CN112707943A

CN112707943A - Small interfering RNA (ribonucleic acid) combined with 5' -terminal conjugate and neutral/cationic mixed lipid material package and modification method thereof

Info

Publication number: CN112707943A
Application number: CN202011644259.1A
Authority: CN
Inventors: 杨振军; 于丽佳; 周新洋; 潘宇飞
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-27
Anticipated expiration: 2040-12-31
Also published as: CN112707943B

Abstract

The invention discloses a small interfering RNA (ribonucleic acid) combined with a 5' -terminal conjugate and a neutral/cationic mixed lipid preparation entrapment modification and a modification method thereof. The modification method comprises the steps of conjugating one or more linking groups as conjugation groups at the 5' -tail ends of the antisense strand or/and the sense strand of the siRNA, carrying out entrapment by utilizing a neutral lipid material and a cation mixed lipid material, and linking the targeting groups on the tail end active esters of the linking groups through condensation reaction. The siRNA lipid complex obtained by the chemical modification strategy provided by the invention has better biological activity, the cell level shows excellent transmembrane transport capacity and the silencing activity of target mRNA, the animal level shows high-efficiency target organ distribution capacity and the capacity of inhibiting tumor growth, the safety of the application in the cell level and the animal level is good, no obvious toxicity exists, and the basis is laid for the wide clinical application of the siRNA technology.

Description

Small interfering RNA (ribonucleic acid) combined with 5' -terminal conjugate and neutral/cationic mixed lipid material package and modification method thereof

Technical Field

The invention relates to a combined 5' -end conjugate and neutral lipid/cation mixed lipid entrapped modified small interfering RNA (siRNA), a chemical modification method thereof and a carrier delivery strategy. The invention realizes the conjugation of the 5' -end of the siRNA antisense strand, and the existence of the conjugate does not influence the gene silencing activity of the siRNA. The siRNA modified by conjugation and entrapped by the method has the advantages of good stability, high delivery efficiency, high distribution of target organs in vivo, strong cell-entering capability, low toxicity, good biological activity and the like, and can be widely applied to the research of drugs for resisting tumors, viruses and metabolic diseases. The invention belongs to the technical field of biological medicines.

Background

Small interfering RNA (siRNA) is a drug candidate molecule with great clinical prospect, the target of the siRNA is mRNA in cells, and the expression of the target gene can be directly silenced at the gene level, so that the occurrence and the development of diseases are fundamentally prevented. Native siRNA is cleaved intracellularly by Dicer to the 21-25mer by dsRNA, usually with a 5 '-phosphate group and a 3' -hydroxyl group. The 5 '-hydroxyl of the synthesized siRNA is rapidly phosphorylated in cells by Clp1 cell kinase, which is also a necessary condition for the siRNA to exert activity, because the siRNA can enter RISC to exert gene silencing activity only after 5' -phosphorylation. Compared with the traditional micromolecule medicine, the siRNA has the advantages of higher specificity, lower toxicity and side effect, easy preparation and the like. But clinical use is limited due to poor serum stability, difficulty in transmembrane, susceptibility to off-target, immune response stimulation, and other drawbacks.

In 2018, FDA (US food and drug administration) approved the first siRNA drug

(Patisiran), which is a candidate therapy for hATTR (hereditary transthyretin amyloidosis), is delivered using liposomes composed of DLin-MC3-DMA, DSPC, PEG-DMG, and cholesterol. In vitro experiments showed that 10nM siRNA knockdown 95% TTR mRNA expression in HepG2 cells, ED in rodent models₅₀It was 0.03 mg/kg. The siRNA drug has better safety than the antisense nucleic acid drug Inotersen of hATTR

2019, FDA approved a second siRNA drug GIVLAARI^TM(givosiran), which is the only effective treatment for acute hepatic porphyria other than intravenous heme, and heme is extremely difficult to obtain. Givosiran was delivered using GalNAc conjugation at an effective dose of 1-3mg/kg in rodent models. With the rapid development of genomics, the success of RNAi (RNA interference) drugs suggests the beginning of a new era of personalized therapy, but unfortunately current marketed drugs can only be delivered hepatologically using LNP delivery systems and GalNAc conjugates targeting the hepatic asialoglycoprotein receptor. Moreover, since none of the siRNAs currently under clinical phase III study is encapsulated by a carrier, the application of cationic lipid materials relying on charge action has been trapped.

The cationic lipid material is combined with the siRNA by means of coulomb force action between positive and negative charges and entraps the siRNA. However, the cationic liposome has strong toxicity, and is easy to combine with negatively charged serum protein under physiological conditions, so that immunogenicity and hepatotoxicity are generated, and siRNA is easy to release outside a target organ. These drawbacks limit the further use of cationic liposomes for drug delivery of entrapped siRNA.

The inventor previously designed and synthesized a basic acetamide glycerol ether molecule DNCA (CN108059619A), which has a head with basic properties and can combine and entrap single-stranded nucleic acid drugs and plasmids through hydrogen bonding and pi-pi stacking (CN 1084478807A). The co-entrapment of 3', 3 "-double peptide-conjugated siRNA with lysine as the head cationic lipid CLD, which was synthesized by combining DNCA and previous design of the inventors, has been successfully applied at the cellular level (Mol Pharm,2019,16, 4920). Here, we optimized the entrapment procedure, reformulated and explored the optimal DNCA/CLD transfection protocol, and added DSPE-PEG to optimize the in vivo application properties of the mixed preparation. By combining a conjugation strategy and a chemical modification method which use a phosphodiester bond as a link between the siRNA chain end and a conjugation group, a high-efficiency and low-toxicity conjugate/neutral/cation mixed lipid siRNA drug entrapment delivery system is obtained.

Disclosure of Invention

In order to improve the specificity of target organ delivery, the cell-entering efficiency and the effectiveness of intracellular release in the in vivo delivery process of small interfering RNA (siRNA), the invention provides a chemical modification method for combining siRNA coated by a 5' -end conjugate and a neutral lipid material/cationic lipid material mixed carrier and a carrier delivery strategy. The invention realizes the rapid synthesis of the conjugate by using a solid phase synthesizer through preparing the conjugate group into the phosphoramidite monomer. The invention uses the mixed lipid material of neutral lipid material and cation to encapsulate the siRNA conjugate, so as to realize more effective, safe and nontoxic siRNA delivery in vivo, thereby improving the drug property of siRNA.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention relates to a chemical modification method of small interfering RNA (siRNA), which comprises 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, and carrying out entrapment by using a neutral lipid material and a cation mixed lipid material, wherein the structural formula of the linking groups is shown in chemical formula I or chemical formula II, the structure shown in the chemical formula I is a six-carbon unit linking group, and the structure shown in the chemical formula II is a three-carbon unit linking group;

wherein the mixed lipid material comprises neutral cytidine lipid material shown in chemical formula III and cationic lipid material shown in chemical formula IV, or comprises neutral cytidine lipid material shown in chemical formula III, cationic lipid material shown in chemical formula IV and PEG2000-DSPE shown in chemical formula V:

wherein, preferably, when one or more linking groups are conjugated at the 5' -terminal of the antisense strand or/and the sense strand of the siRNA, a targeting group is linked on the terminal active ester of the linking group through a condensation reaction, and the structural formula of the targeting group is shown in formula VI:

wherein, preferably, before the siRNA is conjugated, the linking groups shown in chemical formulas VII and VIII are respectively prepared into phosphoramidite monomers shown in chemical formulas IX and X, and then the phosphoramidite monomers are conjugated with the siRNA through 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;

preferably, the sequences of the sense strand and the antisense strand of the natural and conjugated siRNA with the linking group are synthesized by a phosphoramidite method by using a solid phase synthesis technology, the sequences are synthesized from the 3 ' -end to the 5 ' -end on an RNA synthesizer, each coupled nucleoside is a cycle, after the natural nucleoside phosphoramidite monomer is coupled for 21 times, one or more phosphoramidite monomers IX or X are coupled at the corresponding positions of the 5 ' -end of the sequences, and each cycle comprises four reactions: DMT removal, coupling, sealing and oxidation.

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

Wherein, preferably, the mixed lipid material consists of 72.7 percent of neutral cytidine lipid material shown in chemical formula III and 27.3 percent of cationic lipid material shown in chemical formula IV according to molar percentage, wherein, preferably, the molar 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, wherein the mol ratio of total lipid to siRNA is preferably 5.3: 1.

Among them, the preferable method of encapsulating with the mixed lipid material of the neutral lipid material and the cationic lipid material is: 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 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, performing ultrasonic treatment, filtering the mixture by using a 0.22 mu m filter membrane, and obtaining a filtrate containing the modified siRNA, wherein the unbuffered solution is preferably physiological saline, water or any transfection optimization solution, and the buffer solution contains acetate, citrate, carbonate, phosphate or any combination thereof.

The chemical modification strategy also comprises the common use with other chemical modification strategies, including 2 '-O-methyl (2' -OMe), 2 '-fluoro (2' -F), 2 '-O-methoxyethyl (2' -O-MOE), Locked Nucleotide (LNA), phosphorus-sulfur skeleton modification and other end conjugation modes of siRNA and the like.

Further, the invention also provides a binding 5' -end conjugate synthesized according to any one of the chemical modification methods and a neutral/cation mixed lipid carrier for encapsulating the modified small interfering RNA.

Compared with the conjugation of the 3 '-terminal, the 5' -terminal conjugation mode has the following characteristics:

a. the conjugate group is prepared into a corresponding phosphoramidite monomer, so that the conjugate part can be directly used as a monomer to be synthesized by a solid phase synthesizer, and the synthesis is simple and rapid;

b. active ester is reserved at the tail end of the linking group X and is used as an active reaction site, condensation reaction can be utilized to efficiently link various functional groups, and the scale of the conjugate is rapidly expanded;

c. the linking groups are connected by phosphodiester bonds and can be hydrolyzed and sheared by enzyme in cells to release unconjugated siRNA single chains with phosphate ends, and finally the capacities of being easier to load RISC compounds and improving siRNA gene silencing activity are achieved.

In particular embodiments of the invention, the modified sequence of the 5-terminal conjugate 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.

The siRNA and the conjugate thereof are RNA double-stranded complexes formed by annealing conjugated or unconjugated modified antisense strands and sense strands according to requirements.

Taking RL1As/RL0S-siRNA As an example, a linking group represented by formula I (the number of the linking groups represented by formula I is represented by a numeral) and a linking group represented by formula II (represented by L) are conjugated to the 5 ' -end of the antisense strand (As) of the siRNA in sequence (3 ' -5 ' direction) by solid phase synthesis, wherein the linking groups are linked by a phosphodiester bond, and then a targeting group cRGD (R is represented by formula VI) is conjugated by condensation reaction, namely RL1 As. A chemical formula II (shown in L) is conjugated at the 5' -end of a sense strand (shown in S) through solid phase synthesis, a targeting group cRGD (shown in R) shown in a chemical formula VI is conjugated through condensation reaction, namely RL0S, then the synthesized and purified antisense strand and the sense strand are annealed according to the molar ratio of 1:1.1 to form RL1As/RL0S-siRNA, and the rest of conjugated siRNA is similar.

In one embodiment of the invention, the siRNA to be modified is targeted to BRAF in the ERK pathway^V600EMutant mRNA siMB3 sequence. The sequence before modification of siMB3 is as follows:

siMB3 sense strand 5 '-GCUACAGAGAAUCUCUCGAUdtdt-3'

Antisense chain 5 '-AUCGAGAUUUCUGUGAGCdtdt-3'

In one embodiment of the present invention, when the siMB3 sequence is encapsulated by a mixture of neutral lipid material of formula III, cationic lipid material of compound of formula IV, and compound of formula V, the mixture can be uniformly distributed in the form of spherical vesicles, and the siRNA lipid complex has uniform particle size and surface charge neutral about 100 nm.

In one embodiment of the invention, siMB3 and its antisense strand end are conjugated with a conjugate group of formula I and formula VI, and the mixed lipid material has better cell-entering efficiency and gene silencing activity when encapsulated with a commercial cationic transfection reagent Lipofectamine2000 or a mixed lipid material consisting of a neutral lipid material of formula III, a compound cationic lipid material of formula IV and a compound of formula V.

In a specific embodiment of the present invention, when three conjugate groups shown in formula I and one conjugate group shown in formula II are sequentially conjugated to the 5-terminal of the antisense strand of the siMB3 sequence, and one targeting group cRGD shown in formula VI is conjugated through an active ester reaction, and a mixed lipid material composed of a neutral lipid material shown in formula III, a compound cationic lipid material shown in formula IV, and a compound shown in formula V is entrapped, the optimal cellular uptake ability is obtained.

In a specific embodiment of the present invention, three conjugation groups of formula I and one conjugation group of formula II are sequentially conjugated to the 5-terminal end of the antisense strand of the siMB3 sequence, and the conjugation is performed using a solid phase synthesizer, and the phosphoramidite monomers used are formula IX and X, wherein the conjugation groups are linked by a phosphodiester bond, and then a targeting group cRGD of formula VI is linked to the active ester of the group of formula X by a condensation reaction, and the mixed lipid material composed of the neutral lipid material of formula III, the cationic lipid material of formula IV and the compound of formula V has optimal gene silencing activity at a cell level.

In a specific embodiment of the present invention, three conjugation groups of formula I and one conjugation group of formula II are sequentially conjugated to the 5-terminal end of the antisense strand of the siMB3 sequence, and the conjugation is performed using a solid phase synthesizer, and the phosphoramidite monomers used are formula IX and X, wherein the conjugation groups are linked by a phosphodiester bond, and then a targeting group cRGD of formula VI is linked to an active ester of the group of formula X by a condensation reaction, and the mixed lipid material composed of a neutral lipid material of formula III, a cationic lipid material of compound of formula IV, and a compound of formula V is entrapped, so that the anti-tumor activity in animals is optimized.

In a specific embodiment of the invention, the 5-terminus of the antisense strand of the siMB3 sequence described above is conjugated with a conjugate group of formula II. The conjugation is completed by using a solid phase synthesizer, the used phosphoramidite monomer is a chemical formula X, and then a targeting group cRGD shown in a chemical formula VI is connected on the active ester of the group shown in the chemical formula X through condensation reaction, and when the mixed lipid material composed of the neutral lipid material shown in the chemical formula III, the compound cation lipid material shown in the chemical formula IV and the compound shown in the chemical formula V is entrapped, the optimal distribution of the tumor tissues in the animal body is achieved.

Compared with the prior art, the invention has the advantages that:

1. according to the chemical modification strategy combining targeting group conjugation and neutral/cation mixed carrier entrapment, the obtained siRNA lipid complex has better biological activity, the cell level shows excellent transmembrane transport capacity and silencing activity of target mRNA, the animal level shows high-efficiency target organ distribution capacity and tumor growth inhibition capacity, the safety of application at the cell level and the animal level is good, no obvious toxicity exists, and a foundation is laid for wide clinical application of the siRNA technology;

2. the neutral nucleoside lipid material has a base head, can be combined with siRNA through hydrogen bond action and pi-pi accumulation action, is more stable in vivo application compared with charge action between a cationic lipid material and siRNA, is not easy to adsorb charged particles or proteins in a circulation process, and avoids disintegration of a lipid complex outside a target organ and release of siRNA. And the dosage of the cationic lipid material in the preparation formula is reduced based on the combination of the non-electric effect, and the nitrogen-phosphorus ratio of the cationic lipid material to the siRNA is only 3/1, which is far lower than the dosage of the cationic lipid material in other reports.

The 3.5 '-conjugation has simple and rapid synthesis, can be used as a substrate to efficiently expand conjugate combination, can release a siRNA antisense chain with a phosphate at the 5' -end during intracellular degradation and metabolism, and is more easily introduced into a RISC compound to play the gene silencing activity. The invention proves that the conjugation of the 5' -end of the antisense strand does not influence the gene silencing activity of siRNA, expands the conjugation site of siRNA and enriches the modification strategy of siRNA.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) photograph of siRNA/DNCA/CLD/PEG-DSPE lipid complex and empty carrier;

FIG. 2 is the cellular uptake (10nM,4h) of the 5' -terminally single conjugated siRNA neutral/cationic lipid complex of the antisense strand;

FIG. 3 shows the intracellular distribution (50nM,4h) of siRNA neutral/cationic lipid complex single-conjugated at the 5' -end of the antisense strand, wherein blue indicates the nucleus, red indicates Cy3 labeled siRNA, green indicates the lysosome, and the scale is 10 μm;

FIG. 4 shows target gene silencing activity (50nM,48h) of 5 '-terminally mono-conjugated and 5', 5 "-di-conjugated siRNA neutral/cationic lipid complexes;

FIG. 5 shows the antitumor activity and carrier cytotoxicity (100nM,72h) of 5 '-terminally single-conjugated and 5', 5 "-double-conjugated siRNA neutral/cationic lipid complexes;

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

FIG. 7 shows the anti-tumor activity in animals with siRNA (RL0As/S) singly conjugated to the 5' -end of the antisense strand (Balb/c-nude mice, axillary inoculation A375);

a. when the tumor volume reaches about 50mm³Day 0, the formulation was administered by intravenous injection on

days

1, 3, 5, 8, and tumor volumes were measured and calculated daily; b. body weight change profile of mice throughout the treatment; c. the mice were euthanized 48h after the last dose, tumors were collected and the tumors were weighed; d. intratumoral BRAF removal^V600EmRNA expression; e. immunohistochemical investigation of BRAF in removed tumor tissue^V600EExpression, tan speck for BRAF^V600EProtein expression, blue for nuclei, black scale 50 μm. Data are expressed using mean ± SD, n ═ 5, ×) p<0.001；

FIG. 8 shows the anti-tumor activity in animals with siRNA (RL2As/S) singly conjugated to the 5' -end of the antisense strand (Balb/c-nude mice, axillary inoculation A375);

days

1, 3, 5, 7, 9, tumor volume was measured and calculated daily, and each mouse was normalized to day 0 tumor volume of 1; b. the mice were euthanized 48h after the last dose, tumors were collected and the tumors were weighed; c. taking a picture of the taken tumor for observation; d. intratumoral BRAF removal^V600EmRNA expression; e. immunohistochemical investigation of BRAF in removed tumor tissue^V600EExpression, tan speck for BRAF^V600EProtein expression, blue represents cell nucleus, black scale is 50 μm; f. body weight profile of mice throughout the treatment. Data are expressed using mean ± SD, n ═ 5, p<0.05，**p<0.01，***p<0.001，****p<0.0001；

FIG. 9 shows the biochemical indicators of liver and kidney when the antisense strand 5' -end single-conjugated siRNA (RL0As/S) is applied in animals;

figure 10 shows intratumoral target gene silencing activity after a single administration of 48h of siRNA (RL2As/S) single conjugated to the 5' -end of the antisense strand, at a dose of 1.5mg/kg, 2nmol siRNA per mouse, data expressed as mean ± SD, n ═ 4, × p <0.01, × p < 0.001.

Detailed Description

The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description of the specific embodiments proceeds. The 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.

Example 15 Single Strand solid phase Synthesis of end-conjugated siRNA

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

Normal nucleotidic phosphorylated monomers (dT, rGibu, rABz, rCAc, rU) were purchased from Huaren technologies, Inc., of Upa, Upa; CPG (CPG-dT), CAP-A and CAP-B, Oxidation I₂Liquid, Cl₃CCOOH was purchased from biotechnology, okac, beijing; A0.25M solution of 5-ethylmercapto-1H-tetrazole was purchased from Shanghai Zhi research Tech Co., Ltd.

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

the compound of formula VII (8.4g, 4.2mmol) and triethylamine (0.43g, 4.2mmol) were dissolved in 10mL of anhydrous DCM. DMTrCl (1.41g, 4.2mmol) dissolved in DCM was then 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 (1.45g, 41%). Bis (diisopropylamino) (2-cyanoethoxy) phosphine (0.64g,2.1mmol) was added to a mixture of product (0.45g, 1.1mmol) and tetrazole (0.18g, 2.6mmol) under argon and dissolved with 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 represented by formula IX (0.40g, 60.6%).

The compound of formula VIII (1.5g, 12.7mmol) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl) (2.7g, 15.1mmol) were dissolved in 10mL of anhydrous DCM (CH)₂Cl₂) In (1). N-hydroxysuccinimide (NHS) (1.6g, 14.3mmol) was then 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 (1.9g, 69.6%). Bis (diisopropylamino) (2-cyanoethoxy) phosphine (0.8g, 2.6mmol) was added to a mixture of the product (0.3g, 1.4mmol) and tetrazole (0.21g, 3mmol) under argon and dissolved with anhydrous DCM (5mL) the reaction mixture was stirred at room temperature for 2h and purified by column chromatography (PE: EA ═ 10:1) to give the compound of formula X (0.38g, 66%).

The synthesis scale is as follows: about 1.0. mu. mol

Preparation of nucleoside phosphoramidite monomer solution: weighing under the protection of argon, adding anhydrous acetonitrile, wherein 1g of monomer corresponds to 20mL of acetonitrile;

preparing a connecting arm phosphorous acylation monomer solution: the compounds of formula IX, X were weighed under argon, anhydrous acetonitrile was added, 1g monomer corresponding to 20mL acetonitrile.

Natural and conjugated siRNA antisense and sense strand sequences are synthesized by a solid phase synthesis method. On an RNA synthesizer, the sequence is synthesized from the 3 ' -end to the 5 ' -end, one cycle is taken for each coupled nucleoside, after 21 times of coupling of the natural nucleoside phosphoramidite monomers, one or more of the linked arm phosphoramidite monomers are coupled at the corresponding position of the 5 ' -end of the sequence, and each cycle comprises four reactions: DMT removal, coupling, sealing and oxidation.

Synthesizing a sequence:

use of siRNA to be modified in this example to target BRAF in the ERK pathway^V600EsiMB3 of mRNA, the sequence before modification is as follows:

siMB3: sense strand: 5 '-GCUACAGAGAAAUCU CGAUdtdt-3'

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

The modification strategy selects any one of the following:

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

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

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

(4) the 5 ' -end of the above-mentioned siMB3 sequence antisense strand or sense strand is conjugated with one or several linking groups shown in chemical formula I and chemical formula II in sequence (3 ' -5 ' direction), the conjugation is completed by using a solid phase synthesizer, the phosphorous monomers used are chemical formulas IX and X, wherein the conjugation groups are linked through a phosphodiester bond, and then the targeting group cRGD shown in chemical formula VI is connected on the active ester of chemical formula X through a condensation reaction.

The sirnas obtained after the above modification strategy are shown in table 1 below:

TABLE 1

The synthesis steps are as follows: approximately 33mg of CPG-dT was weighed each time and loaded into the synthesis column, following the RNA standard procedure of the ABI394 nucleic acid synthesizer, using the DMT-ON synthesis strategy. Taking the synthesis RL2As as an example, a synthesis sequence is set on the synthesizer sequence panel. Here, the linker arms of the formulas IX and X are also considered to encode phosphorous monomers in the sequence, i.e., 2 linker arms of the formulas IX and 1 linker arm of the formula X are encoded at the 5' -end of the As chain. And (3) manually and independently reacting the cRGD functional group after the synthesis is finished, transferring the powder in the synthesis column to a 1.5mL glass reaction bottle after the synthesis is finished, adding 1mg of the compound shown in the chemical formula XI, 100. mu.l of DIPEA and 1mL of DMF, stirring and reacting at room temperature for 24 hours, then removing the supernatant, washing twice with 1mL of ethanol, washing once with 1mL of diethyl ether, and then drying in the air.

Cleavage and deprotection of RNA: adding 750 μ l methylamine alcohol and 750 μ l methylamine water into the air dried powder, cutting, placing on a shaker, shaking at 60 deg.C and 80rpm for 90 min. The supernatant was then removed, washed three times with DEPC water, aliquoted into two 1.5mL EP tubes and finally lyophilized with a lyophilizer. Add 100. mu.l DMSO and 125. mu.l triethylamine trihydrofluoride to each tube, entangle the sealing film, 80rpm,shake at 65 ℃ for 90 min. After cooling, 100. mu.l of 3M CH was added₃And (4) adding COONa solution, swirling, adding 1mL of n-butanol solution, swirling, and standing at-80 ℃ for 30 min. Then taking out and centrifuging at 12500rpm for 10min, discarding the supernatant, leaving a white precipitate, adding 0.75mL of anhydrous ethanol, swirling, standing at-80 deg.C for 30min, centrifuging at 12500rpm for 10min, discarding the supernatant, repeating the operation once more, and freeze-drying.

Separation and purification of RNA: the sample was diluted with 400. mu.l DEPC water and dissolved, filtered through a 0.22 μm filter head, and washed twice with 200. mu.l DEPC water to a final total volume of about 800. mu.l. The sample is drawn up by a sample injection needle, and the volume of each needle is 120 mu l. Using Xbridge^TM Oligonucleotide BEH C18 OBD^TM Prep Column

10X50mm 2.5 μm nucleic acid isolation column gradient elution (0min, 98% TEAB, 2% CH)₃CN；25min，85％TEAB,15％CH₃CN, v ═ 1 mL/min). After the fractions were collected, they were lyophilized to a powder. Dissolving with 700 μ L DEPC water, Desalting with Sephadex column (HiTrap 5mL desaling) for about 2min, collecting the fraction, lyophilizing again to obtain pure product, and storing at-80 deg.C.

Example 2 neutral/cationic Mixed preparations entrapping siRNA to form lipid complexes (siRNA/DNCA/CLD/PEG2000-DSPE)

The siRNA was mixed with a mixed material composed of 39.7% of the compound of formula III (DNCA, neutral), 59.6% of the compound of formula IV (CLD, cationic), and 0.7% of the compound of formula V (PEG 2000-DSPE). These ratios refer to the mole% of the total of all lipids. The molar ratio of lipid to siRNA was 5.3: 1. DNCA was synthesized according to the method of the reference (CN108059619A), CLD was synthesized according to the method of the reference (New JChem,2014,38(10),4952-4962), and PEG2000-DSPE was purchased from Yuye corporation.

Briefly, a compound of formula III (DNCA, neutral), a compound of formula IV (CLD, cationic) and a compound of formula V were dissolved in an ethanol solution, siRNA was dissolved in DEPC water, mixed using genotti (beijing michael), and then sonicated at 70 ℃ for 10min at a sonication frequency of 150W, 40 kHz. After completion of sonication, the mixture was returned to room temperature, and then filtered through a 0.22 μm filter, whereby the filtrate contained lipid complexes.

For example, in one particular method, a fresh stock lipid solution is prepared in ethanol: 156.5mg of DNCA, 315mg of CLD, 10.5mg of PEG2000-DSPE were weighed and dissolved in 20mL of ethanol to form a lipid stock solution, which was sonicated at 37 ℃ for 4 minutes to form a homogeneous lipid mixture. Subsequently, 20. mu.l of the above stock solution was added to 480. mu.l GenOpti to form 500. mu.l of working lipid stock solution. This amount of lipid was used to form siRNA lipid complexes with 10 nmol. The siRNA dry powder was dissolved in DEPC water to form a 200. mu.M siRNA stock. Mu.l of the stock solution was added to 450. mu.l Genopti to form 500. mu.l of working siRNA stock solution, 500. mu.l of working lipid stock solution was mixed with 500. mu.l of working siRNA stock solution, centrifuged at low speed briefly, and sonicated at 70 ℃ for 10min to form lipid complexes. In vivo and in vitro experiments the formulations were filtered using 0.22 μm filters (Merck millipore) and diluted to 1 × siRNA lipid complex solution using PBS solution prior to use.

Fig. 1 shows exemplary electron micrographs (TEMs) of lipid complexes of siRNA prepared by these methods and empty carriers without siRNA. These liposomes contain siRNA targeting the BRAF in the ERK pathway unconjugated^V600EsiMB3 of mRNA. The empty carrier without siRNA had loose particles and the surface of the particles shriveled. The lipid complex added with siRNA can form spherical nanoparticles with uniform particles, and has a smooth edge and a good shape.

Dynamic light scattering showed that the mean diameter of the siRNA-loaded lipid complex was 138.7. + -. 6.6 (by density), the polydispersity was 0.188. + -. 0.051, and the surface charge was-16.9. + -. 0.4 mV; the empty carriers had an average diameter of 363.7. + -. 20.1 (in terms of density), a polydispersity of 0.635. + -. 0.045 and a surface charge of 24.3. + -. 2.8 mV. Because the sample is dehydrated and dried before TEM shooting, the particles shrink due to water loss, and DLS measures the water and particle size of the nanoparticles, so that the particle size measured by TEM is smaller than that measured by DLS.

Example 3 examination of cellular uptake of antisense strand 5-terminally single conjugated siRNA neutral/cationic lipid complexes by flow cytometry

1. Sample preparation:

As/Cy3-S-simB3, RL0As/Cy3-S-simB3, RL1As/Cy3-S-simB3, RL2As/Cy3-S-simB3, RL3As/Cy3-S-simB3, and RL4As/Cy3-S-simB 3. The 5-terminus of the sense strand of siMB3 was labeled with the fluorescent dye Cy 3. See example 1 for synthesis of antisense strand conjugates and example 2 for preparation of siRNA lipid complex siRNA/DNCA/CLD, except that the mixed formulation consists of 72.7% neutral cytidine lipid material of formula III and 27.3% cationic lipid material of formula IV. These ratios refer to the mole% of the total of all lipids. The molar ratio of total lipid to siRNA was 10: 1.

2. The method comprises the following steps:

a375 was plated in 24-well plates at 6 ten thousand/well and transfected 18h after incubation. The administration concentration of siMB3 and its conjugate was 10nM, and the amount of Lipofectamine2000 used for transfection was 0.5ul, and the transfection complex was prepared by mixing with siRNA according to the instructions for use. The compound is added into a culture plate, after 4 hours of culture, the cell surface is rinsed by using DMEM, the cells are collected by trypsinization, the fluorescence intensity in the cells is measured by a PE channel of a flow cytometer, and the cell uptake capacity of the simB3 and the conjugate thereof is examined, wherein the experimental result is shown in figure 2.

3. As a result:

compared with Lipofectamine2000, the mixed vector DNCA/CLD has more efficient capability of delivering siRNA into cells, which indicates that the presence of serum protein has little influence on the uptake of DNCA/CLD/simB3 nano-complexes even though the serum contains potential transfection competitors such as vitronectin and fibronectin.

Comparison of uptake of cRGD conjugates linked to different length linker arms revealed that uptake increased progressively as the linker arms extended (RL0As/S to RL3 As/S). The lower ability of cells to uptake RL4As/S suggests that continued extension of the linker arm length has not been able to enhance the cellular uptake capacity of the siMB3 conjugate, presumably because increasing the linker arm length while introducing more negatively charged phosphodiester linkages may affect the formation of the siMB3 conjugate/DNCA/CLD lipid complex.

Example 4 examination of the intracellular distribution of the 5-terminal monoconjugated siRNA neutral/cationic lipid complexes of the antisense strand using confocal laser microscopy

1. Sample preparation:

As/Cy3-S-simB3, RL0As/Cy3-S-simB3, RL1As/Cy3-S-simB 3. The 5-terminus of the sense strand of siMB3 was labeled with the fluorescent dye Cy 3. See example 1 for synthesis of antisense strand conjugates and example 3 for preparation of siRNA lipid complex siRNA/DNCA/CLD.

2. The method comprises the following steps:

a375 was plated at 6 ten thousand/well in a confocal dish and transfected 18h after incubation. The administration concentration of siMB3 and its conjugate was 50nM, and the amount of Lipofectamine2000 used for transfection was 1 μ l, and the transfection complex was prepared by mixing with siRNA according to the instructions for use. The complexes were added to plates and after 4h incubation, nuclei and lysosomes were stained using Hoechst, Lysobrite NIR, respectively. Incubate 30 minutes after dye addition. Cell surfaces were rinsed with DMEM to remove free dye and photographed using a confocal microscope. The intracellular distribution of siMB3 and its conjugate was examined and the results are shown in figure 3.

3. As a result:

the confocal results were consistent with the flow cytometry results, and the efficiency of delivering siRNA into cells by Lipofectamine2000 was much lower than that of DNCA/cld (mix). After the cRGD was conjugated to the 5' -end of the antisense strand, there was a significant increase in cellular uptake, indicating that the partially conjugated cRGD may not be inside the DNCA/CLD/siMB3 nanocomplex, thereby increasing the ability of α v β 3 positive cells a375 to take up lipid complexes by targeting α v β 3 integrin into the cells. Cy 3-labeled siRNA (red) is spotted on and within the cell membrane, and may be distributed in the cell membrane transport vesicles and endosomes, thereby undergoing intracellular transport. The siRNA is hardly co-localized with lysosomes (green) after being encapsulated by a DNCA/CLD mixed preparation, which suggests that the lipid complex can effectively avoid entering the lysosomes or can quickly escape from the lysosomes, thereby reducing acidification degradation of the siRNA in the lysosomes to the greatest extent.

Example 5 investigation of Gene silencing Activity of 5-terminal Single/double conjugated siRNA neutral/cationic lipid Complex Using RT-qPCR technology

1. Sample preparation:

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-simB 3. See example 1 for synthesis of the above 5-terminal mono-and di-conjugates.

RT-qPCR experiment:

a375 was plated in 12-well plates at 8 ten thousand/well and transfected 18h after incubation. The administration concentration of siMB3 and its conjugate was 50nM, and the amount of Lipofectamine2000 used for transfection was 1ul, and the transfection complex was prepared by mixing with siRNA according to the instructions for use. Lipid complex siRNA/DNCA/CLD was prepared according to the method of example 3. The complex is added into a culture plate, after 48 hours of culture, 0.5ml of TRizol reagent is added into each hole to extract total RNA, the RNA is reversely transcribed into cDNA by using a reverse transcription kit, real-time fluorescence quantitative PCR is carried out, and the silencing capability of target mRNA of the simB3 and the conjugate thereof is examined, and the experimental result is shown in figure 4.

3. Results

First, the efficiency of transfection of siRNA using the commercial transfection reagent and the mixed DNCA/CLD preparation was compared, and it was found that the siRNA gene silencing activity of the mixed lipid DNCA/CLD-loaded siRNA was superior to that of the lipo-loaded siRNA using the commercial transfection reagent Lipofectamine2000 (lipo group) and had significant differences (p < 0.0001).

The antisense chain 5' -single-conjugate gene silencing activity examination shows that the end-conjugated short connecting arms (L0As/S and L1As/S) have no significant difference (p >0.05) in the gene silencing capability with the unconjugated simB3 (As/S). Continuing to examine the gene silencing activity of RL0-RL4, a series of conjugates with ends conjugated with a bulky group cRGD, the results indicate that there is some difference in gene silencing activity and that, in relation to the length of the linking arm, 5 '-mono-conjugates with longer chain arms (RL2As/S, RL3As/S and RL4As/S) are more active than conjugates with shorter chain arms (RL1As/S and RL0As/S), which may be due to the fact that the insertion of a longer linking arm between the 5' -end of the antisense strand and the cRGD makes the structure of the conjugated moiety more loose, reducing steric hindrance when the siRNA conjugate is loaded into RISC. The lesser difference in activity between RL2As/S-siMB3 and siMB3 is likely due to the fact that it can adopt the appropriate conformation to avoid steric clashes with RISC and therefore has similar gene silencing activity as the unconjugated.

The single conjugation of cRGD at the 5' -end of the sense strand had no significant effect on gene silencing activity (As/RL0S-simB3), even with the shortest linking arm. While the activity of gene silencing was slightly reduced for the double conjugate compared to the unconjugated siMB3, the difference in activity was not significant at this concentration, and when the 5 '-end of the sense strand was conjugated to cRGD via a shorter linker, the activity of the 5' -conjugated large group of the antisense strand was better than that of the smaller group.

Example 6 examination of the antitumor Activity and empty vector cytotoxicity of 5-terminal Mono/Biconjugated siRNA neutral/cationic lipid Complex

1. Sample preparation:

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 and L1As/RL0S-simB 3. The above 5-terminal mono-and-bi-conjugates were prepared as described in example 1, and siRNA lipid complex siRNA/DNCA/CLD was prepared as described in example 3.

Cck-8 experiment

A375 was plated at 1 ten thousand per well in 96-well plates and transfected 18h after incubation. The administration concentration of siMB3 and its conjugate was 100nM, the siRNA lipid complex was added to the plate, after 72h of incubation, the medium was discarded, 10% cck-8 reagent was added, incubation was performed at 37 ℃ for 2h, and absorbance at 450nM was measured using a plate reader. As a result, the control group was homogenized so that the absorbance thereof was 1. The antitumor activity of siMB3 and its conjugate and the cytotoxicity of the empty vector were examined and the results of the experiment are shown in fig. 5.

3. Results

Evaluation of the antitumor activity at the cellular level of the mono-conjugated siMB3/DNCA/CLD lipid complex revealed that conjugation of the 5' -end of the antisense strand reduced its antitumor activity in vitro, regardless of the size of the conjugated group. After the 5' -end of the sense chain is conjugated, the antitumor activity is reduced, and in the double conjugate, the gene silencing activity of only the double conjugate L1As/L0S is similar to that of the non-conjugate As/S, and the L0As/RL0S also has better antitumor activity. The DNCA/CLD empty vector (mix group) has low cytotoxicity, which indicates that the lipid complex applied in vitro is safe and low in toxicity.

Example 7 examination of the in vivo distribution of 5-terminally singly conjugated siRNA neutral/cationic lipid complexes in animals

1. Sample preparation:

As/Cy7-S-simB3, RL0As/Cy7-S-simB3, and the 5' -end of the sense strand of simB3 were labeled with a fluorescent dye Cy 7. See example 1 for synthesis of antisense strand conjugates and example 2 for preparation of siRNA lipid complex siRNA/DNCA/CLD/PEG 2000-DSPE.

2. The method comprises the following steps:

the axilla of a 6-week-old nude mice were inoculated with a375 cells, and the tumor volume reached about 500mm after 10 days³. Subsequently, a dose of 1.5mg/kg of siRNA was administered by tail vein injection of Cy 7-labeled simB3(As/S) or cRGD conjugate (RL0As/S) entrapped in DNCA/CLD/PEG 2000-DSPE. The fluorescence signal of the siRNA was measured by an in vivo imaging system 1.5h to 36h after administration. 36h after dosing, animals were euthanized and tumor tissues and organs were removed and photographed. The results of the experiment are shown in FIG. 6.

3. As a result:

accumulation of RL0As/S in the tumor was clearly observed throughout the imaging, but only a weak fluorescence signal was observed for the As/S not conjugated with cRGD (FIG. 6 a). Quantification of intratumoral fluorescence signals revealed that tumor accumulation was about 90% higher for cRGD-conjugated siMB3 than for unconjugated siMB3(p <0.05), mixed vectors were effective in delivering siRNA to tumors, and intratumoral fluorescence signals were significantly higher for As/S group than for BLANK group (p < 0.01). However, due to autofluorescence of food in the gastrointestinal tract, siRNA signals in the liver and kidney are difficult to observe, but are not related to fluorescence signals within tumors.

Ex vivo results it can be found (fig. 6b) that Cy 7-labeled siMB3 mainly accumulated in tumors and liver, RL0As/S fluorescence signal was stronger in tumors than As/S, ex vivo intratumor fluorescence quantification results corresponded to in vivo distributed tumor quantification results, cRGD-conjugated siMB3(RL0As/S) had stronger intratumoral accumulation capacity than unconjugated siMB3(p < 0.05). The above results indicate that the DNCA/CLD/PEG2000-DSPE mixed vector can deliver siMB3 and its conjugates to tumor tissues with high efficiency and still have significant intratumoral and hepatic distribution 36 hours after administration, and this combined conjugation and delivery strategy provides a platform for systemic delivery of siRNA for in vivo tumor treatment.

Example 8 examination of the antitumor Activity and mechanism thereof of 5-terminal Mono-conjugated siRNA neutral/cationic lipid Complex in animals

1. Sample preparation:

As/S-simB3, RL0As/S-simB3, RL2As/S-simB 3. siRNA and conjugate synthesis is described in example 1, and preparation of siRNA lipid complex siRNA/DNCA/CLD/PEG2000-DSPE is described in example 2.

2. The method comprises the following steps:

160w of melanoma cells A375 were inoculated to the axilla of 3-week-old nude mice, and the nude mice were divided into 5 groups after 7 days of tumor formation, at which time the average tumor volume was about 50mm³(day 0), 5 of each group. Tail vein injection of siMB3 and conjugate cocktail formulation was treated with BLANK (tail vein injection formulation solvent genotti) as BLANK control, and siMB3 (unencapsulated siMB3) as negative control, mix (DNCA/CLD/PEG 2000-DSPE empty vector without siRNA), mix-NC (mix-encapsulated siFL targeting firefly luciferase mRNA). On

days

1, 3, 5, 8 (RL0As/S) or

days

1, 3, 5, 7, 9 (RL2As/S), the siRNA is administered by tail vein injection at a dose of 2mg/kg (RL0As/S) or 1.48mg/kg (RL2 As/S). The tumor size and mouse weight are taken daily or every two days during the administration treatment period, and the tumor volume V is equal to the length x the width according to the formula²X 0.5 tumor volume was calculated. After 48h of the last dose, 1ml of blood was taken, the mice were subsequently euthanized, tumor tissue isolated and weighed and recorded by photography. A part of tumor tissues (50-100mg) are taken and added with TRizol, homogenized and extracted to obtain total RNA, the RNA is reversely transcribed into cDNA by using a reverse transcription kit, real-time fluorescence quantitative PCR is carried out, and the intratumor mRNA silencing capability of the simB3 and the conjugate thereof is examined. Fixing another part of tumor tissue (50-100mg) in paraformaldehyde overnightSubsequently embedded in paraffin and sectioned for nuclei and BRAF^V600EAnd (3) protein marker staining treatment. The blood of the mouse is kept still at 4 ℃ overnight, and centrifuged at low speed for 15 minutes to separate the serum, and then the biochemical indexes of the liver and the kidney in the serum are detected. The experimental results are shown in fig. 7, 8, 9 and 10.

3. As a result:

the in vivo antitumor activity of the shorter linked arm linked RL0As/S-siMB3 was first examined (FIG. 7). The results show that encapsulation of siMB3 and its conjugates significantly inhibited tumor growth throughout the treatment (fig. 7a, p)<0.001), but the ability of conjugate RL0As/S to inhibit tumor growth was weaker than that of the non-conjugate As/S, despite a p-value of 0.25, with no significant difference. The mice grew steadily throughout the dosing treatment, with no difference between groups, indicating that conjugation did not affect the safety of the siRNA lipid complex (fig. 7 b). Mice were euthanized 48h after the last dose and tumors were removed and weighed (fig. 7c), with the minimum mix-As/S tumor volume and a certain reduction in mix-RL0As/S tumor volume compared to the blank and negative controls, but the cure failure rate was 2/5 in the whole group, with two of the five mice having similar tumor volumes to the control group. RT-qPCR assay for intratumoral BRAF^V600EmRNA levels found that As/S was more effective in silencing target mRNA than RL0As/S (FIG. 7 d). Immunohistochemical results were consistent with qPCR results (fig. 7e), with significant reduction of BRAF in both siMB3 and conjugate treated groups in paraffin sections of tumors in mice^V600EExpression of protein (brown spots).

In vivo antitumor experiments show that although RL0As/S with cRGD targeting group has better tumor accumulation capacity, the spatial distance between the conjugation group and the antisense strand is too close to greatly influence the gene silencing activity, so the therapeutic effect of RL0As/S conjugate is weaker than that of unconjugated simB 3. In combination with the cell experiments, RL2As/S was examined for anti-tumor activity in vivo (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), also lower than that of the unencapsulated siRNA (p)<0.001) and mix-NC group (p)<0.001) (fig. 8 a). Tumor volumes of blank and negative control groups were day 0By more than 25 times, the mix-As treated group increased 12 times, while 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 treated group increased only about 6-fold. The siMB3 lipid complex with the 5' -end of the antisense strand linked to the cRGD conjugate group via a longer linking arm can be more effective in treating tumors than the unconjugated siMB3 lipid complex. After 48h of the last dose, the mice were euthanized, tumor tissue isolated weighed (fig. 8b) and photographically recorded (fig. 8 c). As shown in FIG. 8b, the tumor weight in the mix-As/S group was significantly less than that in the null and negative control (p)<0.05), the tumor weight of the conjugate mix-RL2As/S group was also significantly reduced compared to the blank and negative control groups (p)<0.01). The conjugate did not differ significantly from the unconjugated tumor (p)>0.5), but the mean tumor weight was less for the mix-RL2As/S group than for the mix-As/S group. The size of the ex vivo tumor volume of each group was consistent with the tumor growth curve (fig. 8 c). RT-qPCR assay for intratumoral BRAF^V600EAs/S and RL2As/S were found to silence target mRNA expression by about 40% more efficiently at the mRNA level (FIG. 8d), and were significantly different from blank and negative controls (p)<0.001). Simultaneous assessment of intratumoral BRAF by immunohistochemical experiments^V600EAt the protein level of (4), yellow spots for BRAF^V600EExpression of the protein (FIG. 8 e). Compared with NC, both simB3 and RL2As/S down-regulate BRAF^V600EWhile RL2As/S silences with similar efficiency As/S. The excellent antitumor capacity of RL2As/S is related to the conjugation of a targeting group to increase the accumulation of siMB3 in tumor tissues, and the extension of a linking arm of the conjugation group and the 5' -end of an antisense strand to restore the gene silencing activity of siRNA. Since at the end of the in vivo experiment, the growth rate of the tumors treated with RL0As/S was greater than that of unconjugated siMB3 (fig. 7a), although there was no significant difference in tumor volume between the two (p ═ 0.25). These results indicate that the tumor targeting conjugate group cRGD increases nanoparticle accumulation of siMB3 conjugate in tumor tissues, and that the extended spatial distance between cRGD and 5 '-terminus (L2 compared to L0) is the key point for the excellent anti-tumor efficiency of the antisense strand 5' -terminus conjugate. Furthermore, both steadily changing body weight and no mouse death during treatment indicated that all conjugates were sufficiently safe for in vivo use (fig. 8 f).

To verify hepatorenal toxicity of the conjugates and formulations, whole blood was collected 48 hours after the last dose and biochemical indicators of the blood of the mice were measured (fig. 9). The biochemical indexes of CREA-J, UA, UREA representing the kidney function and ALT, AST, ALP, TP and ALB representing the liver function of each group have no obvious difference from the non-administration group, which shows that the conjugate and the DNCA/CLD/PEG2000-DSPE mixed lipid preparation have good safety and almost no hepatotoxicity and kidney toxicity, and the simB3/DNCA/CLD/PEG2000-DSPE lipid complex can be used as a safe and effective mouse tumor therapy.

To validate the mechanism of siMB3 conjugate for tumor treatment, intratumoral BRAF was determined after a single administration^V600EThe expression level of mRNA. Balb/c-nude 4-week-old mice were used, the right axilla was inoculated with 160w of A375 cells, and the tumor volume was about 500-800mm 20 days after inoculation³The preparation is divided into four groups at random, and administered via tail vein injection. 48h after administration, the animals were euthanized, and 50-100mg of tumor tissue was taken to quantify the expression level of mRNA. As shown in fig. 10, siMB3 silences intratumoral BRAF after one administration^V600EExpression of mRNA. Unconjugated As/S silenced 14% of target mRNA, RL2As/S with the 5' -end of the antisense strand conjugated cRGD silenced 22% of target mRNA expression, and significant difference compared with mix-NC group (NC vs<0.05；NC vs.RL2As/S,p<0.001). The slightly lower level of target gene inhibition in siRNA tumors was probably due to the larger tumor size when administered, the inability of siRNA to transfect all cells in tumor tissues completely, and the more pronounced gene silencing activity after multiple administrations (FIG. 8 d). RL2As/S inhibits tumor growth and BRAF at animal level^V600EmRNA silencing is involved.

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

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, and carrying out entrapment by using a mixed lipid material of a neutral lipid material and a cationic lipid material, 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;

2. the chemical modification method of claim 1, further comprising linking a targeting group to an active ester at the end of the linking group by condensation reaction while conjugating one or more linking groups to the 5' -end of the antisense strand or/and the sense strand of the siRNA, wherein the targeting group has a structural formula shown in formula VI:

3. the chemical modification method according to claim 2, 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;

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

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

6. The chemical modification method according to claim 1, wherein the mixed lipid material comprises, in terms of mole percent, 72.7% of the neutral cytidine lipid material represented by formula III and 27.3% of the cationic lipid material represented by formula IV, or 39.7% of the neutral cytidine lipid material represented by formula III, 59.6% of the cationic lipid material represented by formula IV, and 0.7% of PEG2000-DSPE represented by formula V.

7. The chemical modification method according to claim 6, 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 conjugated with one or more linking groups through a phosphodiester bond in DEPC water, mixing the solution by using an unbuffered solution or a buffer solution, performing ultrasonic treatment, and filtering the solution by using a 0.22 mu m filter membrane, wherein the filtrate contains modified siRNA nano-particles, wherein preferably, the buffer solution is physiological saline, water or any transfection optimization solution, and the buffer solution contains acetate, citrate, carbonate, phosphate or any combination thereof.

8. A chemical modification method as claimed in any one of claims 1 to 7, wherein said 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), phospho-sulphur backbone modifications and other end conjugation approaches of siRNA, etc.

9. 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 8 entrap the modified small interfering RNA.

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