CELL-PENETRATING PEPTIDE-MULTIARM POL YETHYLENE GLYCOL-DRUG CONJUGATE HAVING TARGETING PROPERTY AND APPLICATION THEREOF

Abstract

The present invention provides a cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula I, II, III, IV or V. As compared with linear-chain-type PEG-cell-penetrating-peptide conjugates, the multi-arm PEG has multiple end groups, and has introduction sites for multiple functional groups, which can connect to multiple different active groups. In addition, the cell-penetrating-peptide multi-arm-PEG medicine conjugate to enable the medicine to enter the pathogenetic cells in a targeting manner, to achieve precise treatment. The present invention further provides use of a cell-penetrating-peptide multi-arm-PEG medicine conjugate having targeting ability in preparation of a targeting medicine, especially use of a cell-penetrating-peptide multi-arm-PEG medicine conjugate having targeting ability in the treatment of eye age-related macular degeneration, asthma and pulmonary fibrosis.

Claims

1. A cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula I: ##STR00025## wherein R is a center molecule, and is selected from a polyhydroxy structure, a poly-amino structure or a poly-carboxyl structure; the PEGs are the same or different —(CH.sub.2CH.sub.2O).sub.m—, and an average value of m is an integer of 3-250; C is a cell penetrating peptide (CPP), and is selected from a transcribed trans-activator, VP22, transportan, a membrane-type amphiphilic peptide, a signal transduction peptide and an arginine-sequence-rich peptide; D is a medicine molecule, and the medicine molecule is selected from: a small-molecule medicine, a dye, a polypeptide, an antibody, a plasmid DNA, nucleic acid, liposome, bacteriophage particles, superparamagnetic particles, a fluorescent stain, nanoparticles, a virus, quantum dots and a magnetic-resonance-imaging contrast medium; X is a linking bond between the PEG and CPP, and the linking bond is formed by one or two or more of an amido bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a mercapto-maleimide bond, --triazole-, a carbon-sulphur bond and an ether bond; Y is a linking bond between the PEG and the medicine molecule D, and the linking bond is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond or a mercapto-maleimide bond; and n is a quantity of branches or a quantity of arms, and n is an integer greater than or equal to 3; and k is a quantity of branches or arms that are linked to a CPP terminal, and 1≤k≤n.

2. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 1, characterized in that R is selected from: a pentaerythritol or polypentaerythritol structure, a glycerol or polyglycerol structure, methyl glucoside and sucrose; C is selected from: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPGα, M918, R6Pen, penetratin, Pep-1-K, ARF1-22, Tp10, POD, polylysine formed by 3-100 lysine residues, and polyarginine formed by 4-9 arginine residues; D is selected from a small-molecule medicine, a polypeptide, an antibody and nucleic acid; X is selected from one or a combination of two or more of —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, -triazole- and a mercapto-maleimide bond, wherein j is an integer of 0-10; Y is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, a thioester bond and a mercapto-maleimide bond; and n is an integer of 3-22; and k is an integer of 1-14.

3. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 2, characterized in that R is selected from: ##STR00026## wherein l is an integer greater than or equal to 1 and smaller than or equal to 10; C is LMWP, or polyarginine formed by 8 arginines; D is selected from Omalizumab, Nintedanib, Bevacizumab, Pembrolizumab, Trastuzumab, Nivolumab, VEGF-siRNA, IL-v-siRNA, Syk-siRNA and GATA-3-siRNA, wherein v is selected from 4, 5, 8 and 13; X is selected from —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, -triazole- and a mercapto-maleimide bond, wherein j is an integer of 0-5; and n is an integer of 3-14; and k is an integer of 1-6.

4. The cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula I according to claim 1, having a structure of: ##STR00027##

5-8. (canceled)

9. A cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula IV: ##STR00028## wherein R is a center molecule, and is selected from a polyhydroxy structure, a poly-amino structure or a poly-carboxyl structure; the PEGs are the same or different —(CH.sub.2CH.sub.2O).sub.m—, and an average value of m is an integer of 3-250; C is a CPP, and is selected from a transcribed trans-activator, VP22, transportan, a membrane-type amphiphilic peptide, a signal transduction peptide and an arginine-sequence-rich peptide; D is a medicine molecule, and the medicine molecule is selected from: a small-molecule medicine, a dye, a polypeptide, an antibody, a plasmid DNA, nucleic acid, liposome, bacteriophage particles, superparamagnetic particles, a fluorescent stain, nanoparticles, a virus, quantum dots and a magnetic-resonance-imaging contrast medium; T is a targeting group, and T is selected from: a protein, an antibody, an antibody fragment or a derivative thereof, a small-molecule peptide, a polypeptide, glucose, galactose, folic acid and hyaluronic acid; X is a linking bond between the PEG and CPP, and the linking bond is formed by one or two or more of an amido bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a mercapto-maleimide bond, -triazole-, a carbon-sulphur bond and an ether bond; Y is a linking bond between the PEG and the medicine molecule D, and the linking bond is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond or a mercapto-maleimide bond; B is a linking bond between the PEG and the targeting group, and the linking bond is formed by one or two or more of an amido bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a mercapto-maleimide bond, a carbon-sulphur bond and an ether bond; and n is a quantity of branches or a quantity of arms, and n is an integer greater than or equal to 3; k is a quantity of branches or arms that are linked to a CPP terminal, and 1≤k≤n; and g is a quantity of branches or arms that are linked to the targeting group, and 1≤g≤n.

10. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 9, characterized in that R is selected from: a pentaerythritol or polypentaerythritol structure, a glycerol or polyglycerol structure, methyl glucoside and sucrose; C is selected from: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPGα, M918, R6Pen, penetratin, Pep-1-K, ARF1-22, Tp10, POD, polylysine formed by 3-100 lysine residues, and polyarginine formed by 4-9 arginine residues; D is selected from a small-molecule medicine, a polypeptide, an antibody and nucleic acid; the antibody in T is a monoclonal antibody, and the antibody fragment or the derivative thereof is a single chain of an Fv or Fab fragment; X is selected from one or a combination of two or more of —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-10; Y is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, a thioester bond and a mercapto-maleimide bond; B is selected from one or a combination of two or more of —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-10; and n is an integer of 3-22; k is an integer of 1-14; and g is an integer of 1-8.

11. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 10, characterized in that R is selected from: ##STR00029## wherein l is an integer greater than or equal to 1 and smaller than or equal to 10; C is LMWP, or polyarginine formed by 8 arginines; D is selected from a monoclonal antibody and siRNAs having a length of oligonucleotide of 19-23 bp; T is selected from: folic acid, RGD, cRGD, hyaluronic acid, glucose and galactose; X is selected from —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-5; B is selected from —CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-5; and n is an integer of 3-14; k is an integer of 1-6; and g is an integer of 1-4.

12. The cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula IV according to claim 9, having a structure of: ##STR00030##

13. A cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula V: ##STR00031## wherein R is a center molecule, and is selected from a polyhydroxy structure, a poly-amino structure or a poly-carboxyl structure; the PEGs are the same or different —(CH.sub.2CH.sub.2O).sub.m—, and an average value of m is an integer of 3-250; C is a CPP, and is selected from a transcribed trans-activator (Tat), VP22, transportan, a membrane-type amphiphilic peptide (MAP), a signal transduction peptide and an arginine-sequence-rich peptide; D and D′ are independently selected from: a small-molecule medicine, a dye, a polypeptide, an antibody, a plasmid DNA, nucleic acid, liposome, bacteriophage particles, superparamagnetic particles, a fluorescent stain, nanoparticles, a virus, quantum dots and a magnetic-resonance-imaging contrast medium; X is a linking bond between the PEG and CPP, and the linking bond is formed by one or two or more of an amido bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a mercapto-maleimide bond, -triazole-, a carbon-sulphur bond and an ether bond; Y is a linking bond between the PEG and the medicine molecule D, and the linking bond is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, an ether bond, a carbonyl bond, a thioester bond or a mercapto-maleimide bond; Z is a linking bond between the PEG and the medicine molecule D′, and the linking bond is formed by one or two or more of an amido bond, a disulfide bond, a hydrazone bond, an ester bond, a thioester bond, a mercapto-maleimide bond, a carbon-sulphur bond and an ether bond; and n is a quantity of branches or a quantity of arms, and n is an integer greater than or equal to 3; k is a quantity of branches or arms that are linked to a CPP terminal, and 1≤k≤n; and p is a quantity of branches or arms that are linked to the medicine molecule D′, and 1≤p≤n.

14. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 13, characterized in that R is selected from: a pentaerythritol or polypentaerythritol structure, a glycerol or polyglycerol structure, methyl glucoside and sucrose; C is selected from: LMWP, Tat48-60, Tat48-60-P10, CAI, HIV-TAT, MAP, MPGα, M918, R6Pen, penetratin, Pep-1-K, ARF1-22, Tp10, POD, polylysine formed by 3-100 lysine residues, and polyarginine formed by 4-9 arginine residues; D is selected from a small-molecule medicine; D′ is selected from nucleic acid; X is selected from one or a combination of two or more of —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-10; Y is selected from: a disulfide bond, a hydrazone bond, an amido bond, an ester bond, a thioester bond and a mercapto-maleimide bond; Z is selected from one or a combination of two or more of —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-10; and n is an integer of 3-22; k is an integer of 1-14; and p is an integer of 1-8.

15. The cell-penetrating-peptide multi-arm-PEG medicine conjugate according to claim 13, characterized in that R is selected from: ##STR00032## wherein l is an integer greater than or equal to 1 and smaller than or equal to 10; C is LMWP, or polyarginine formed by 8 arginines; D is selected from a small-molecule medicine; D′ is selected from a monoclonal antibody and siRNAs having a length of oligonucleotide of 19-23 bp; X is selected from —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-5; Z is selected from —(CH.sub.2).sub.jCONH(CH.sub.2).sub.j—, —(CH.sub.2).sub.j—S—S—(CH.sub.2).sub.j—, —(CH.sub.2).sub.jNH—N═C(CH.sub.2).sub.j—, —(CH.sub.2).sub.jCOO(CH.sub.2).sub.j— and —(CH.sub.2).sub.j—S—(CH.sub.2).sub.j—, wherein j is an integer of 0-5; and n is an integer of 3-14; k is an integer of 1-6; and p is an integer of 1-4.

16. The cell-penetrating-peptide multi-arm-PEG medicine conjugate having a general formula V according to claim 13, having a structure of: ##STR00033##

17-19. (canceled)

Description

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0111] The technical solutions of the present invention will be described clearly and completely below with reference to the embodiments of the present invention. Apparently, the described embodiments are merely certain embodiments of the present invention, rather than all of the embodiments. All of the other embodiments that a person skilled in the art obtains on the basis of the embodiments in the present invention without paying creative work fall within the protection scope of the present invention.

Example 1

Preparation Method of Four-Arm PEG-LMWP Conjugate (MW10000)

[0112] ##STR00012##

[0113] LMWP was dissolved in 20 mM of a KH.sub.2PO.sub.4 buffer solution. 4arm PEG-1 arm NHS-3arm Opss was dissolved in DMSO, and then added dropwise into the buffer solution. The reaction was performed at room temperature for 2 hours. The product was filtered, purified by using a heparin column, and freeze-dried or precipitated.

Example 2

Preparation Method of Four-Arm PEG-LMWP-MAL Conjugate (MW10000)

[0114] ##STR00013##

[0115] The above-prepared 4arm PEG-1 arm LMWP-3arm OPSS was dissolved in dichloromethane. Proper amounts of TEA and MAL-NH2 were added. The reaction was performed at room temperature for 12 hours. The reaction liquor was concentrated. The product was purified and freeze-dried or precipitated by using isopropanol.

Example 3

Preparation Method of Four-Arm PEG-LMWP-MAL-RGD Conjugate (MW10000)

[0116] ##STR00014##

[0117] The above-prepared 4arm PEG-1 arm LMWP-MAL-3arm OPSS was dissolved in dichloromethane. A proper amount of RGD was added. The reaction was performed at room temperature for 12 hours. The reaction liquor was concentrated. The product was purified and freeze-dried or precipitated by using isopropanol.

Example 4

Preparation Method of Four-Arm PEG-LMWP-MAL-RGD-3arm-VEGF-siRNA Conjugate (MW10000)

[0118] ##STR00015##

[0119] A 1M DTT solution was added into the above-prepared 4arm PEG-1arm LMWP-MAL-RGD-3 arm OPSS. The reaction was performed at room temperature, to obtain 4arm PEG-1arm LMWP-MAL-RGD-3arm Thiol. Then a mutant SH-VEGF-siRNA was dissolved in 10 mM KH.sub.2PO.sub.4 and 0.15M NaCl, and added dropwise into a proper amount of 4arm PEG-1 arm LMWP-MAL-RGD-3arm Thiol purified by using a heparin column under stirring. The mixture was reacted under continuous stirring for 2 hours. After the reaction has completed, the unreacted SH-VEGF-siRNA was removed by cation-column purification. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

[0120] The sequences of the positive-sense strand and the antisense strand of the SH-VEGF-siRNA are as follows:

TABLE-US-00002 Positive-sense strand: 5′-GAUAGAGCAAGACAAGAAAUU-3′ Antisense strand: 3′-UUCUAUCUCGUUCUGUUCUUU-5′

Example 5

Preparation Method of Four-Arm PEG-LMWP-(VEGF-siRNA)3 Conjugate (MW10000)

[0121] ##STR00016##

[0122] The above-prepared 4arm PEG-1 arm LMWP-3arm OPSS solution was directly added dropwise into a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of pH=6.9 containing a certain amount of SH-VEGF-siRNA under stirring. The system was reacted at 40° C. for 30 min to 1 h, and when detection by using gel electrophoresis showed that the SH-VEGF-siRNA has completely reacted, the reaction was stopped. The reaction liquor was purified by using a DEAE column, wherein the system of the mobile phase was formed by a phase A: a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of PH=6.9, and a phase B: a 20 mM NaH.sub.2PO.sub.4+2M NaCl+1 mM EDTA buffer solution of PH=6.9. The sample was collected at 45% of the phase B, and the gel electrophoresis shown that the product was of a strip. The sample was desalted by using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

Example 6

Preparation Method of Four-Arm PEG-(LMWP)2 Conjugate (MW10000)

[0123] ##STR00017##

[0124] LMWP was dissolved in a 20 mM KH.sub.2PO.sub.4 buffer solution. 4arm PEG-2arm NHS-2arm Opss was dissolved in DMSO, and then added dropwise into the buffer solution. The reaction was performed at room temperature for 2 hours. The product was filtered, purified by using a heparin column, and freeze-dried. The solution may also be directly used for the next step of reaction.

Example 7

Preparation Method of Four-Arm PEG-(LMWP)2-(VEGF-siRNA)2 Conjugate (MW10000)

[0125] ##STR00018##

[0126] The above-prepared 4arm PEG-2arm LMWP-2arm OPSS solution was directly added dropwise into a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of pH=6.9 containing a certain amount of SH-VEGF-siRNA under stirring. The system was reacted at 40° C. for 30 min to 1 h, and when detection by using gel electrophoresis showed that the SH-VEGF-siRNA has completely reacted, the reaction was stopped. The reaction liquor was purified by using a DEAE column, wherein the system of the mobile phase was formed by a phase A: a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of PH=6.9, and a phase B: a 20 mM NaH.sub.2PO.sub.4+2M NaCl+1 mM EDTA buffer solution of PH=6.9. The sample was collected at 45% of the phase B, and the gel electrophoresis shown that the product was of a strip. The sample was desalted by using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

Example 8

Preparation Method of Four-Arm PEG-(LMWP)3 Conjugate (MW10000)

[0127] ##STR00019##

[0128] LMWP was dissolved in a 20 mM KH.sub.2PO.sub.4 buffer solution. 4arm PEG-3arm NHS-1arm Opss was dissolved in DMSO, and then added dropwise into the buffer solution. The reaction was performed at room temperature for 2 hours. The product was filtered, purified by using a heparin column, and freeze-dried. The solution may also be directly used for the next step of reaction.

Example 9

Preparation Method of Four-Arm PEG-(LMWP)3-VEGF-siRNA Conjugate (MW10000)

[0129] ##STR00020##

[0130] The above-prepared 4arm PEG-3 arm LMWP-1arm OPSS solution was directly added dropwise into a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of pH=6.9 containing a certain amount of SH-VEGF-siRNA under stirring. The system was reacted at 40° C. for 30 min to 1 h, and when detection by using gel electrophoresis showed that the SH-VEGF-siRNA has completely reacted, the reaction was stopped. The reaction liquor was purified by using a DEAE column, wherein the system of the mobile phase was formed by a phase A: a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of PH=6.9, and a phase B: a 20 mM NaH.sub.2PO.sub.4+2 M NaCl+1 mM EDTA buffer solution of PH=6.9. The sample was collected at 45% of the phase B, and the gel electrophoresis shown that the product was of a strip. The sample was desalted by using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

Example 10

Preparation Method of Eight-Arm PEG-LMWP Conjugate (MW10000)

[0131] ##STR00021##

[0132] LMWP was dissolved in a 20 mM KH.sub.2PO.sub.4 buffer solution. 8arm PEG-1arm NHS-7arm Opss was dissolved in DMSO, and then added dropwise into the buffer solution. The reaction was performed at room temperature for 2 hours. The product was filtered, purified by using a heparin column, and freeze-dried or precipitated.

Example 11

Preparation Method of Eight-Arm PEG-(LMWP)1-(VEGF-siRNA)7 Conjugate (MW10000)

[0133] ##STR00022##

[0134] The above-prepared 8arm PEG-1arm LMWP-7arm OPSS solution was directly added dropwise into a 20mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of pH=6.9 containing a certain amount of

[0135] SH-VEGF-siRNA under stirring. The system was reacted at 40° C. for 30 min to 1 h, and when detection by using gel electrophoresis showed that the SH-VEGF-siRNA has completely reacted, the reaction was stopped. The reaction liquor was purified by using a DEAE column, wherein the system of the mobile phase was formed by a phase A: a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of PH=6.9, and a phase B: a 20 mM NaH.sub.2PO.sub.4+2M NaCl+1 mM EDTA buffer solution of PH=6.9. The sample was collected at 45% of the phase B, and the gel electrophoresis shown that the product was of a strip. The sample was desalted by using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

Example 12

Preparation Method of Eight-Arm PEG-(LMWP)2 Conjugate (MW10000)

[0136] ##STR00023##

[0137] LMWP was dissolved in a 20 mM KH.sub.2PO.sub.4 buffer solution. 8arm PEG-2arm NHS-6arm Opss was dissolved in DMSO, and then added dropwise into the buffer solution. The reaction was performed at room temperature for 2 hours. The product was filtered, purified by using a heparin column, and freeze-dried or precipitated.

Example 13

Preparation Method of Eight-Arm PEG-(LMWP)2-(VEGF-siRNA)6 Conjugate (MW10000)

[0138] ##STR00024##

[0139] The above-prepared 8arm PEG-2arm LMWP-6arm OPSS solution was directly added dropwise into a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of pH=6.9 containing a certain amount of SH-VEGF-siRNA under stirring. The system was reacted at 40° C. for 30 min to 1 h, and when detection by using gel electrophoresis showed that the SH-VEGF-siRNA has completely reacted, the reaction was stopped. The reaction liquor was purified by using a DEAE column, wherein the system of the mobile phase was formed by a phase A: a 20 mM NaH.sub.2PO.sub.4+1 mM EDTA buffer solution of PH=6.9, and a phase B: a 20 mM NaH.sub.2PO.sub.4+2M NaCl+1 mM EDTA buffer solution of PH=6.9. The sample was collected at 45% of the phase B, and the gel electrophoresis shown that the product was of a strip. The sample was desalted by using an ultrafiltration centrifuge tube with a cut-off molecular weight of 3000. The product was obtained by freeze drying or precipitation, and was stored at −20° C.

Example 14

Comparison of the Cell Effect Inhibitions Between LMWP-PEG5000-VEGF-siRNA and the Four-Arm PEG-LMWP-(VEGF-siRNA)3 (MW 10000)

[0140] Cell transfection was performed on LMWP-PEG5000-VEGF-siRNA and the four-arm PEG-LMWP-(VEGF-siRNA).sub.3 (MW 10000) prepared in Example 5 on 293T cell, and the expression of the VEGFA in the sample was detected by using RT-PCR. The result showed that the cell effect of the four-arm PEG-LMWP-(VEGF-siRNA).sub.3 (MW 10000) is obviously better than that of LMWP-PEG5000-VEGF-siRNA.

[0141] The above particular embodiments are merely illustrative explain on the present invention, and are not limiting the present invention. A person skilled in the art can understand that the particular structures in the present invention may have other variations.