CN112707943B - Combining 5'-terminal conjugate and neutral/cationic mixed lipid-encapsulated small interfering RNA and its modification method - Google Patents

Abstract

The invention discloses a modified small interfering RNA combined with a 5'-terminal conjugate and a neutral/cationic mixed lipid preparation and a modification method thereof. The modification method includes conjugating one or more linking groups at the 5′-end of the antisense strand or/and the sense strand of the siRNA as a conjugating group, and using neutral lipid material and cationic mixed lipid material for encapsulation. It also includes linking the targeting group through a condensation reaction on the terminal active ester of the linking group. The siRNA lipid complex obtained by the chemical modification strategy provided by the present invention has better biological activity, exhibits excellent transmembrane transport ability and silencing activity of target mRNA at the cellular level, and exhibits efficient target organ distribution at the animal level. The ability to inhibit tumor growth, and the safety of application at the cell level and animal level, without obvious toxicity, has laid a foundation for the wide application of siRNA technology in clinical practice.

Description

Combining 5'-terminal conjugates and small interferences entrapped in neutral/cationic mixed lipids RNA and its modification method

technical field

The present invention relates to a modified small interfering RNA (siRNA) combined with a 5'-terminal conjugate and a neutral lipid/cationic mixed lipid material, as well as a chemical modification method and a carrier delivery strategy. The present invention realizes conjugation to the 5'-end of the antisense strand of siRNA, and the presence of the conjugate does not affect the gene silencing activity of siRNA. The siRNA conjugated, modified and encapsulated by the method of the present invention has the advantages of good stability, high delivery efficiency, high target organ distribution in vivo, strong cell entry ability, low toxicity and good biological activity, and can be widely used in anti-tumor, Antiviral and metabolic diseases drug research. The invention belongs to the technical field of biomedicine.

Background technique

Small interfering RNA (siRNA) is a class of drug candidate molecules with great clinical prospects. Its target is intracellular mRNA, which can directly silence the expression of target genes at the gene level and fundamentally prevent the occurrence and development of diseases. Native siRNA is cleaved intracellularly by Dicer from dsRNA to 21-25mer, usually with 5'-phosphate and 3'-hydroxyl groups. The 5′-hydroxyl group of the synthesized siRNA will be rapidly phosphorylated by the Clp1 cell kinase in the cell, which is also a necessary condition for the siRNA to exert its activity, because the siRNA can enter RISC to exert its gene silencing activity only after 5′-phosphorylation. Compared with traditional small molecule drugs, siRNA has the advantages of higher specificity, lower toxicity and side effects, and easy preparation. However, its clinical application is limited due to its poor serum stability, difficulty in transmembrane transmembrane, easy off-target, stimulation of immune response and other defects.

(patisiran)，它是hATTR(遗传性运甲状腺素蛋白淀粉样变性)的候选疗法，使用由DLin-MC3-DMA、DSPC、PEG-DMG和胆固醇组成的脂质体进行递送。体外实验表明在HepG2细胞中，10nM siRNA可敲低95％TTR mRNA表达，在啮齿动物模型中的ED₅₀为0.03mg/kg。这一siRNA药物的安全性优于hATTR的反义核酸药物Inotersen

2019年，FDA批准了第二例siRNA药物GIVLAARI^TM(givosiran)，它是除了静脉注射血红素以外唯一有效的急性肝卟啉症的治疗方法，且血红素极难获得。Givosiran使用了GalNAc缀合进行递送，在啮齿动物模型中的有效剂量是1-3mg/kg。随着基因组学的快速发展，RNAi(RNA干扰)药物的成功表明个体化治疗新时代的开始，但遗憾的是目前的上市药物使用靶向肝脏脱唾液酸糖蛋白受体的LNP递送体系和GalNAc缀合物而仅能进行肝脏递送。而且鉴于目前正处于临床III期研究的siRNA均未使用载体包载，依赖电荷作用的阳离子脂材的应用已经陷入了困境。In 2018, the FDA (US Food and Drug Administration) approved the first siRNA drug

(patisiran), a candidate therapy for hATTR (hereditary transthyretin amyloidosis), delivered using liposomes consisting of DLin-MC3-DMA, DSPC, PEG-DMG and cholesterol. In vitro experiments showed that 10 nM siRNA knocked down 95% of TTR mRNA expression in HepG2 cells, with an ED ₅₀ of 0.03 mg/kg in a rodent model. The safety of this siRNA drug is better than that of hATTR antisense nucleic acid drug Inotersen

In 2019, the FDA approved a second siRNA drug, GIVLAARI ^™ (givosiran), which is the only effective treatment for acute hepatic porphyria other than intravenous heme, which is extremely difficult to obtain. Givosiran is delivered using GalNAc conjugation, and effective doses in rodent models are 1-3 mg/kg. With the rapid development of genomics, the success of RNAi (RNA interference) drugs indicates the beginning of a new era of personalized therapy, but unfortunately the current marketed drugs use LNP delivery systems targeting liver asialoglycoprotein receptors and GalNAc Conjugates are only capable of liver delivery. And since none of the siRNAs currently under clinical phase III studies are encapsulated with carriers, the application of charge-dependent cationic lipids has fallen into a predicament.

Cationic lipids bind and encapsulate siRNA by the Coulomb force between the positive and negative charges of siRNA. However, cationic liposomes are highly toxic and easily bind to negatively charged serum proteins under physiological conditions, resulting in immunogenicity, liver toxicity, and easy release of siRNA outside target organs. These defects limit the further application of cationic liposomes to encapsulate siRNA into drugs.

The inventor designed and synthesized the base acetamide glyceryl ether molecule DNCA (CN108059619A) in the early stage. Because of its base head, it can be combined by hydrogen bonding and π-π stacking and encapsulate single-stranded nucleic acid drugs and plasmids (CN1084478807A). Combining DNCA and the cationic lipid CLD with lysine as the head designed and synthesized by the inventors to co-encapsulate 3′,3″-dipeptide-conjugated siRNA has been successfully applied at the cellular level (Mol Pharm, 2019, 16, 4920) Here, we optimized the encapsulation method, reformulated and explored the optimal DNCA/CLD transfection recipe, and added DSPE-PEG to optimize the properties of the mixed formulation for in vivo applications. Combining the use of phosphodiester bonds as siRNA The conjugation strategy and chemical modification method of linking the chain end with the conjugating group have obtained a conjugate/neutral/cationic mixed lipid siRNA drug-encapsulated delivery system with high efficiency and low toxicity.

SUMMARY OF THE INVENTION

In order to improve the specificity of target organ delivery, cell entry efficiency and intracellular release effectiveness of small interfering RNA (siRNA) in vivo delivery, the present invention provides a combination of 5'-terminal conjugates and neutral lipids/ Chemical modification method and carrier delivery strategy of siRNA encapsulated in cationic lipid mixed carrier. In the present invention, the conjugated group is prepared as a phosphoramidite monomer, and a solid-phase synthesizer is used to realize the rapid synthesis of the conjugate. The present invention utilizes neutral lipid material and cationic mixed lipid material to encapsulate the siRNA conjugate to achieve more effective, safe and non-toxic delivery of siRNA in vivo, thereby improving the druggability of siRNA.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

A chemical modification method for small interfering RNA (siRNA) of the present invention, comprising conjugating one or more linking groups as a conjugating group at the 5'-end of the sense strand or/and antisense strand of the siRNA, and Neutral lipid material and cationic mixed lipid material are used for encapsulation, wherein the structural formula of the linking group is shown in chemical formula I or chemical formula II, wherein the structure shown in chemical formula I is a six-carbon unit linking group, chemical formula The structure shown in II is a linking group of three carbon units;

Wherein, the mixed resin is composed of neutral cytidine resin represented by chemical formula III, cationic resin represented by chemical formula IV, or neutral cytidine resin represented by chemical formula III, chemical formula IV The composition of cationic lipid material and PEG2000-DSPE shown in chemical formula V:

Among them, preferably, when the antisense strand or/and the 5′-end of the sense strand of the siRNA are conjugated with one or more linking groups, it also includes linking the targeting group through a condensation reaction on the terminal active ester of the linking group group, the structural formula of the targeting group is shown in chemical formula VI:

Among them, preferably, before conjugating the siRNA, the linking groups shown in chemical formula VII and chemical formula VIII are prepared into phosphoramidite monomers shown in chemical formula IX and chemical formula X, respectively, and then the siRNA is linked to the siRNA through a phosphodiester bond. Carry out conjugation; then connect the targeting group shown in chemical formula VI through a condensation reaction on the active ester of the phosphoramidite monomer shown in chemical formula IX or chemical formula X;

Among them, it is preferred to use solid-phase synthesis technology to synthesize the natural and conjugated linking group siRNA sense strand and antisense strand sequence by phosphoramidite method. On RNA synthesizer, the sequence is from 3'-end to 5' -Synthesis is carried out at the end, each coupling of a nucleoside is a cycle, after 21 couplings of the natural nucleoside phosphoramidite monomer, one or several of the phosphoramidite monomers IX or X in the sequence 5 Coupling is carried out at the corresponding position of the '-end, and each cycle includes four reactions: de-DMT, coupling, blocking, and oxidation.

Among them, preferably, the coupling time after the injection of the phosphoramidite monomer in each cycle is 600 seconds/time, and the coupling is performed 3 times.

Wherein, preferably, calculated according to the mole percentage, the mixed resin is composed of 72.7% of the neutral cytidine resin shown by the chemical formula III and 27.3% of the cationic resin shown by the chemical formula IV, wherein, preferably, the total lipid The molar ratio of mass to siRNA was 10:1. Or the mixed resin material is composed of 39.7% neutral cytidine resin shown in chemical formula III, 59.6% cationic resin material shown in chemical formula IV and 0.7% PEG2000-DSPE shown in chemical formula V, wherein, the preferred , the molar ratio of total lipid to siRNA was 5.3:1.

Among them, preferably, the method for encapsulation by using the mixed lipid material of neutral lipid material and cationic lipid material is: dissolving the mixed lipid material in an ethanol solution, and 5' of the antisense strand or/and the sense strand - siRNA whose ends are conjugated with one or more linking groups through a phosphodiester bond is dissolved in DEPC water, mixed with unbuffered solution or buffered solution, sonicated, filtered through a 0.22 μm filter, and the filtrate contains the modified siRNA, Wherein, preferably, the unbuffered solution is physiological saline, water or any transfection optimized solution, and the buffered solution comprises acetate, citrate, carbonate, phosphate or any combination thereof.

Wherein, the chemical modification strategy also includes the co-use with other chemical modification strategies, including 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'- siRNA of siRNA -O-methoxyethyl (2'-O-MOE), locked nucleotide (LNA), modification of phosphorus-sulfur backbone and other terminal conjugation methods, etc.

Further, the present invention also proposes a 5'-terminal conjugate synthesized according to any one of the chemical modification methods described above and a neutral/cationic mixed lipid material carrier to encapsulate the modified small interfering RNA.

Compared with the 3'-end conjugation, the 5'-end conjugation method of the present invention has the following characteristics:

a. The conjugated group is prepared into the corresponding phosphoramidite monomer, so that the conjugated part can be directly synthesized as a monomer using a solid-phase synthesizer, and the synthesis is simple and fast;

b. The end of the linking group X retains an active ester as an active reaction site, which can efficiently link a variety of functional groups by condensation reaction and rapidly expand the scale of the conjugate;

c. The linking groups are connected by phosphodiester bonds, which can be hydrolyzed and cleaved by enzymes in the cell to release unconjugated siRNA single-strands with phosphate ends, which are ultimately easier to load into RISC complexes and improve siRNA genes. The ability to silence activity.

In a specific embodiment of the present invention, the modified sequence of the 5-terminal conjugate is selected from the group consisting of the following sequences:

(1) An antisense strand or a sense strand obtained by conjugating a linking group shown in chemical formula II to the 5'-end of the sense strand or antisense strand of the small interfering RNA sequence;

(2) The antisense obtained by conjugating one or more linking groups shown in chemical formula I and one chemical formula II to the 5′-end of the sense strand or antisense strand of the small interfering RNA sequence in turn in the 3′-5′ direction sense chain or sense chain;

(3) Conjugate a linking group shown in chemical formula II to the 5′-end of the sense strand or antisense strand of the small interfering RNA sequence, and the conjugation is completed by a solid-phase synthesizer, and the phosphoramidite monomer used is chemical formula X, then on the active ester of the group shown in chemical formula X, the targeting group shown in chemical formula VI is connected by condensation reaction, and the obtained sense strand or antisense strand;

(4) Conjugate one or more linking groups shown in chemical formula I and one chemical formula II to the sense strand or 5'-end of the antisense strand of the small interfering RNA sequence in turn in the 3'-5' direction, and the conjugation uses The solid-phase synthesizer is completed, the phosphoramidite monomers used are chemical formula IX and X, and then the target group shown in chemical formula VI is connected by condensation reaction on the active ester of the group shown in chemical formula X, and the obtained reaction a sense strand or a sense strand conjugate;

The antisense strand and the sense strand in the above (1)-(4) are in the form of free combination.

The siRNA and its conjugates of the present invention are RNA double-stranded complexes formed by annealing a conjugated or unconjugated modified antisense strand and a sense strand according to requirements.

Taking RL1As/RLOS-siRNA as an example, at the 5'-end (3'-5' direction) of the antisense strand (represented by As) of the siRNA, a chemical formula I (the number represents the link shown in chemical formula I) is conjugated by solid-phase synthesis. number of groups) and a linking group shown in chemical formula II (represented by L), wherein the conjugating groups are linked by phosphodiester bonds, and then a targeting group cRGD shown in chemical formula VI is conjugated through a condensation reaction (R represents), namely RL1As. A chemical formula II (represented by L) is conjugated to the 5'-end of the sense strand (represented by S) by solid-phase synthesis, and then a targeting group cRGD (represented by R) represented by chemical formula VI is conjugated by a condensation reaction, namely RLOS, Then, the synthesized and purified antisense strand and the sense strand are annealed at a molar ratio of 1:1.1 to form RL1As/RLOS-siRNA, and the rest of the conjugated siRNA is deduced by analogy.

In a specific embodiment of the present invention, the siRNA to be modified is the siMB3 sequence targeting the BRAF ^V600E mutated mRNA in the ERK pathway. The sequence of siMB3 before modification is as follows:

siMB3: sense strand: 5′-GCUACAGAGAAAUCUCGAUdtdt-3′

Antisense strand: 5'-AUCGAGAUUUCUCUGUAGCdtdt-3'

In a specific embodiment of the present invention, the above-mentioned siMB3 sequence is encapsulated by a mixed lipid material consisting of a neutral lipid material represented by chemical formula III, a compound cationic lipid material represented by chemical formula IV, and a compound represented by chemical formula V. When , the siRNA lipoplexes can be uniformly distributed in the form of spherical vesicles, and the particle size of the siRNA lipoplexes is uniform.

In a specific embodiment of the present invention, siMB3 and its antisense chain end are conjugated with a conjugation group shown in chemical formula I and a chemical formula VI, and use commercial cationic transfection reagent Lipofectamine2000 or by chemical formula III. When encapsulated by the mixed lipid material composed of the cationic lipid material, the compound of the chemical formula IV, and the compound of the chemical formula V, the mixed lipid material has better cell entry efficiency and gene silencing activity.

In a specific embodiment of the present invention, three conjugating groups shown in chemical formula I and one conjugating group shown in chemical formula II are sequentially conjugated to the 5-terminal of the antisense strand of the siMB3 sequence described above. group, and a targeting group cRGD shown in chemical formula VI is conjugated through active ester reaction, and is composed of neutral lipid material shown in chemical formula III, compound cationic lipid material shown in chemical formula IV and compound shown in chemical formula V. When encapsulated in the mixed lipid material, it has the optimal cellular uptake capacity.

In a specific embodiment of the present invention, three conjugating groups shown in chemical formula I and one conjugating group shown in chemical formula II are sequentially conjugated to the 5-terminal of the antisense strand of the siMB3 sequence described above. The conjugation was completed using a solid-phase synthesizer, and the phosphoramidite monomers used were chemical formulas IX and X, wherein the conjugation groups were linked by phosphodiester bonds, and then on the active ester of the group shown in chemical formula X A targeting group cRGD represented by chemical formula VI is connected through a condensation reaction, and a mixed lipid material package composed of a neutral lipid material represented by chemical formula III, a compound cationic lipid material represented by chemical formula IV and a compound represented by chemical formula V When loaded, it has the best cell-level gene silencing activity.

In a specific embodiment of the present invention, three conjugating groups shown in chemical formula I and one conjugating group shown in chemical formula II are sequentially conjugated to the 5-terminal of the antisense strand of the siMB3 sequence described above. The conjugation was completed using a solid-phase synthesizer, and the phosphoramidite monomers used were chemical formulas IX and X, wherein the conjugation groups were linked by phosphodiester bonds, and then on the active ester of the group shown in chemical formula X A targeting group cRGD represented by chemical formula VI is connected through a condensation reaction, and a mixed lipid material package composed of a neutral lipid material represented by chemical formula III, a compound cationic lipid material represented by chemical formula IV and a compound represented by chemical formula V When loaded, it has the best anti-tumor activity in animals.

In a specific embodiment of the present invention, the 5-terminus of the antisense strand of the above-mentioned siMB3 sequence is conjugated with a conjugating group shown in chemical formula II. The conjugation is completed using a solid-phase synthesizer, and the phosphoramidite monomer used is chemical formula X, and then a targeting group cRGD shown in chemical formula VI is connected by condensation reaction on the active ester of the group shown in chemical formula X, and When encapsulated by the mixed lipid material consisting of the neutral lipid material represented by the chemical formula III, the compound cationic lipid material represented by the chemical formula IV, and the compound represented by the chemical formula V, it has the optimal tumor tissue distribution in animals.

Compared with the prior art, the advantages of the present invention are:

1. The chemical modification strategy of combining targeting group conjugation and neutral/cationic mixed carrier encapsulation provided by the present invention, the obtained siRNA lipid complex has better biological activity, and exhibits excellent transmembrane transport at the cellular level The ability and silencing activity of target mRNA, animal level shows efficient target organ distribution ability and tumor growth inhibition ability, and it has good safety and no obvious toxicity in cell level and animal level, which is widely used in clinical application of siRNA technology Foundation;

2. The neutral nucleoside lipid material has a base head, which can be combined with siRNA through hydrogen bonding and π-π stacking, and is more stable in in vivo application than the charge interaction between cationic lipid material and siRNA , it is not easy to adsorb charged particles or proteins during the circulation process, avoiding the disassembly of lipid complexes outside the target organ and the release of siRNA. And the combination based on non-electric effect reduces the dosage of cationic resin in the formulation. The nitrogen-phosphorus ratio of cationic resin and siRNA is only 3/1, which is far lower than the dosage of cationic resin in other reports.

3. 5'-conjugation is simple and fast to synthesize, can be used as a substrate to efficiently expand the conjugate combination, and can release the siRNA antisense strand with 5'-terminal phosphoric acid during intracellular degradation and metabolism, making it easier to enter the RISC complex to play genes Silencing activity. The present invention proves that the 5'-end conjugation of the antisense strand does not affect the gene silencing activity of siRNA, expands the siRNA conjugation site, and enriches the modification strategy of siRNA.

Description of drawings

Figure 1 is a transmission electron microscope (TEM) photograph of siRNA/DNCA/CLD/PEG-DSPE lipoplex and empty carrier;

Figure 2 is the cellular uptake (10 nM, 4h) of siRNA neutral/cationic lipid complexes monoconjugated at the 5'-end of the antisense strand;

Figure 3 shows the intracellular distribution of siRNA neutral/cationic lipid complexes mono-conjugated at the 5′-end of the antisense strand (50nM, 4h), in which blue represents the nucleus, red represents Cy3-labeled siRNA, and green represents soluble siRNA Enzyme, the scale bar in the figure is 10 μm;

Figure 4 is the target gene silencing activity of 5'-terminal mono-conjugated and 5',5"-diconjugated siRNA neutral/cationic lipid complexes (50nM, 48h);

Figure 5 shows the antitumor activity and carrier cytotoxicity of 5'-terminal monoconjugated and 5',5"-diconjugated siRNA neutral/cationic lipid complexes (100 nM, 72h);

Figure 6 shows the distribution of neutral/cationic lipid complexes of siRNA mono-conjugated at the 5'-end of the antisense strand in vivo (a) and in vitro (b), photographed using a small animal in vivo imaging system, with excitation light is 745nm, and the emission light is 800nm;

Figure 7 is the anti-tumor activity of 5'-terminal mono-conjugated siRNA (RLOAs/S) of the antisense strand in animals (Balb/c-nude mice, inoculated with A375 in the armpit);

a. When the tumor volume reached about 50mm3, it was the 0th day, the preparation was administered by intravenous injection on the 1st, ^3rd , 5th, and 8th days, and the tumor volume was measured and calculated every day; b. The weight change curve of the mice during the whole treatment process ; c. Euthanize the mice 48h after the last administration, collect the tumor and record the weight of the tumor; d. BRAF ^V600E mRNA expression in the tumor taken out; e. BRAF ^V600E expression in the tumor tissue taken out by immunohistochemical investigation, yellowish brown Dots represent BRAF ^V600E protein expression, blue represents nuclei, and the black bar is 50 μm. Data are represented by mean±SD, n=5, ***p<0.001;

Figure 8 is the anti-tumor activity of antisense strand 5'-terminal monoconjugated siRNA (RL2As/S) in animals (Balb/c-nude mice, inoculated with A375 in the armpit);

a. When the tumor volume reached about 50mm3, it was the 0th day. The preparation was administered by intravenous injection on the 1st, ^3rd , 5th, 7th, and 9th days. The tumor volume was measured and calculated every day. The tumor volume was normalized to 1; b. The mice were euthanized 48 hours after the last administration, and the tumors were collected and weighed for recording; c. The removed tumors were photographed and observed; d. BRAF ^V600E mRNA expression in the removed tumors ; e. The expression of BRAF ^V600E in the removed tumor tissue was investigated by immunohistochemistry, the yellow-brown spots represent the protein expression of BRAF ^V600E , the blue represents the nucleus, and the black scale is 50 μm; f. The weight change curve of the mice during the whole treatment process. Data are expressed as mean±SD, n=5, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001;

Fig. 9 is the biochemical indexes of liver and kidney when the 5'-end single-conjugated siRNA of antisense strand (RLOAs/S) is applied in animals;

Figure 10 shows the target gene silencing activity in the tumor after a single administration of siRNA (RL2As/S) at the 5'-end of the antisense strand for 48 hours. The dose of siRNA was 1.5 mg/kg, and each mouse was 2 nmol siRNA , data are expressed as mean±SD, n=4, **p<0.01, ***p<0.001.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become more apparent with the description of the specific embodiments. However, the embodiments are only exemplary 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 technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Single-stranded solid-phase synthesis of 5-terminally conjugated siRNA

RNA synthesis was performed using an Applied Biosystems model 394 RNA solid-phase synthesizer.

Normal nucleotide phosphorylated monomers (dT, rGibu, rABz, rCAc, rU) were purchased from Anhui Wuhu Huaren Technology Co., Ltd.; CPG (CPG-dT), CAP-A and CAP _- B, oxidized I solution, Cl ₃ CCOOH was purchased from Beijing Aoke Biotechnology Company; 0.25M 5-ethylmercapto 1H-tetrazolium solution was purchased from Shanghai Zhiyan Technology Co., Ltd. (Shanghai).

According to the method of the literature (Biomacromolecules 2018,19,7,2526-2534), the linking groups shown in chemical formula I/chemical formula II are prepared into phosphoramidite monomers shown in chemical formula IX and chemical formula X, namely:

The compound of formula VII (8.4 g, 4.2 mmol) and triethylamine (0.43 g, 4.2 mmol) were dissolved in 10 mL of anhydrous DCM. Then DMTrCl (1.41 g, 4.2 mmol) dissolved in DCM was slowly added to the above mixture. The reaction mixture was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure and purified by column chromatography (PE:EA=5:1) to give the product (1.45 g, 41%). Under argon, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.64 g, 2.1 mmol) was added to the product (0.45 g, 1.1 mmol) and tetrazolium (0.18 g, 2.6 mmol) was dissolved in dry DCM (5 mL). The reaction mixture was stirred at room temperature for 2 hours. Purified by column chromatography (PE:EA=10:1) to obtain the compound of formula IX (0.40 g, 60.6%).

Compound of formula VIII (1.5 g, 12.7 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) (2.7 g, 15.1 mmol) were dissolved in 10 mL of dry DCM ( _{CH2Cl2 )} . Then N-hydroxysuccinimide (NHS) (1.6 g, 14.3 mmol) was added and the mixture was stirred overnight. The solvent was removed under reduced pressure and purified by column chromatography (PE:EA=1:1) to give the product (1.9 g, 69.6%). Under argon, bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.8 g, 2.6 mmol) was added to the product (0.3 g, 1.4 mmol) and tetrazolium (0.21 g, 3 mmol) The mixture was dissolved in anhydrous DCM (5 mL). The reaction mixture was stirred at room temperature for 2 hours. Purification by column chromatography (PE:EA=10:1) gave the compound of formula X (0.38 g, 66%) .

Synthesis scale: ~1.0 μmol

Preparation of nucleoside phosphorylated monomer solution: weigh under argon protection, add anhydrous acetonitrile, 1g monomer corresponds to 20mL acetonitrile;

Preparation of linker arm phosphorylated monomer solution: weigh the compounds represented by chemical formulas IX and X under argon protection, add anhydrous acetonitrile, and 1 g of monomer corresponds to 20 mL of acetonitrile.

Native and conjugated siRNA antisense and sense strand sequences were synthesized by solid-phase synthesis. On an RNA synthesizer, the sequence is synthesized from the 3'-end to the 5'-end, and each coupling of one nucleoside is a cycle. After 21 couplings of the natural nucleoside phosphoramidite monomer, one or several The linking arm phosphoramidite monomer is coupled at the corresponding position at the 5'-end of the sequence, and each cycle includes four reactions: de-DMT, coupling, blocking, and oxidation.

Synthetic sequence:

In this example, the siRNA to be modified is used as siMB3 targeting BRAF ^V600E mRNA in the ERK pathway, and the sequence before modification is as follows:

siMB3: sense strand: 5′-GCUACAGAGAAAAUCU CGAUdtdt-3′

Antisense strand: 5'-AUC GAGAUU UCU CUG UAG C dtdt-3'

Choose any of the following for the grooming strategy:

(1) Conjugating a linking group of chemical formula II at the 5'-end of the antisense strand or the sense strand of the siMB3 sequence described above;

(2) Conjugate one or more linking groups of chemical formula I and one chemical formula II in sequence (3'-5' direction) at the 5'-end of the antisense strand or sense strand of the siMB3 sequence described above, wherein the conjugation The groups are linked by phosphodiester bonds;

(3) Conjugate a linking group shown in chemical formula II to the 5'-end of the antisense strand or sense strand of the siMB3 sequence described above, and the conjugation is completed by a solid-phase synthesizer, and the phosphorous monomer used is chemical formula X, the targeting group cRGD shown in chemical formula VI is connected by condensation reaction on the active ester of the linking group shown in chemical formula X;

(4) Conjugate one or more linking groups shown in chemical formula I and one chemical formula II to the 5'-end of the antisense or sense strand of the siMB3 sequence described above in turn (3'-5' direction), The combination is completed by a solid-phase synthesizer, and the phosphorous monomers used are chemical formulas IX and X, wherein the conjugated groups are linked by phosphodiester bonds, and then the active ester of chemical formula X is connected by condensation reaction shown in chemical formula VI. Targeting group cRGD.

The siRNA obtained after the above modification strategy is shown in Table 1 below:

Table 1

Synthesis steps: About 33 mg of CPG-dT was weighed each time and loaded into the synthesis column, and the synthesis strategy of DMT-ON was adopted according to the RNA standard procedure of ABI394 nucleic acid synthesizer. Taking the synthesis of RL2As as an example, set the synthesis sequence on the synthesizer sequence panel. Wherein, the linking arms shown by chemical formulas IX and X are also regarded as phosphorous monomers programmed into the sequence, that is, two linking arms represented by chemical formula IX and one chemical formula X are added to the 5'-end of the As chain. After the synthesis, the cRGD functional group was manually reacted separately. After the synthesis, the powder in the synthesis column was transferred to a 1.5 mL glass reaction flask, and 1 mg of the compound represented by chemical formula XI, 100 μl of DIPEA, and 1 mL of DMF were added to stir and react at room temperature for 24 hours. The supernatant was discarded, washed twice with 1 mL of ethanol and once with 1 mL of ether, and then air-dried.

RNA cleavage and deprotection: add 750 μl methylamino alcohol and 750 μl methylamine water to the dried powder for cleavage, place on a shaker, 60° C., 80 rpm, and shake for 90 min. After that, the supernatant was taken out, washed three times with DEPC water, evenly distributed into two 1.5mL EP tubes, and finally lyophilized by a freeze dryer. 100 μl of DMSO and 125 μl of triethylamine trihydrofluoric acid were added to each tube, wrapped with parafilm, 80 rpm, 65° C., and shaken for 90 min. After cooling, 100 μl of 3M CH ₃ COONa solution was added, vortexed, 1 mL of n-butanol solution was added, and after vortexing, it was placed at -80° C. for 30 min. Then take out and centrifuge at 12500rpm for 10min, discard the supernatant, leaving a white precipitate, add 0.75mL absolute ethanol, vortex, put it at -80°C for 30min, centrifuge at 12500rpm for 10min, discard the supernatant, repeat this operation again, freeze-dried.

10x50mm 2.5μm核酸分离柱梯度洗脱(0min，98％TEAB,2％CH₃CN；25min，85％TEAB,15％CH₃CN，v＝1mL/min)。馏分收集完毕后，冻干成粉末。再用700μL DEPC水溶解，用葡聚糖凝胶柱(HiTrap 5mL Desalting)进行脱盐(DEPC水，v＝1.5mL/min)出峰时间在2min左右，收集馏分，再次冻干，得纯产物，-80℃保存。Isolation and purification of RNA: The sample was diluted and dissolved with 400 μl of DEPC water, filtered with a 0.22 μm filter, and washed twice with 200 μl of DEPC water, the final total volume was about 800 μl. Aspirate with a syringe, and inject a volume of 120 μl per needle. Using XBridge ^™ Oligonucleotide BEH C18 OBD ^™ Prep Column

Gradient elution on a 10x50 mm 2.5 μm nucleic acid separation column (0 min, 98% TEAB, 2% CH ₃ CN; 25 min, 85% TEAB, 15% CH ₃ CN, v=1 mL/min). After the fractions were collected, they were lyophilized to powder. Dissolved in 700 μL of DEPC water, desalted with a Sephadex column (HiTrap 5mL Desalting) (DEPC water, v=1.5mL/min), the peak time was about 2min, the fractions were collected and lyophilized again to obtain the pure product, Store at -80°C.

Example 2 Neutral/cationic mixed material encapsulating siRNA to form lipid complex (siRNA/DNCA/CLD/PEG2000-DSPE)

The siRNA was mixed with a mixed material consisting of 39.7% compound of formula III (DNCA, neutral), 59.6% compound of formula IV (CLD, cationic), 0.7% compound of formula V Composition of the compound shown (PEG2000-DSPE). These ratios refer to mole % of the total amount of all lipids. The molar ratio of lipid to siRNA was 5.3:1. DNCA was synthesized according to the method in the literature (CN108059619A), CLD was synthesized according to the method in the literature (New JChem, 2014, 38(10), 4952-4962), and PEG2000-DSPE was purchased from Yuanye Company.

Briefly, compounds of formula III (DNCA, neutral), compounds of formula IV (CLD, cationic), and compounds of formula V were dissolved in ethanol solution, and siRNA was dissolved in DEPC water , mixed with GenOpti (Beijing Maichen), and then ultrasonicated at 70°C for 10min, ultrasonic frequency 150W, 40kHz. After the ultrasound was completed and returned to room temperature, it was filtered through a 0.22 μm filter, and the filtrate contained lipid complexes.

For example, in one specific method, a fresh lipid stock solution is prepared in ethanol: 156.5 mg DNCA, 315 mg CLD, 10.5 mg PEG2000-DSPE are weighed and dissolved in 20 mL ethanol to form a lipid stock solution, which is Sonicate for 4 min at 37°C to form a homogeneous lipid mixture. Subsequently, 20 [mu]l of the above stock solution was added to 480 [mu]l GenOpti to form a 500 [mu]l working lipid stock. This amount of lipid was used to form siRNA lipoplexes with 10 nmol. Dissolve the siRNA dry powder in DEPC water to form a 200 μM siRNA stock solution. 50 μl of the above stock solution was added to 450 μl of Genopti to form 500 μl of working siRNA stock solution, 500 μl of working lipid stock solution was mixed with 500 μl of working siRNA stock solution, briefly centrifuged at low speed, and sonicated at 70°C for 10 min to form lipoplexes. The formulations used in the in vivo and in vitro experiments were filtered through a 0.22 μm filter (merck millipore) and diluted to a 1× siRNA lipoplex solution with a PBS solution before use.

Figure 1 shows exemplary electron micrographs (TEM) of lipoplexes of siRNA prepared by these methods and an empty carrier without siRNA. The siRNA contained in these liposomes was unconjugated siMB3 targeting BRAF ^V600E mRNA in the ERK pathway. The particles of the empty carrier without siRNA were looser, and the particle surface shrunken. The lipoplex after adding siRNA can form spherical nanoparticles with uniform particles, and the edge is smooth and the shape is better.

Dynamic light scattering showed that the mean diameter of the siRNA-loaded lipoplex was 138.7±6.6 (according to density), the polydispersity coefficient was 0.188±0.051, and its surface electrical property was -16.9±0.4mV; the mean diameter of the empty vector was 363.7 ± 20.1 (based on density), the polydispersity coefficient is 0.635 ± 0.045, and its surface electrical properties are 24.3 ± 2.8 mV. Since the sample needs to be dehydrated and dried before TEM shooting, the particles will shrink due to the loss of water, and DLS measures the water and particle size of the nanoparticles, so the particle size measured by the TEM method is smaller than that by the DLS method.

Example 3 Cellular uptake of antisense 5-terminal monoconjugated siRNA neutral/cationic lipoplexes by flow cytometry

1. Sample:

As/Cy3-S-siMB3, RL0As/Cy3-S-siMB3, RL1As/Cy3-S-siMB3, RL2As/Cy3-S-siMB3, RL3As/Cy3-S-siMB3, RL4As/Cy3-S-siMB3. The 5-terminus of the sense strand of siMB3 was labeled with the fluorescent dye Cy3. The synthesis of the antisense strand conjugate is shown in Example 1, and the preparation of the siRNA lipoplex siRNA/DNCA/CLD is shown in Example 2. The difference is that the mixed material is composed of 72.7% neutral cytidine lipid material represented by chemical formula III, 27.3% of the cationic resin composition of formula IV. These ratios refer to mole % of the total amount of all lipids. The molar ratio of total lipid to siRNA was 10:1.

2. Method:

A375 was plated in a 24-well plate at 60,000/well, and transfected after culturing for 18 h. The administration concentration of siMB3 and its conjugates was 10 nM, and the amount of Lipofectamine 2000 used for transfection was 0.5 ul, and the transfection complex was prepared by mixing with siRNA according to its instruction manual. The complex was added to the culture plate, and after 4 h of culture, the cell surface was rinsed with DMEM, the cells were collected by trypsin digestion, and the intracellular fluorescence intensity was measured by the PE channel of the flow cytometer to investigate the cellular uptake ability of siMB3 and its conjugates. The experimental results are shown in Figure 2.

3. Results:

Compared with Lipofectamine2000, the mixed vector DNCA/CLD has a more efficient ability to deliver siRNA into cells, indicating that the presence of serum protein has little effect on the uptake of DNCA/CLD/siMB3 nanocomplexes, even if the serum contains potential transfection competition , such as vitronectin and fibronectin.

Comparing the uptake of cRGD conjugates linked by linking arms of different lengths, it was found that the uptake gradually increased as the linking arm lengthened (RL0As/S to RL3As/S). The ability of cells to uptake RL4As/S was low, suggesting that continuing to extend the link arm length could not enhance the cellular uptake ability of siMB3 conjugates, which may be due to the introduction of more negatively charged phosphodiesteric bisphosphonates while increasing the link arm length. Ester bond, which may affect the formation of the siMB3 conjugate/DNCA/CLD lipoplex.

Example 4 Intracellular distribution of antisense 5-terminal monoconjugated siRNA neutral/cationic lipoplexes using confocal laser microscopy

1. Sample:

As/Cy3-S-siMB3, RL0As/Cy3-S-siMB3, RL1As/Cy3-S-siMB3. The 5-terminus of the sense strand of siMB3 was labeled with the fluorescent dye Cy3. See Example 1 for the synthesis of the antisense strand conjugate and Example 3 for the preparation of the siRNA lipoplex siRNA/DNCA/CLD.

2. Method:

A375 was plated in confocal dishes at 60,000/well, and transfected after culturing for 18 hours. The administration concentration of siMB3 and its conjugates was 50 nM, and the amount of Lipofectamine 2000 used for transfection was 1 μl, and the transfection complex was prepared by mixing it with siRNA according to its instruction manual. The complex was added to the culture plate, and after 4 hours of culture, the nucleus and lysosome were stained with Hoechst and Lysobrite NIR, respectively. Incubate for 30 minutes after adding dye. Cell surfaces were rinsed with DMEM to remove free dye and photographed using a confocal microscope. The intracellular distribution of siMB3 and its conjugates was investigated, and the experimental results are shown in Figure 3.

3. Results:

Confocal results were consistent with flow cytometry results, and the efficiency of Lipofectamine2000 in delivering siRNA into cells was much lower than that of DNCA/CLD(Mix). After cRGD was conjugated to the 5′-terminus of the antisense strand, cellular uptake was significantly increased, suggesting that the partially conjugated cRGD may not be inside the DNCA/CLD/siMB3 nanocomplex, thereby increasing intracellular entry by targeting αvβ3 integrins. Capacity of αvβ3-positive cells A375 to uptake lipid complexes. Cy3-labeled siRNA (red) was punctately aggregated on the cell membrane and in the cell, and may be distributed in the cell membrane transport vesicles and endosomes for intracellular transport. The siRNA hardly co-localized with lysosomes (green) after being encapsulated in the DNCA/CLD mixture, suggesting that this lipid complex can effectively avoid entering or rapidly escape from lysosomes, thereby minimizing the Acidification and degradation of siRNA in lysosomes.

Example 5 Investigating the gene silencing activity of 5-terminal single/double conjugated siRNA neutral/cationic lipoplexes by RT-qPCR technology

1. Sample:

As/S-siMB3, L0As/S-siMB3, L1As/S-siMB3, RL0As/S-siMB3, RL1As/S-siMB3, RL2As/S-siMB3, RL3As/S-siMB3, RL4As/S-siMB3, L1As/ L0S-siMB3, As/RL0S-siMB3, RL0As/RL0S-siMB3, RL1As/RL0S-siMB3, L0As/RL0S-siMB3, L1As/RL0S-siMB3. See Example 1 for the synthesis of the above 5-terminal monobiconjugates.

2. RT-qPCR experiment:

A375 was plated in 12-well plates at 80,000/well, and transfected after culturing for 18 hours. The administration concentration of siMB3 and its conjugates was 50 nM, and the amount of Lipofectamine 2000 used for transfection was 1 ul, and the transfection complex was prepared by mixing with siRNA according to its instruction manual. Lipoplex siRNA/DNCA/CLD was prepared according to the method of Example 3. The complex was added to the culture plate. After culturing for 48 hours, 0.5 ml of TRizol reagent was added to each well to extract total RNA. The RNA was reverse transcribed into cDNA using a reverse transcription kit, and real-time fluorescent quantitative PCR was performed to investigate the activity of siMB3 and its conjugates. Target mRNA silencing ability, the experimental results are shown in Figure 4.

3. Results

Firstly, the efficiency of siRNA transfection using commercial transfection reagent and DNCA/CLD mixed preparation was compared, and it was found that the gene silencing activity of siRNA encapsulated by mixed lipid material DNCA/CLD was better than that encapsulated using commercial transfection reagent Lipofectamine 2000 (lipo group), and had Significant difference (p<0.0001).

The gene silencing activity of 5′-single conjugates of antisense strands was investigated, and it was found that the gene silencing ability of end-conjugated short linkers (LOAs/S and L1As/S) and unconjugated siMB3 (As/S) had no significant gene silencing ability Sex difference (p>0.05). Continued to investigate the gene silencing activity of R0-RL4, a series of conjugates with a larger bulk group cRGD at the end, the results showed that the gene silencing activity was different and related to the length of the linking arm, with a longer linking arm (RL2As /S, RL3As/S and RL4As/S) 5′-monoconjugates were more active than conjugates linked by shorter linking arms (RL1As/S and RL0As/S), this result may be due to The insertion of a longer linker arm between the 5'-end of the sense strand and cRGD will loosen the structure of the conjugated moiety and reduce the steric hindrance when loading the siRNA conjugate into RISC. RL2As/S-siMB3 showed less difference in activity from siMB3, probably because it could adopt an appropriate conformation to avoid steric conflict with RISC and thus have similar gene silencing activity to the unconjugated conjugate.

Single-conjugated cRGD at the 5'-end of the sense strand had no apparent effect on gene silencing activity (As/RLOS-siMB3), even with the shortest linker arm. Compared with unconjugated siMB3, the gene silencing activity of the biconjugate decreased slightly, but the difference in activity was not obvious at this concentration. When cRGD was conjugated to the 5′-end of the sense strand through a shorter linking group Afterwards, the antisense strand 5'-conjugated large groups were more active than conjugated smaller groups.

Example 6 Investigate the antitumor activity and empty vector cytotoxicity of 5-terminal single/double conjugated siRNA neutral/cationic lipid complexes

1. Sample:

As/S-siMB3, L0As/S-siMB3, L1As/S-siMB3, RL0As/S-siMB3, RL1As/S-siMB3, L1As/L0S-siMB3, RL0As/RL0S-siMB3, RL1As/RL0S-siMB3, As/ RL0S-siMB3, L0As/RL0S-siMB3, L1As/RL0S-siMB3. See Example 1 for the preparation of the above 5-terminal single-double conjugate, and see Example 3 for the preparation of siRNA lipoplex siRNA/DNCA/CLD.

2. cck-8 experiment

A375 was plated in a 96-well plate at 10,000/well, and transfected after culturing for 18 hours. The administration concentration of siMB3 and its conjugates was 100nM, and the siRNA lipid complex was added to the culture plate. After culturing for 72h, the medium was discarded, 10% cck-8 reagent was added, and the siRNA was incubated at 37°C for 2h. The absorbance at 450 nm was measured by the instrument. The results were normalized by taking the absorbance of the control group as 1. The antitumor activity of siMB3 and its conjugates and the cytotoxicity of the empty carrier were investigated. The experimental results are shown in Figure 5.

3. Results

The cell-level antitumor activity of single-conjugated siMB3/DNCA/CLD lipoplexes was evaluated, and it was found that conjugation to the 5′-terminus of the antisense strand reduced its antitumor activity in vitro, and was associated with the conjugation group. Size doesn't matter. After the 5′-end of the sense strand was conjugated, the antitumor activity also decreased. Among the biconjugates, only the gene silencing activity of the biconjugate L1As/LOS was similar to that of the non-conjugate As/S, and the same was true for L0As/RLOS Has good antitumor activity. The cytotoxicity of DNCA/CLD empty vector (mix group) was low, suggesting that the application of lipid complexes in vitro is safe and low in toxicity.

Example 7 Investigating the distribution of 5-terminal monoconjugated siRNA neutral/cationic lipid complexes in animals

1. Sample:

As/Cy7-S-siMB3, RL0As/Cy7-S-siMB3, the 5'-end of the sense strand of siMB3 was labeled with the fluorescent dye Cy7. See Example 1 for the synthesis of the antisense strand conjugate, and Example 2 for the preparation of the siRNA lipoplex siRNA/DNCA/CLD/PEG2000-DSPE.

2. Method:

A375 cells were inoculated in the armpit of 6-week-old nude mice, and the tumor volume reached approximately 500 mm ³ after 10 days. Subsequently, DNCA/CLD/PEG2000-DSPE-encapsulated Cy7-labeled siMB3 (As/S) or cRGD conjugate (RL0As/S) was injected via tail vein, and the dose of siRNA was 1.5 mg/kg. The fluorescence signal of siRNA was measured by in vivo imaging system from 1.5h to 36h after administration. 36h after administration, animals were euthanized, and tumor tissues and organs were removed and photographed. The experimental results are shown in Figure 6.

3. Results:

The intratumoral accumulation of RL0As/S could be clearly observed during the whole photographing process, but the As/S without cRGD had only a weak fluorescence signal (Fig. 6a). The intratumoral fluorescence signal was quantified, and the results showed that the tumor accumulation of cRGD-conjugated siMB3 was about 90% higher than that of unconjugated siMB3 (p<0.05). The internal fluorescence signal was significantly higher than that of the BLANK group (p<0.01). However, due to autofluorescence of food in the gastrointestinal tract, it was difficult to observe the siRNA signal in liver and kidney, but it was not related to the fluorescence signal in the tumor.

In vitro results (Figure 6b) can be found that Cy7-labeled siMB3 mainly accumulates in tumors and livers, and the fluorescence signal of RL0As/S in tumors is stronger than that of As/S. Correspondingly, the intratumoral accumulation ability of cRGD-conjugated siMB3 (RLOAs/S) was stronger than that of unconjugated siMB3 (p<0.05). The above results show that the DNCA/CLD/PEG2000-DSPE mixed carrier can efficiently deliver siMB3 and its conjugates to tumor tissues, and there is still obvious intratumor and liver distribution 36 hours after administration. The conjugation and delivery of this combination The strategy provides a platform for systemic delivery of siRNA to treat tumors in vivo.

Example 8 Investigate the antitumor activity of 5-terminal monoconjugated siRNA neutral/cationic lipid complex in animals and its mechanism

1. Sample:

As/S-siMB3, RL0As/S-siMB3, RL2As/S-siMB3. See Example 1 for the synthesis of siRNA and conjugate, and Example 2 for the preparation of siRNA lipoplex siRNA/DNCA/CLD/PEG2000-DSPE.

2. Method:

160w melanoma cells A375 were inoculated into the armpits of 3-week-old nude mice. After 7 days of tumor formation, the nude mice were divided into 5 groups. At this time, the average tumor volume was about 50 mm ³ (day 0), with 5 mice in each group. Tail vein injection of siMB3 and conjugate mixture for treatment, blank control is BLANK (tail vein injection preparation solvent Genopti), negative control is siMB3 (unencapsulated siMB3), mix (DNCA/CLD/PEG2000 without siRNA) -DSPE empty vector), mix-NC (mix-encapsulated siFL targeting firefly luciferase mRNA). The siRNA was administered at a dose of 2 mg/kg (RL0As) by tail vein injection on days 1, 3, 5, and 8 (RL0As/S) or on days 1, 3, 5, 7, and 9 (RL2As/S). /S) or 1.48 mg/kg (RL2As/S). During the treatment period, the tumor size and the mouse body weight were measured every day or every two days, and the tumor volume was calculated according to the formula tumor volume V=length× ^width2 ×0.5. 48h after the last administration, 1 ml of blood was collected, and then the mice were euthanized, and the tumor tissue was isolated, weighed, and photographed for recording. A portion of tumor tissue (50-100 mg) was added to TRizol and homogenized to extract total RNA. The RNA was reverse transcribed into cDNA using a reverse transcription kit, and real-time quantitative PCR was performed to investigate the intratumoral mRNA silencing ability of siMB3 and its conjugates. Another part of tumor tissue (50-100 mg) was fixed in paraformaldehyde overnight, then embedded in paraffin, and sectioned, and stained for nuclei and BRAF ^V600E protein marker. The blood of mice was kept at 4°C overnight, and the serum was separated by low-speed centrifugation for 15 minutes, and then the biochemical indicators of liver and kidney in the serum were detected. The experimental results are shown in Figure 7, Figure 8, Figure 9, and Figure 10.

3. Results:

The in vivo antitumor activity of RLOAs/S-siMB3 linked by the shorter linker arm was first examined (Figure 7). The results showed that siMB3 and its conjugates could significantly inhibit tumor growth during the entire treatment process after encapsulation (Fig. 7a, p<0.001), but the ability of conjugate RLOAs/S to inhibit tumor growth was weaker than that of non-conjugate As/S, despite the p-value of 0.25, was not significantly different. During the whole administration and treatment process, the body weight of the mice increased steadily, and there was no difference between the groups, indicating that the conjugation did not affect the safety of the siRNA lipoplex (Fig. 7b). The mice were euthanized 48 hours after the last administration and the tumors were taken out and weighed (Fig. 7c). The tumor volume of mix-As/S was the smallest, and the tumor volume of mix-RL0As/S was also reduced to a certain extent compared with the blank and negative controls. The cure failure rate was 2/5, and two of the five mice had tumors similar in size to the control group. RT-qPCR assay of intratumoral BRAF ^V600E mRNA levels found that As/S could silence target mRNA more effectively than RLOAs/S (Figure 7d). The immunohistochemical results were consistent with the qPCR results (Fig. 7e), in tumor paraffin sections of mice, both siMB3 and conjugate-treated groups significantly reduced the expression of BRAF ^V600E protein (brown spots).

In vivo anti-tumor experiments show that although RLOAs/S with cRGD targeting groups have better tumor accumulation ability, the spatial distance between their conjugating groups and antisense strands is too close, which greatly affects gene expression. silencing activity, so the therapeutic effect of the RLOAs/S conjugate was weaker than that of the unconjugated siMB3. Combined with cell experiments, the in vivo antitumor activity of RL2As/S was investigated (Fig. 8).

After 5 tail vein administrations, the tumor growth rate of mix-siMB3 and its conjugate mix-RL2As/S was significantly lower than that of BLANK (p<0.0001) and lower than that of unencapsulated siRNA (p<0.001) and mix-NC group (p<0.001) (Fig. 8a). The tumor volume of the blank and negative control groups was more than 25 times that of day 0, the mix-As treatment group increased by 12 times, and the tumor growth rate of the mix-RL2As/S group was much slower than that of the mix-As/S group ( p<0.05), the tumor volume in the mix-RL2As/S treatment group increased only about 6-fold. The siMB3 lipoplex with the cRGD-conjugating group attached to the 5'-terminus of the antisense strand via a longer linking arm can treat tumors more effectively than the unconjugated siMB3 lipoplex. 48h after the last administration, the mice were euthanized, and the tumor tissues were isolated and weighed (Fig. 8b) and recorded by photographing (Fig. 8c). As shown in Figure 8b, the tumor weight of the mix-As/S group was significantly smaller than that of the blank and negative control groups (p<0.05), and the tumor weight of the conjugate mix-RL2As/S group was also significantly lower than that of the blank and negative control groups. small (p<0.01). There was no significant difference between conjugated and unconjugated tumors (p>0.5), but the mean tumor weight of the mix-RL2As/S group was smaller than that of the mix-As/S group. The size of the ex vivo tumor volume in each group was consistent with the tumor growth curve (Fig. 8c). RT-qPCR assay of intratumoral BRAF ^V600E mRNA levels found that As/S and RL2As/S could more effectively silence about 40% of the target mRNA expression (Fig. 8d), and were significantly different from the blank and negative control groups (p<0.001). ). The protein level of BRAF ^V600E in the tumor was simultaneously assessed by immunohistochemical experiments, and the yellow spots represented the expression of BRAF ^V600E protein (Fig. 8e). Compared with NC, both siMB3 and RL2As/S down-regulated BRAF ^V600E , while the silencing efficiency of RL2As/S and As/S was similar. The excellent anti-tumor ability of RL2As/S is not only related to the conjugation of the targeting group, which increases the accumulation of siMB3 in tumor tissues, but also to the extension of the linking arm between the conjugation group and the 5′-end of the antisense strand, which makes the siRNA more effective. Gene silencing activity is restored. Because at the end of the in vivo experiments, RLOAs/S-treated tumors grew faster than unconjugated siMB3 (Fig. 7a), although there was no significant difference in tumor volume (p=0.25). These results suggest that the tumor-targeting conjugation group cRGD increases the accumulation of siMB3-conjugated nanoparticles in tumor tissues, and that the extended spatial distance between cRGD and the 5′-terminus (L2 compared to L0) is Key points for excellent antitumor efficacy of antisense strand 5'-end conjugates. Furthermore, stable body weights and no mouse death during treatment indicated that all conjugates were sufficiently safe for in vivo application (Fig. 8f).

In order to verify the liver and kidney toxicity of the conjugates and preparations, 48 hours after the last administration, whole blood was collected, and the blood biochemical indexes of the mice were measured (Fig. 9). The blood biochemical indexes of CREA-J, UA, UREA representing renal function and ALT, AST, ALP, TP, and ALB of liver function in each group were not significantly different from those of the non-administered group, indicating that the conjugation and DNCA/CLD/PEG2000-DSPE The mixed lipid preparation has good safety and almost no liver and kidney toxicity. The siMB3/DNCA/CLD/PEG2000-DSPE lipid complex can be used as a safe and effective tumor therapy in mice.

To verify the mechanism of siMB3 conjugates in treating tumors, the expression level of intratumoral BRAF ^V600E mRNA was determined after a single dose. Balb/c-nude 4-week-old mice were used to inoculate 160w of A375 cells in the right armpit. 20 days after inoculation, when the tumor volume was about 500-800mm ³ , the mice were randomly divided into four groups and administered by tail vein injection respectively. 48h after administration, the animals were euthanized, and 50-100 mg of tumor tissue was taken to quantify the mRNA expression. As shown in Figure 10, siMB3 silenced intratumoral BRAF ^V600E mRNA expression after a single administration. Unconjugated As/S silenced 14% of the target mRNA, and RL2As/S with cRGD conjugated to the 5′-end of the antisense strand silenced 22% of the target mRNA expression, which was significantly different from the mix-NC group (NC). vs. As/S, p<0.05; NC vs. RL2As/S, p<0.001). The slightly lower level of intratumoral target gene inhibition by siRNA may be due to the large tumor volume at the time of administration, and the siRNA could not be fully transfected into all cells in the tumor tissue, and the gene silencing activity was more pronounced after multiple administrations (Figure 8d). The inhibition of tumor growth by RL2As/S at the animal level is associated with silencing of BRAF ^V600E mRNA.

The information shown and described in detail herein is sufficient to accomplish the foregoing objects of the invention, and thus preferred embodiments of the invention represent the subject matter of the invention, which is broadly encompassed by the invention. The scope of the present invention fully encompasses other embodiments apparent to those skilled in the art, therefore, the scope of the present invention is not to be limited by anything other than the appended claims, wherein the singular form of elements used is used unless explicitly stated otherwise. It does not mean "one and only", but "one or more". All known structural, compositional and functional equivalents of the above-described preferred embodiments and parts of the additional embodiments to those of ordinary skill in the art are hereby incorporated by reference and are intended to be encompassed by the present claims.

Furthermore, no particular apparatus or method is required to express each and every problem solved by the present invention, for they are intended to be encompassed within the claims of the present invention. Furthermore, no matter whether or not all parts, elements, or method steps of the present disclosure are expressly recited in the claims, they are not dedicated to the public. However, it will be apparent to those of ordinary skill in the art that various changes and modifications in form, reagents and details of synthesis can be made without departing from the spirit and scope of the invention as set forth in the appended claims .

Claims (9)

1. A chemical modification method of small interfering RNA (siRNA) is characterized by comprising the steps of conjugating one or more linking groups as conjugation groups at the 5' -end of a sense strand or/and an antisense strand of the siRNA, linking targeting groups on active ester at the end of the linking groups through condensation reaction, and carrying by using mixed lipid materials of neutral lipid materials and cationic lipid materials, wherein the structural formula of the linking groups is shown as a chemical formula I or a chemical formula II, the structure shown as the chemical formula I is a six-carbon unit linking group, and the structure shown as the chemical formula II is a three-carbon unit linking group; the structural formula of the targeting group is shown as a chemical formula VI;

wherein, calculated according to the mole percentage, the mixed lipid material consists of 72.7 percent of neutral cytidine lipid material shown in a chemical formula III and 27.3 percent of cationic lipid material shown in a chemical formula IV, and the mole ratio of the total lipid to the siRNA is 10: 1; or the mixed lipid material consists of 39.7 percent of neutral cytidine lipid material shown in a chemical formula III, 59.6 percent of cationic lipid material shown in a chemical formula IV and 0.7 percent of PEG2000-DSPE shown in a chemical formula V, and the molar ratio of the total lipid to the siRNA is 5.3: 1:

2. the chemical modification method of claim 1, wherein the linking groups of formula VII and VIII are prepared as phosphoramidite monomers of formula IX and X, respectively, prior to the conjugation of the siRNA, and then conjugated to the siRNA via a phosphodiester bond; then connecting a targeting group shown in a chemical formula VI on the active ester of the phosphoramidite monomer shown in a chemical formula IX or a chemical formula X through a condensation reaction;

3. the chemical modification method of claim 2, wherein the natural and linking group-conjugated siRNA sense and antisense strand sequences are synthesized by phosphoramidite method using solid phase synthesis technique, the sequences are synthesized from 3 ' -end to 5 ' -end on RNA synthesizer, one cycle for each coupled nucleoside, after 21 couplings of natural nucleoside phosphoramidite monomers, one or several of said phosphoramidite monomers IX or X are coupled at the corresponding position of 5 ' -end of the sequence, each cycle comprising four reactions: DMT removal, coupling, sealing and oxidation.

4. A chemical modification process as claimed in claim 3, wherein the coupling time after each cycle of phosphoramidite monomer injection is 600 seconds per cycle and 3 couplings.

5. The chemical modification method according to claim 1, wherein the method of encapsulating with a mixed lipid material of a neutral lipid material and a cationic lipid material comprises: dissolving the mixed lipid material in an ethanol solution, dissolving siRNA of which the 5' -end of the antisense strand or/and the sense strand is/are conjugated with one or more linking groups through a phosphodiester bond in DEPC water, mixing the siRNA with an unbuffered solution or a buffer solution, carrying out ultrasonic treatment, filtering the mixture by using a 0.22 mu m filter membrane, and obtaining a filtrate containing modified siRNA nano-particles.

6. A chemical modification method as claimed in claim 5, wherein the buffer solution is physiological saline, water or any transfection optimisation solution, the buffer solution comprising acetate, citrate, carbonate, phosphate or any combination thereof.

7. The chemical modification method of any one of claims 1-6, wherein the chemical modification strategy further comprises co-use with other chemical modification strategies, including 2 '-O-methyl (2' -OMe), 2 '-fluoro (2' -F), 2 '-O-methoxyethyl (2' -O-MOE), Locked Nucleotides (LNA), phosphosulfur backbone modifications, and other end conjugation modalities for siRNA.

8. The binding 5' -end conjugate and the neutral/cationic mixed lipid preparation synthesized according to the chemical modification method of any one of claims 1 to 7 entrap the modified small interfering RNA.

9. The small interfering RNA of claim 8, wherein the 5' -end conjugate modified small interfering RNA sequence is selected from the group consisting of:

(1) the antisense strand or the sense strand obtained by conjugating a linking group shown in a chemical formula II at the 5' -end of the sense strand or the antisense strand of the small interfering RNA sequence;

(2) the antisense strand or the sense strand obtained by conjugating one or more linking groups shown in a chemical formula I and a chemical formula II along the 3 ' -5 ' direction at the 5 ' -end of the sense strand or the antisense strand of the small interfering RNA sequence;

(3) the method comprises the steps of conjugating a linking group shown in a chemical formula II at the 5' -end of a sense strand or an antisense strand of a small interfering RNA sequence, completing conjugation by using a solid phase synthesizer, using a phosphoramidite monomer shown in a chemical formula X, and connecting a targeting group shown in a chemical formula VI on an active ester of a group shown in the chemical formula X through a condensation reaction to obtain the sense strand or the antisense strand;

(4) the antisense strand or sense strand conjugate is prepared by sequentially conjugating one or more linking groups shown in chemical formula I and one linking group shown in chemical formula II along the 3 ' -5 ' direction at the sense strand or the 5 ' -terminal of the antisense strand of the small interfering RNA sequence by using a solid phase synthesizer, using phosphoramidite monomers shown in chemical formulas IX and X, and then connecting a targeting group shown in chemical formula VI on an active ester of a group shown in chemical formula X through condensation reaction;

the antisense strand and the sense strand in the above (1) to (4) are in a freely combined form.

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