments that are known to those of ordinary skill in the art are therefore incorporated herein by reference and are intended to be encompassed by the present claims.

Moreover, no apparatus or method is required to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. It will be apparent, however, to one skilled in the art that various changes and modifications in form, reagents and synthetic details may be made without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims (6)

1. A nucleic acid delivery system, which is characterized by comprising a liposome of a neutral base lipid carrier and a metal salt, wherein the structural formula of the neutral base lipid carrier is shown as a formula I:

formula I

Wherein the metal salt is calcium salt.

2. The nucleic acid delivery system of claim 1, wherein the calcium salt is CaCl₂。

3. Use of a nucleic acid delivery system according to claim 1 or 2 for the preparation of a nucleic acid delivery reagent.

4. The use of claim 3, wherein said nucleic acid comprises an oligonucleotide and plasmid DNA.

5. The use of claim 4, wherein said oligonucleotide comprises an aptamer, siRNA.

6. The use according to claim 4, wherein the nucleic acid is a nucleic acid analogue modified by at least one of D-, L-isonucleoside modification, deoxyinosine modification, peptide conjugation modification and phosphorylation modification.

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