CN106046119B

CN106046119B - Preparation of 1-methyl-beta-carboline-3-RGDS as VEGF-siRNA delivery vector

Info

Publication number: CN106046119B
Application number: CN201610362687.2A
Authority: CN
Inventors: 崔纯莹; 王玉记; 赵雯
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2016-05-26
Filing date: 2016-05-26
Publication date: 2019-12-31
Anticipated expiration: 2036-05-26
Also published as: CN106046119A

Abstract

The invention relates to an amphiphilic carrier membrane material with antitumor activity, which comprises the following components in part by weight: 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine (CRS) and a preparation method of the amphiphilic carrier membrane material.

Description

Preparation of 1-methyl-beta-carboline-3-RGDS as VEGF-siRNA delivery vector

Technical Field

The invention relates to a CRS prepared from a newly synthesized 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine amphiphilic membrane material, a CRS carrying VEGF-siRNA, in particular to a CRS of the CRS and the VEGF-siRNA and a construction method thereof, and also relates to an application of a drug carrier in preparing the CRS of the VEGF-siRNA, belonging to the field of biological medicine and pharmacology.

Background

In recent years, genetic-level tumor therapy has received much attention. Among various means of gene intervention, small interfering RNA (siRNA) can specifically inhibit gene expression, and achieves the anti-tumor effect by inhibiting high expression genes affecting the growth of tumor cells. Vascular Endothelial Growth Factor (VEGF), which is reported to be abundant in most solid tumors, binds to vascular endothelial growth factor receptor 2 (VEGFR-2), promotes angiogenesis around tumor cells, thereby accelerating tumor growth and invasion. However, naked siRNA does not function effectively in vivo due to water solubility and electronegativity, it is degraded rapidly in circulation and it hardly penetrates cell membrane. Therefore, researchers have been working on efficient carriers of siRNA.

Liposomes, as well as being a mature and widely studied carrier, are used not only for the entrapment of chemical drugs, but also in their cationized form for the carrying of siRNA. The cationic liposome can adsorb the siRNA with electronegativity through electric adsorption and is combined with the extracellular membrane with electronegativity, so that the siRNA is entrapped and transfected. However, many related studies in recent years have shown that cationic liposomes lack targeting and have some toxicity. Therefore, researchers have sought a vector with electropositive properties, cell affinity, low toxicity and targeting properties.

RGD (Arg-Gly-Asp) targeting peptides act as transmembrane glycoproteins, mediating cell adhesion by binding to integrin receptors on the cell surface. Among various targeting peptides, RGDS (Arg-Gly-Asp-Ser) targeting peptide has high affinity with integrin receptors on the surface of tumor cells and can inhibit cell adhesion phenomenon, thereby playing roles in anti-inflammation and anti-angiogenesis. And (3) the effect of resisting tumor invasion. Nowadays, due to the tumor targeting property, long circulation property and hydrophilicity of RGDS, many researchers use it as a modification and optimization pharmacophore of antitumor drugs.

Among traditional tumor chemotherapy drugs, DNA intercalating compounds are being developed for anti-tumor therapy. In the study of the 20 th century and the 80 th century, researchers confirmed that indole alkaloids with antitumor effect in peganum harmala seeds are mainly beta-carboline and dehydro-beta-carboline. The lipophilic beta-carboline exerts potential antitumor activity by intercalating into DNA. Research shows that-CH is introduced into the 1-position₃The amino acid substance introduced into the 3-position can improve the antitumor activity of the carboline substance and reduce the toxicity.

Based on the above, in the present study we linked RGDS targeting to 1-methyl- β -carboline-3-carboxylic acid compounds to form entirely new compounds. The compound has the property similar to that of phospholipid in liposome material, has certain hydrophilicity and lipophilicity, and can form a nanoscale electropositive structure. We studied the properties of this material as a carrier. VEGF-siRNA capable of silencing VEGF gene is entrapped by methods such as gene compression and orthogonal experiments, the anti-tumor and gene silencing effects of cell level, protein level and nucleotide level of the VEGF-siRNA are researched, and the targeting property, low carrier toxicity and tumor inhibition effects in vivo of the VEGF-siRNA are verified by an S180 Holland sarcoma mouse.

Disclosure of Invention

One of the objectives of the present invention is to provide an amphiphilic carrier membrane material with anti-tumor activity: 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine (or simply CRS).

The invention also aims to provide a preparation method of an amphiphilic carrier membrane material CRS (1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine), which comprises the following steps:

1) synthesis of 1-methyl-beta-carboline-3-carboxylic acid

Wherein, the first is CH₃CHO/H⁺,②:SOCl₂/CH₃OH,③:KMnO₄/CH₃COCH₃,④:NaOH.

2) The synthetic route of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine is as follows:

⑤:DCC/HOBt/NMM,⑥:TFA/TfOH(TFA:trifluoroacetic acid；TfOH:Trifluoromethanesulfonic acid),⑦:HCl-EtOAc

the invention also aims to provide a pharmaceutical composition, which comprises the following components: an anti-tumor gene therapy drug VEGF-siRNA, and 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine.

Preferably, the pharmaceutical composition of the present invention comprises the following components: antineoplastic gene therapy drugs VEGF-siRNA, cholesterol, calf thymus DNA, protamine and 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine. Further, lecithin may be added as required.

Further preferably, the pharmaceutical composition of the present invention comprises the following components by mass percent:

more preferably, the pharmaceutical composition of the present invention comprises the following components by mass percent:

the fourth purpose of the invention is to provide a preparation method of the pharmaceutical composition, which comprises the following steps:

dissolving 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine in chloroform and methanol, removing the solvent by rotary evaporation, adding 1mL of DEPC water for hydration, and performing ultrasonic treatment on a probe for 15 minutes to obtain a CRS nano solution. Following VEGF-siRNA/CRS (μ L): and (3) absorbing the liposome nano solution with a corresponding volume by using a pipette gun, adding an RNase-free EP tube, diluting and uniformly mixing by using an RNase-free water, adding the liposome nano solution into the VEGF-siRNA aqueous solution by using a gene compression method, gently blowing and beating the solution for 10 times, and standing the solution at room temperature for 20min to obtain the VEGF-siRNA liposome nano-liposome.

Preferably, the preparation method of the pharmaceutical composition of the present invention comprises the following steps:

dissolving 10mg of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine (CRS) and 1mg of cholesterol in chloroform and methanol, removing the solvent by rotary evaporation, adding 1mL of DEPC water for hydration, and performing ultrasonic treatment on a probe for 15 minutes to obtain a CRS nano solution. Sucking 60 μ L of CRS nano solution with a pipette, adding RNase-free EP tube, diluting with RNase-free water, mixing, adding 15 μ L of protamine (120 μ g), mixing and incubating for 10min, adding 1.2 μ L of calf thymus DNA (1.60 μ g) into 9 μ L of LVEGF-siRNA (15.96 μ g) aqueous solution, mixing and incubating for 10min, mixing CRS complex solution and VEGF-siRNA complex solution, gently blowing for 10 times, and standing at room temperature for 20min to obtain the final product.

The invention also aims to provide a CRS carrier, which mainly comprises lecithin, cholesterol and 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine.

The fifth purpose of the invention is to provide a gene compression method for carrying CRS genes, which compresses CRS and VEGF-siRNA through protamine and calf thymus simultaneously and improves the gene entrapment rate.

The sixth purpose of the invention is to provide the application of the medicine in preparing the medicine for treating the tumor.

The invention relates to a method for evaluating the anti-tumor activity of VEGF-siRNA/CRS and CRS by using a cervical cancer HeLa cell strain as a model and using an MTT method, and the result shows that the VEGF-siRNA/CRS has more excellent anti-tumor activity.

The invention relates to a method for determining gene silencing efficiency of protein level by ELISA and mRNA level by RT-PCR by using a cervical cancer HeLa cell strain as a model, and the result shows that VEGF-siRNA/CRS has higher gene silencing efficiency compared with a commercially available transfection reagent and naked VEGF-siRNA, thereby indicating that the VEGF-siRNA/CRS has more excellent anti-tumor activity.

The invention relates to a method for determining CRS targeting by using an S180 sarcoma mouse and a healthy mouse as models, determining CRS targeting by using animal fluorescence, determining CRS safety by using an acute toxicity experiment, and determining VEGF-siRNA/CRS tumor inhibition by using a tumor inhibition experiment. The results show that VEGF-siRNA/CRS and CRS have obvious tumor inhibition effect and stronger VEGF-siRNA/CRS effect compared with positive drugs and naked VEGF-siRNA.

Compared with the existing products, the invention has the following beneficial effects:

the CRS carrier is prepared by synthesizing 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine from 1-methyl-beta-carboline-3-carboxylic acid and RGDS through a liquid phase peptide connection method, and has obvious anti-tumor effect compared with common liposome and cationic liposome. The CRS designed by the subject is designed for the research work of siRNA delivery carriers, is not reported in domestic and foreign documents, and has innovation in structural design.

The new CRS is loaded with VEGF-siRNA tumor neovascularization siRNA, the tumor targeting and transfection efficiency of the siRNA are improved, and the siRNA cell uptake capacity and gene silencing effect are promoted. The research on the aspect is not reported in the literature.

The related terms described in the present invention are further explained:

CRS: 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine

Gene-carrying CRS: medicaments containing VEGF-siRNA and CRS

VEGF: vascular endothelial growth factor

VEGF-siRNA: the small interfering RNA of the vascular endothelial growth factor belongs to the existing products and can be purchased in the market

RGDS (Arg-Gly-Asp-Ser): arginine-glycine-aspartic acid-serine tetrapeptide

DEPC water: purified water treated with diethylpyrocarbonate and sterilized at high temperature and high pressure

HeLa human cervical cancer cell line

RNase-free EP tube: EP tube without RNA degrading enzyme

RNase-free water: pure water without RNA degrading enzyme

THF tetrahydrofuran

DCC: dicyclohexylcarbodiimide

DCU: dicyclohexylurea

HOBt: 1-hydroxybenzotriazoles

NMM: n-methyl morpholine

TFA: trifluoroacetic acid

TfOH: trifluoromethanesulfonic acid

HCl-EtOAc: saturated ethyl acetate solution for dissolving hydrochloric acid gas

Boc-Arg (tos): arginine with naked carboxyl

HCl Gly-OBzl: glycine with bare amino group

Boc-Arg (tos) -Gly-OBzl arginine-glycine dipeptide

Boc-Arg (tos) -Gly: carboxy-naked arginine-glycine dipeptide

Boc-Asp (OBzl): aspartic acid with naked carboxyl

HCl & Ser-OBzl: serine with naked amino group

Boc-Asp (OBzl) -Ser-OBzl: aspartic acid-serine dipeptides

HCl. Asp (OBzl) -Ser-OBzl: amino-naked aspartic acid-serine dipeptide

Boc-arg (tos) -Gly-asp (OBzl) -Ser-OBzl: arginine-glycine-aspartic acid-serine tetrapeptide with all protected groups

HCl. Arg (tos) -Gly-Asp (OBzl) -Ser-OBzl: arginine-glycine-aspartic acid-serine tetrapeptide with naked amino

PC: phospholipids

CH: cholesterol

DMEM: culture medium containing various amino acids and glucose for cell culture

lipo^TM2000: commercially available transfection reagents

CCK-8: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt for cell viability assay

BCA: disodium 2, 2-biquinoline-4, 4-dicarboxylate for determination of total protein concentration

calibretor Diluent RD 5K: dilution of calibrator for VEGF protein concentration determination

wash buffer: washing solution for VEGF protein concentration determination

VEGF conjugate: VEGF conjugates for determination of VEGF protein concentration

substrate solution: visualization reagent for VEGF protein concentration determination

stop solution: reaction stop solution for VEGF protein concentration determination

VEGF standard: VEGF standard solution for determining VEGF protein concentration

And (3) Trizol: cell lysate for RNA extraction

VEGF-mRNA: vascular endothelial growth factor messenger ribonucleic acid

RT EnzymeMix: assay for VEGF messenger ribonucleic acid

Optical additive film: sealing plate film

Gene expression Master Mix: assay for VEGF messenger ribonucleic acid

Gene Expression Assays: assay for VEGF messenger ribonucleic acid

To-pro: fluorescent dye containing Cy3

Drawings

FIG.1 particle size and potential comparison of CRS with VEGF-siRNA/CRS (n ═ 3)

FIG. 2 Hela cells under inverted microscope

FIG. 3 comparison of survival rate of Hela cells with CRS and VEGF-siRNA/CRS-loaded cells (n ═ 3)

FIG. 4 comparison of silencing levels of protein on Hela cells by CRS and VEGF-siRNA/CRS vector (n. 3)

FIG. 5 comparison of mRNA silencing levels of Hela cells by CRS and VEGF-siRNA/CRS-loaded cells (n. 3)

FIG. 6 comparison of fluorescence at different concentrations of CRS, Cy3 fluorescein and Cy3-CRS

FIG. 7 fluorescence analysis of CRS S180 Holland sarcoma mice in vivo

FIG. 8 tumor weight comparison of CRS with VEGF-siRNA/CRS-loaded tumor (n ═ 10)

FIG. 9 comparison of tumor volumes for CRS and VEGF-siRNA/CRS-loaded tumor (n ═ 10)

FIG. 10 murine weight changes in CRS in acute toxicity experiments (n ═ 8)

FIG. 11 comparison of the body ratio of CRS in acute toxicity test (n. 8)

FIG. 12 comparison of mouse organs for CRS in acute toxicity test (n ═ 8)

Detailed Description

To further illustrate the invention, a series of examples are given below. These examples are purely illustrative and are intended to be a detailed description of the invention only and should not be taken as limiting the invention.

EXAMPLE 1 preparation of isoquinoline derivatives

The CRS preparation method comprises the following steps:

1) synthesis of 1-methyl-beta-carboline-3-carboxylic acid

400mL of water was placed in a 500mL eggplant flask, 98% concentrated sulfuric acid (0.2mL) was slowly added dropwise thereto, I (5.0g,24.5mmol) was added to the mixture, the mixture was dissolved by sonication, and 40% acetaldehyde (4.5mL,49.0mmol) was added dropwise and reacted overnight. When the solution turns milk white, adjusting pH to 6-7 with strong ammonia water, standing for 1H, filtering, washing with water to remove salt, and air drying the filter cake to obtain 1(4.0g, yield 80%, ESI/MS (M/z)231[ M + H ] +). Slowly adding 3.0mL of thionyl chloride into vigorously stirred 37.5mL of methanol under a ice salt bath, stirring for 40min under an ice bath, adding II (4.0g and 17.4mmol), reacting at room temperature overnight, displaying that a raw material point disappears by thin-layer chromatography, enabling the solution to be in a light yellow suspension state, pumping out methanol by a water pump, dissolving ethyl acetate, neutralizing by a large amount of 10% Na2CO3 until the pH value is 6-7, separating by a separating funnel, remaining an ethyl acetate layer, drying for 2h by anhydrous Na2SO4, carrying out suction filtration, and spin-drying the ethyl acetate layer to obtain a product III (2.8g and 11.5 mmol). Under ice bath, 2(2.8g,11.5mmol, yield 70%, ESI/MS (M/z)245[ M + H ] +) is dissolved in 200mL acetone, KMnO4(2.5g,15.9mmol) is slowly added, stirring is carried out for 1H in ice bath, reaction is carried out overnight at room temperature, thin layer chromatography shows that the raw material point disappears, the solution is in a dark brown suspension state, rotary drying acetone and methanol are dissolved, suction filtration is carried out, filtrate is dried in a rotary manner, column chromatography (dichloromethane: methanol) is carried out gradient separation, and product IV (1.7g,6.9mmol, yield 60%, ESI/MS (M/z)241[ M + H ] +) is obtained. Dissolving IV (1.7g,6.9mmol) in 2N NaOH (41.4mL,8.3mmol), carrying out oil bath reaction at 60 ℃ for 3H, showing that the raw material point disappears by thin layer chromatography, the solution is a yellow brown solution, standing to room temperature, neutralizing with concentrated hydrochloric acid until the pH value is 7, precipitating a large amount of yellow green solid, cooling, standing for 1H, and carrying out suction filtration to obtain V (1.36g, 5.9mmol, the yield is 80%, ESI/MS (M/z)227[ M + H ] +).

The synthetic route of 1-methyl-beta-carboline-3-carboxylic acid is CH3CHO/H⁺,②:SOCl₂/CH₃OH,③:KMnO₄/CH₃COCH₃,④:NaOH.

2) Fully protected RGDS synthesis

Reacting Boc-Arg (tos) (5)0g,11.7mmol) was placed in a 250mL eggplant flask, THF was dissolved with stirring, HOBt (1.6g,11.7mmol) was added, DCC (2.9g,14.0mmol) dissolved in THF was added under ice bath conditions, activation was performed for 30min, HCl · Gly-OBzl (2.4g,11.7mmol) was slowly added, NMM adjusted PH 8-9, reaction was performed overnight, thin layer chromatography showed disappearance of the starting material spot, DCU was filtered off, THF was spun off, and the residue was dissolved in 150mL ethyl acetate. The resulting solution was sequentially treated with saturated NaHCO₃Three times washing with aqueous solution, three times washing with saturated NaCl aqueous solution and 5% KHSO₄Three times of washing with aqueous solution, three times of washing with saturated NaCl aqueous solution and saturated NaHCO₃Three washes with aqueous solution and three washes with saturated aqueous NaCl solution. Separating with separating funnel to obtain ethyl acetate layer, and adding anhydrous Na₂SO₄Drying, filtering, and concentrating the filtrate under reduced pressure to dryness to obtain Boc-Arg (tos) -Gly-OBzl (6.1g,10.6mmol, yield 90%, ESI/MS (M/z)576[ M + H ] as pale yellow solid powder](+) of; the same procedure was followed using Boc-Asp (OBzl) (5.0g,15.5mmol), DCC (3.8g,18.6mmol), HOBt (2.1g,15.5mmol) and HCl Ser-OBzl (3.6g,15.5mmol) to synthesize Boc-Asp (OBzl) -Ser-OBzl (6.9g,13.8mmol, 89% yield, ESI/MS (M/z)538[ M + H ] as a pale yellow paste]⁺). Boc-Arg (tos) -Gly-OBzl (6.1g,10.6mmol) was placed in a 250mL eggplant flask, dissolved in 200mL methanol, added with 0.6g Pd/C (10%) and charged with H₂(0.02Mba), stirred at room temperature until the thin layer chromatography showed disappearance of the starting material spot. Pd/C is filtered off, the filtrate is decompressed and rotary evaporated to be dry, and pure white solid powder Boc-Arg (tos) -Gly (4.6g,9.5mmol, yield 90%, ESI/MS (M/z)486[ M + H ] is obtained]⁺) (ii) a Boc-Asp (OBzl) -Ser-OBzl (6.9g,13.8mmol) was dissolved in 35mL of 4mol/L hydrogen chloride-ethyl acetate solution, stirred at room temperature for 4 hours, TLC showed the disappearance of the starting material spot, ethyl acetate was pumped dry, and the residue was repeatedly pumped dry with a small amount of diethyl ether to remove hydrogen chloride gas. Finally, the residue was ground to a white powder of HCl. Asp (OBzl) -Ser-OBzl (4.1g,10.3mmol, 75% yield, ESI/MS (M/z)435[ M-H ] with a small amount of ether]^-). Boc-Arg (tos) -Gly (5.0g,10.3mmol) and HCl. Asp (OBzl) -Ser-OBzl (4.1g,10.3mmol) were subjected to liquid phase condensation according to the previous ligation method, DCC (2.5g,12.36mmol), HOBt (1.4g,10.3mmol) to form Boc-Arg (tos) -Gly-Asp (OBzl) -Ser-OBzl (6.3g,7.2mmol, yield 70%, ESI/MS (M/z)902[ M-H/z ]]^-) Column chromatography (dichloro: methanol) was fractionally pure.

3) Synthesis of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine final product

The purified Boc-Arg (tos) -Gly-Asp (OBzl) -Ser-OBzl (1.0g,1.2mmol) was dissolved in 5mL of 4mol/l hydrogen chloride-ethyl acetate solution, stirred at room temperature for 4 hours, TLC showed the disappearance of the starting material spot, ethyl acetate was pumped off, and the residue was repeatedly pumped off with a small amount of diethyl ether to remove hydrogen chloride gas. Finally, the residue was ground to a white powder of HCl.Arg (Tos) -Gly-Asp (OBzl) -Ser-OBzl (0.62g,0.8mmol, 70% yield, ESI/MS (M/z)241[ M + H ] with a small amount of diethyl ether]+). VI (0.2g,0.8mmol) is dissolved in DMF, HOBt (0.1g,0.8mmol) is added with stirring, DCC (0.2g,0.96mmol) dissolved in DMF is added under ice bath condition, activation is carried out for 30min, HCl.Arg (tos) -Gly-Asp (OBzl) -Ser-OBzl (0.62g,0.8mmol) is slowly added, NMM adjusts pH to 8-9, reaction is carried out overnight, thin layer chromatography shows that the raw material point disappears, DCU is filtered off, DMF is dried, the residue is dissolved by 150mL of methanol, and decompression and spin-drying are carried out to obtain the yolk color product VII (0.3g,0.8mmol, the yield is 40 percent, ESI/MS (M/z)241[ M + H/Z ] 241]⁺) Column chromatography (dichloro: methanol), dissolved in 6mL trifluoroacetic acid under ice bath, added with 1.5mL trifluoromethanesulfonic acid, reacted for 30min, added with ether and washed with water repeatedly, and finally pumped to dryness with a water pump to obtain the final product VIII (0.2g,0.1mmol, yield 87%) as a fluorescent yellow powder.

A synthetic route of a 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine final product,

⑤:DCC/HOBt/NMM,⑥:TFA/TfOH,⑦:HCl-EtOAc(TFA:trifluoroacetic acid；TfOH:Trifluoromethanesulfonic acid).

¹H NMR(300MHz,DMSO-d6)δ/ppm＝12.02(s,1H),8.35(d,7.8Hz,2H),8.28(d,＝8.1Hz,1H),7.95(d,7.8Hz,1H),7.62(m,1H),7.95(dd,＝4.5Hz,1H),7.29(m,2H),4.69(m,2H),4.25(m,1H),4.04(m,1H),3.89(m,1H),3.81(m,1H),3.63(m,2H),3.15(m,2H),2.86(s,3H),1.84(m,1H),1.57(m,2H).

¹³C NMR(125MHz,DMSO-d6)δ/ppm＝174.07,173.95,172.08,170.18,168.19,164.61,157.75,141.58,141.33,138.47,136.47,128.80,127.88,122.60,121.88,120.44,112.71.63.04,55.93,55.36,52.22,51.79,49.06,42.96,31.24,24.68,20.97.

ESI-MS(m/z)640[M-H]^-.

IR(KBr)3343.42,3216.03,1722.30,1651.68,1627.85,1547.34,1515.79,1364.64,1240.57,1223.98,1166.57,1026.54,909.35,813.73,759.97,635.74.

the maximum absorption peak wavelength in the UV spectrum is 271.0nm, and the HPLC determination purity is 95.06%.

Example 2 preparation of Carrier CRS

Preparing a membrane material and a medicinal solution:

CRS is prepared by adopting a thin film dispersion method, and the preparation method of the membrane material and the medicinal solution comprises the following steps:

10mg/mL Phospholipid (PC) in chloroform: dissolving 10mg of PC in a proper amount of chloroform, transferring the solution into a 1mL brown volumetric flask, and fixing the volume by using the chloroform;

10mg/mL Cholesterol (CH) in chloroform: dissolving 10mg of CH in a proper amount of chloroform, transferring the solution into a 1mL brown volumetric flask, and fixing the volume by using the chloroform;

100mg/mL 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine in chloroform-methanol mixed solution: dissolving 100mg of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine in a proper amount of methanol, transferring the solution into a 25mL brown volumetric flask, and metering the volume by using chloroform;

10mg/mL Tween 80 in DEPC: dissolving 100mg of Tween 80 in a proper amount of chloroform, transferring into a 10mL brown volumetric flask, and fixing the volume with DEPC water;

weighing appropriate amount of membrane material solution according to corresponding proportion, adding into eggplant bottle, and screening by pre-experiment with lipid material ratio, membrane material ratio, Tween 80 content and ultrasonic time as screening factors according to L9 (3)⁴) Orthogonal table design a three-factor four-level orthogonal test was performed. Measuring chloroform solution of PC and chloroform and methanol mixed solution of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine according to (A) lipid material ratio (PC: CRS m/m)40:60, 30:70 and 0:100, and measuring chloroform and methanol mixed solution of CH according to (B) membrane material ratio (PC + CRS: CH m/m)5:1, 10:1 and 15:1Adding the solution into eggplant bottle, heating in water bath at 37 deg.C, rotating at 120rpm, and reducing vacuum degree to 1mbar in a gradient of 250mbar, and performing rotary evaporation to remove organic solvent to form uniform film. Hydrating with 0% Tween 80 (Tween 80m/v) and 1% and 2% emulsion DEPC water, ultrasonic treating with probe for 5min, 10min and 15min to obtain CRS carrier,

the grain diameter is about 100-200nm, and the Zeta-Potential-25 mV to 40mV (the more 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine, the more positive the Zeta-Potential).

TABLE 1 CRS three-factor four-level L9 (3)⁴) Quadrature test

Through the orthogonal experiment, the preferable proportion relation is obtained as follows:

phospholipid: 1-methyl- β -carboline-3-arginine-glycine-aspartic acid-serine ═ 0:100 (mg/mg);

(phospholipid + 1-methyl- β -carboline-3-arginine-glycine-aspartic acid-serine): cholesterol 10:1(mg/mg)

Content of emulsifier: 1% (g/100mL)

And (3) probe ultrasonic time: 15min

Example 3 CRS-containing pharmaceutical preparation

The support CRS was prepared according to the method of example 2.

Pipette 60mL of vector CRS (25%, 50%, 75%, 100%), add RNase-free EP tube, add different volumes of protamine (2 mg. mL)^-1) Adding DEPC water to 120 μ L, quickly blowing, and standing at room temperature for 20 min.

Pipette 9. mu.L of VEGF-siRNA solution (10uM), add RNase-free EP tube, add calf thymus DNA (1 mg. multidot.mL) of different volume^-1) Adding DEPC water to 120 μ L, quickly blowing, and standing at room temperature for 10min.

The grain diameter is about 80-150nm, and the Zeta-Potential is about 0-15 mV.

Example 4 pharmaceutical compositions containing CRS and VEGF-siRNA

Dissolving 10mg of 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine (CRS) and 1mg of cholesterol in chloroform and methanol, removing the solvent by rotary evaporation, adding 1mL of DEPC water for hydration, and performing ultrasonic treatment on a probe for 15 minutes to obtain a CRS nano solution. Sucking 60 mu L of CRS nano solution by a pipette, adding an RNase-free EP tube, diluting and uniformly mixing by an RNase-free water, adding 15 mu L of protamine (120 mu g), mixing and incubating for 10min, adding 1.2 mu L of calf thymus DNA (1.60 mu g) into 9 mu L of LVEGF-siRNA (15.96 mu g) aqueous solution, mixing and incubating for 10min, mixing the CRS complex solution and the VEGF-siRNA complex solution, gently blowing for 10 times, and standing for 20min at room temperature to obtain the VEGF-siRNA.

Example 5 evaluation of anti-tumor Activity in vitro of VEGF-siRNA/CRS

Cell culture:

and (3) culture environment: CO with a volume fraction of 5% at 37 ℃₂A constant temperature incubator saturated with humidity;

culture solution: a blank DMEM medium for transfection, a 10% complete DMEM medium (containing 10% inactivated fetal calf serum, the final concentration of penicillin is 100U/mL, and the final concentration of streptomycin sulfate is 100 mug/mL);

the passage method comprises the following steps: every 2-3 days, cell growth was observed, with a degree of fusion of 80-90% trypsinization, 1: passage 3

When observed under an inverted microscope, HeLa cells grow adherent to the wall, are fusiform, have clear edges and cell nucleuses, are uniformly distributed, and have the shape similar to that of paving stones.

Preparing the medicine:

1) VEGF-siRNA/CRS: preparing a proper amount of DEPC aqueous solution of VEGF-siRNA, adding four CRS according to the proportion of an electrophoresis experiment, preparing a compound by a gene compression method, and carrying out gradient dilution on blank DMEM for later use.

2) CRS: reference 1), DEPC solution of VEGF-siRNA was replaced with DEPC water of the same volume, diluted with blank DMEM in gradient, ready for use.

3)VEGF-siRNA/lipo^TM2000: preparing appropriate amount of aqueous DEPC solution of VEGF-siRNA, referenceThe complex was prepared and diluted in DMEM blank in gradient for use.

4) VEGF-siRNA: appropriate amount of DEPC aqueous solution of VEGF-siRNA. Diluted with a blank DMEM gradient for use.

5) Blank group: HeLa cells were cultured normally in wells, and were not administered, but DMEM with equal amounts of DEPC water was substituted.

6) And (3) zero setting group: wells were cell free and other treatment pain groups.

The experimental method comprises the following steps:

HeLa cells were seeded at 75cm²In a culture flask, 10% complete DMEM was cultured at 37 ℃ with 5% CO₂Culturing in saturated humidity. After the cells have grown to log phase, they are trypsinized and plated on a well plate.

Before administration, the medium was carefully removed and 1.2.4 of the prepared solution was added at 100. mu.L/well, 6 wells per group and incubated for 4 h.

After transfection was complete, the supernatant was carefully removed and added to 10% complete medium at 100. mu.L/well in 6 replicate wells per group and incubation continued for 48 h.

After 48h, adding CCK-8 at a concentration of 100 μ L/well, repeating the steps for 6 wells in each group, shaking by a micro-oscillator for 10min at 600r, mixing uniformly, and continuing to culture for 4 h. And measuring the absorbance (OD) value of the detection wavelength of 490nm in a microplate reader, and calculating the cell survival rate. Survival rate { (AS-AB)/(AC-AB) } × 100% AS: administration group; AB, zero-adjusting group; AC blank group

As a result:

as can be seen from tables 1, 2 and 3, VEGF-siRNA/CRS (25%, 40%, 75%, 100% CRS) showed significant tumor cell inhibition compared to free VEGF-siRNA, with higher concentrations showing more significant inhibition and higher CRS content producing strong inhibition at high concentrations, but 100% CRS and 75% CRS do not differ much between 100nM and 125nM, and an analysis of 100nM may be sufficient to achieve VEGF inhibition, with no further change in concentration. CRS (25%, 40%, 75%, 100% CRS) showed significant tumor cell inhibition compared to free VEGF-siRNA, with inhibition being more pronounced at higher concentrations. But the inhibition was weaker than that of VEGF-siRNA/CRS group.

TABLE 2 transfection 4h further incubation 48h control HeLa cell viability (n ═ 3)

TABLE 3 transfection 4h incubation for 48h VEGF-siRNA/CRS group HeLa cell viability (n ═ 3)

TABLE 4 transfection 4h incubation for 48h CRS group HeLa cell viability (n ═ 3)

As can be seen from fig. 3, in the experimental concentration range:

1) the success of the cell model was demonstrated by the high survival rate of the negative control, and the inhibition rate of the positive control group.

2) The difference between blank CRS-liposome and VEGF-siRNA/CRS is not obvious at low concentration, and the difference is obvious at high concentration. The cell inhibition is proved to be not only from the carrier per se, but also to have the silencing effect of VEGF-siRNA.

3) The blank 100% CRS group has weaker cell inhibition effect than other groups, and for analysis reasons, other groups probably all originate from the combined effect of the lipid material and the CRS, while the 100% group only shows the inhibition effect of the CRS.

4) In each group, the difference between the gene carried by 100 percent CRS and the gene carried by no CRS is large, the obvious gene inhibition effect is shown, and the method has obvious advantages compared with the other three groups, and the follow-up research is continued.

EXAMPLE 6 evaluation of the silencing efficiency of VEGF-siRNA/CRS protein levels

Preparing the medicine:

1) VEGF-siRNA/CRS: preparing a proper amount of DEPC aqueous solution of VEGF-siRNA, adding 100% CRS according to the electrophoresis proportion in chapter II, preparing a compound by a gene compression method, and carrying out gradient dilution on blank DMEM for later use.

3)VEGF-siRNA/lipo^TM2000: preparing a proper amount of DEPC aqueous solution of VEGF-siRNA, preparing a compound according to the instruction, and diluting the compound by blank DMEM according to a gradient for later use.

The experimental method comprises the following steps:

1) HeLa cells were seeded at 75cm²In a culture flask, 10% complete DMEM was cultured at 37 ℃ with 5% CO₂Culturing in saturated humidity. After the cells grow to logarithmic phase, digesting with pancreatin, and inoculating by using a pore plate;

2) before administration, the medium was carefully removed, the preparation solution was added at 500. mu.L/well, 3 wells per group, and cultured for 4 h.

3) After transfection, carefully taking out supernatant, adding 10% complete culture medium, 500 mu L/hole, 3 multiple holes in each group, and continuing culturing for 48 h;

4) after 48h, the supernatant from each well was removed separately and transferred to a 2mL EP tube, centrifuged at 4 ℃ and 1000rpm for 5min, and after centrifugation was completed, the centrifuged supernatant was carefully removed and transferred to a new 2mLEP tube, labeled, and worked up on ice throughout the process.

And (3) total protein determination:

1) solutionA was mixed by shaking and the mixture was mixed according to the number of samples as 50: 1 mixing solution A and solution B to prepare fresh BCA working solution, and fully and uniformly mixing. Effective within 24 hours;

2) diluting the standard substance: completely dissolving protein standard (5mg/MlBSA, preserving at-20 deg.C) and diluting 10 μ L with triple distilled water to 100 μ L to obtain final concentration of 0.5 mg/mL;

3) diluting the diluted standard (0.5mg/mL) as mother liquor to 0.25mg/mL,0.1mg/mL,0.05mg/mL, 0.25mg/mL,0.125mg/mL,0.025mg/mL step by step, and determining the volume of the solution to be 20 μ L. Measuring the absorbance at 562nm on a microplate reader, and making a standard curve;

4) adding 20 μ L diluted sample into sample well of 96-well plate, adding 200 μ LBCA working solution into each well, gently blowing with sample gun, vortex mixing to avoid bubble generation, and centrifuging at 1000rpm for 5min to reduce bubble generation and standing at 37 deg.C for 60 min. And cooling the liquid to room temperature, measuring the absorbance at 562nm on a microplate reader, and calculating the total protein concentration of each group according to a standard curve.

VEGF protein assay:

1) moving the various reagents and samples to room temperature for equilibration for at least 30 minutes;

2) preparing a dilution-by-weight standard VEGF standard

3) Add 1mL of calibretor Diluent RD5K to the VEGF standard reagent bottle and shake gently for 15 min. 2mL of 8 EP tubes were taken and 500. mu.L of calibretor Diluent RD5K was added to each tube, and tubes 1 to 8 were named. Adding 500 mu L of VEGF standard solution into the tube 1, then sequentially adding 500 mu L of uniformly mixed solution into the subsequent tube, and diluting by multiple times;

4) sample adding: respectively provided with a blank hole, a standard hole and a sample hole to be detected. Blank wells were spiked with calibretor Diluent RD 5K. Adding 200 μ L of standard substance or sample to be tested into each of the other wells, gently shaking and mixing, covering with plate paste, and incubating at room temperature for 2 h;

5) preparing 1 xwash buffer, namely diluting 25 xwash buffer by 25 times with triple distilled water, and calculating the required amount according to 400 mu L of each hole and the washing times;

6) discard the liquid in the well, spin dry, wash with 400 μ L wash buffer 3 times, and tap the last time upside down on clean filter paper until no liquid is present. VEGF conjugate was added at 200. mu.L per well, plated and incubated at room temperature for 2 h. And (4) discarding liquid in the holes, spin-drying and washing the plate for three times. Soaking for 2 minutes each time, drying at 400 mu L/hole;

7) light-shielding, adding 200. mu.L of substrate solution to each well, covering with a plate, and incubating at room temperature for 20 min.

8) The reaction was stopped by adding 50. mu.L of stop solution per well. If the color changes are inconsistent, the plate is tapped to mix the color. The optical density of each well was measured at a wavelength of 450nm with a microplate reader within 30 minutes from the termination of the reaction.

The experimental results are as follows:

as can be seen from fig. 4:

1) no statistical difference exists in the secretion contents of Blank, VEGF-siRNA and 100% CRS group VEGF protein, which indicates that the three kinds of administration can not play a role in down-regulating protein expression.

2)VEGF-siRNA/lipo^TM2000 histone expression was significantly reduced, and VEGF-siRNA group was unchanged, demonstrating the success of the cell model.

3) The VEGF-siRNA/100% CRS group has obvious and obvious difference with VEGF-siRNA, and with increasing, the protein expression is lower, which accords with the rule of gene silencing.

4) When the difference between VEGF-siRNA/100% CRS was not significant at 100nM and 125nM, as in the cell viability assay, it is further shown that silencing by siRNA was roughly complete at 100nM and that increasing concentrations did not cause much change.

5) VEGF-siRNA/100% CRS can achieve protein down-regulation effect similar to VEGF-siRNA/lipoTM2000 group at siRNA concentration of 100nM, and shows high gene silencing effect of the new compound VEGF-siRNA/100% CRS.

6) The 100% CRS group and Blank group have no statistical difference, which indicates that the carrier can not generate the function of down-regulating protein expression under the condition of not carrying VEGF-siRNA, and the toxicity effect of the carrier is proved to be from the chemical anti-tumor effect of the membrane material part by combining with the experimental analysis of cell survival rate.

EXAMPLE 7 evaluation of the silencing efficiency of VEGF-siRNA/CRSmRNA levels

Preparing the medicine:

2)VEGF-siRNA/lipo^TM2000: preparing a proper amount of DEPC aqueous solution of VEGF-siRNA, preparing a compound according to the instruction, and diluting the compound by blank DMEM according to a gradient for later use.

3) VEGF-siRNA: appropriate amount of DEPC aqueous solution of VEGF-siRNA. Diluted with a blank DMEM gradient for use.

4) Blank group: HeLa cells were cultured normally in wells, and were not administered, but DMEM with equal amounts of DEPC water was substituted.

The experimental method comprises the following steps:

extracting cell whole RNA:

1) taking a cell culture plate which is cultured for 48 hours, carefully and fully taking and discarding supernatant, adding Trizol lysate (1 mL/hole), repeatedly and softly blowing and beating the sample completely by a sampler until the cells are fully lysed, enabling the bottom of a hole plate to be transparent, sucking the lysate, transferring the lysate to an EP (ultraviolet) tube, standing for 5min, and storing on ice.

2) Chloroform according to volume ratio: chloroform was added to Trizol ═ 0.2:1, the two phases were mixed by vortexing, the mixture was allowed to stand on ice for 5min, at 4 ℃ 12000 Xg, and centrifuged for 15min, after which the liquid in the tube was divided into three layers, the upper colorless phase was an aqueous phase, the lower red phase was a phenol-chloroform phase, and there was a turbid float in the middle. The required whole RNA was present in the aqueous phase, which was carefully aspirated and transferred to a new EP tube.

3) Isopropanol according to volume ratio: adding isopropanol (0.5: 1) into Trizol, mixing the two phases by vortex, standing on ice for 10min, standing at 4 deg.C and 12000 Xg, centrifuging for 10min, and precipitating white flocculent precipitate at the tube wall and bottom after centrifugation to obtain RNA.

4) And (3) sucking and cleaning the supernatant, wherein the volume ratio of the supernatant to the ethanol is 75 percent: trizol ═ 1:1, wash RNA pellet, vortex mix well, 4 ℃, 7500 × g, centrifuge for 5 min.

5) Sucking supernatant, air drying for 10min to precipitate RNA, resuspending DEPC, gently blowing, mixing, standing for 10min, and dissolving in 55-60 deg.C water bath for 10min.

6) The sample was allowed to stand at room temperature, and 2. mu.L of Nanodrop-1000 was aspirated to determine the RNA content.

Reverse transcription:

1) the loading volume required 2ug and was calculated based on the measured RNA concentration.

2) Each reaction well: MicroAmp^TMOptical 96-Well Reaction Plate 10. mu.L 2 XTT Buffer + 1. mu.L 20 XTT enzymeMix + RNA solution per Well in a total volume of 20. mu.L, with DEPC water being insufficient. The plate port was sealed by adding an optically adhesive film. 4 ℃ and 100Centrifuge at 0rmp for 1min to remove air bubbles and allow the liquid to pool to the bottom of the well.

3) Start cDNA in PCR System 9700, conditions: 94 ℃ for 5min,94 ℃ for 30s,75 ℃ for 30s,72 ℃ for 25s,72 ℃ for 7min,4 ℃ infinity. The cycle was 30 times.

4) The sample was allowed to stand at room temperature, and 2. mu.L of Nanodrop-1000 was aspirated to determine the cDNA content.

RT-PCR：

1) The loading amount required 50ng, and the loading volume was calculated based on the measured cDNA concentration.

2)Gene ExpressionMaster Mix、VEGF primers and GAPDH primers in Gene Expression Assays were thawed on ice.

3) Each reaction well: MicroAmp^TMOptical 96-Well Reaction Plate 25 per WellGene expression Master Mix, 2.5. mu.L of primer (VEGF or GAPDH) and cDNA in a total volume of 50. mu.L, with a deficiency of DEPC water. The Optical additive film seals the plate opening. Centrifuge at 1000rmp for 1min at 4 ℃ to remove air bubbles and allow the liquid to pool to the bottom of the well.

4) RT-PCR was started in the Applied Biosystems7500Real-Time PCR System, conditions: hold 50 ℃ for 2min,95 ℃ for 10min. cycles (40cycles) 95 ℃ for 15sec,60 ℃ for 1 min.

5) The experimental process was carried out on ice throughout.

The experimental results are as follows:

as can be seen from fig. 5:

1)VEGF-siRNA/lipo^TMthe relative amount of VEGF mRNA in the 2000 group is obviously reduced, and the naked VEGF-siRNA group has no change, thereby proving the success of the cell model.

2) No statistical difference exists in the VEGF-mRNA relative content of Blank and VEGF-siRNA groups, which indicates that the three administration can not play a role in down-regulating mRNA expression.

3) The VEGF-siRNA/100% CRS group has obvious and obvious difference with naked VEGF-siRNA, and the mRNA relative content is lower along with the increase, which accords with the rule of gene silencing.

4) When the difference between VEGF-siRNA/100% CRS was not significant at 100nM and 125nM, as was the case with the cell viability assay, protein level results, further indicating that silencing of siRNA was roughly complete at 100nM and that increasing concentrations did not cause much change.

5) VEGF-siRNA/100% CRS can reach the same level as VEGF-siRNA/lipo at siRNA concentration of 100nM^TM2000 groups of similar VEGF-mRNA relative content down-regulation effects showed high gene silencing effect of the developed new compound VEGF-siRNA/100% CRS.

Experimental example 8 in vivo targeting verification of CRS in mice

Preparing the medicine:

1) to-pro/100% CRS: preparing a proper amount of 100% CRS solution, adding a To-pro aqueous solution (Cy3 fluorescent labeled dye) according To a proper ratio, uniformly swirling, and incubating at room temperature for 10min for later use.

2) To-pro/DEPC: the aqueous To-pro solution (Cy3 fluorescent-labeled dye) DEPC was diluted To (1) the same concentration in water and vortexed uniformly for use.

The experimental method comprises the following steps:

1) mouse tail vein fixing device, exposing tail, disinfecting with alcohol, selecting one of left and right tail vein, injecting prepared sample, 200 μ L/mouse.

2) After injection, the mice were gas anesthetized at 15min,30min,60min,120min, and 240min, fixed and photographed with an In-vivo Image System FXpRo small animal fluorescence detector (WL16405-10782 WW 32725-.

The experimental results are as follows:

as can be seen in fig. 6:

the blank formulation group and the pure Cy3 fluorescein group have weaker fluorescence intensity, but when the two substances are incubated and loaded, the fluorescence intensity is greatly enhanced, and the FL reaches 25000 or more corresponding to the surface ruler, so that the effective connection of the fluorescein and the carrier is reflected, and conditions are provided for the subsequent in vivo observation of the mice.

As can be seen from fig. 7:

1) at 0min, when no sample was injected, there was no fluorescence distribution in the mice of both the administration group (A) and the control group (B). And (3) at 15min after injection, the drug administration group (A) mice have fluorescence enrichment in the right axilla, the control group (B) mice have no observation, the time lasts for 30min and 60min, the drug administration group (A) drugs are metabolized into the liver, the fluorescence enrichment occurs in the liver, and the control group (B) mice have no phenomenon. From 120min to 240min, the drug is continuously metabolized, and the fluorescence at the tumor site under the right axilla is weakened but still exists.

2) The whole process shows the metabolic pathway of the carrier entering the body of the mouse, and the enrichment of the tumor part under the right axilla shows a certain targeting property of the material.

Experimental example 9 evaluation of antitumor Activity of VEGF-siRNA/CRS

Preparing the medicine:

1) VEGF-siRNA/100% CRS, prepared by gene compression method, the final concentration of VEGF-siRNA in the compound is 0.3 mg/kg:

2) 100% CRS, same as (1), wherein VEGF-siRNA was replaced with DEPC water;

3) VEGF-siRNA, DEPC water dilution to the same concentration (1);

4) DOX, 2 mu mol/kg, and preparing an aqueous solution;

the experimental method comprises the following steps:

1) mice inoculated with ascites tumor in the right axilla were randomly grouped into 10 mice each, for a total of 5 groups, and weighed. The mice of each group are normally raised, and the tumor volume is measured by a vernier caliper until the tumor volume grows to 80-90mm³And administration is started.

2) The administration was tail vein injection once a day for 5 days. 0.2mL of the prepared sample was injected into the tail vein. On day 6, each group of tumor-bearing mice was sacrificed, dissected, and tumors were removed. Weighing the weight and tumor weight according to tumor inhibition ratio (TGI) [ (average tumor weight of negative control group-average tumor weight of treatment group)/average tumor weight of negative control group]X 100% tumor inhibition. Data obtained byAnd (4) representing and carrying out t test.

The experimental results are as follows:

as can be seen from fig. 8 and 9:

1) the NS group and the siRNA group have no significant difference (P is more than 0.01) and have significant difference (P is less than 0.01) with the DOX group, thus proving the success of modeling.

2) The tumor weights of the DOX group, the CRS group and the VEGF-siRNA/CRS group are obviously smaller than those of the NS group and the Naked siRNA group, the tumor inhibition rates respectively reach 52.62 percent, 35.81 percent and 51.21 percent, and good antitumor activity is shown.

3) Compared with the VEGF-siRNA/CRS group, the CRS group has higher tumor inhibition rate, and further proves the tumor inhibition effect of the VEGF-siRNA while proving that the carrier has the anti-tumor activity.

4) The VEGF-siRNA/CRS group has little difference of tumor inhibition rate compared with the DOX group, which indicates that the VEGF-siRNA/CRS group can achieve nearly the same anti-tumor effect as the DOX group in a mouse body.

Experimental example 10 evaluation of safety of CRS

Preparing the medicine:

100% CRS: preparing appropriate amount of 100% CRS solution

The experimental method comprises the following steps:

1) healthy mice were randomly grouped into 8 groups of 2 total groups and weighed.

2) The administration mode is tail vein injection, only 1 time is needed in the first day, and the dosage is 100 times of the effective dosage. Each group of mice was normally bred for 6 days, weighed daily, and observed for changes in behavior and appearance. Weighing, sacrifice, dissection, coring, liver, spleen, kidney, brain, weighing, photographing, and observing morphological changes on day 7.

The experimental results are as follows:

behavior and appearance observation:

after the administration of large dose on day 1, compared with NS group, the mice in the administration group have no obvious abnormal behavior, good action, no lethargy phenomenon, and good response capability to the outside. On day 2, mice in the administered group were observed to be yellowish in color compared to NS group coat, presumably related to the yellow color of the formulation. After 3 to 7 days, there was no abnormality in behavior, the color of the hair became lighter and lighter, and no significant difference was observed between the NS group and the day 7.

As can be seen from fig. 10, 11, and 12:

1) the body weight of the mice in the 100% CRS group decreased on day 2 of the bolus administration, and gradually increased on days 3 to 7 thereafter, to reach the final body weight equivalent to that in the NS group. For analysis reasons, CRS in 100 times dosage still has some toxicity, but does not cause death or produce obvious behavioral effects, and the weight of the mice gradually increases from day 3 to day 7, and is approximately equal to the weight of the normal saline group on day 7.

2) In the comparison of the weight ratios of the organs, the CRS group and the NS group have no significant difference in the body ratios, and the visual observation of Fig.15.3 also shows that the CRS group has the same good appearance as the NS group, no discoloration, no spots and no damage.

Claims

1. The pharmaceutical composition is characterized by comprising the following components in percentage by mass:

2. the pharmaceutical composition according to claim 1, which is composed of the following components in mass percent:

3. a carrier comprising lecithin, cholesterol, and 1-methyl-beta-carboline-3-arginine-glycine-aspartic acid-serine.

4. Use of the pharmaceutical composition of claim 1 for the preparation of a medicament for the treatment of a tumor.