WO2012155328A1 - 包含内皮抑素和rna干扰分子的组合物及其应用 - Google Patents

包含内皮抑素和rna干扰分子的组合物及其应用 Download PDF

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WO2012155328A1
WO2012155328A1 PCT/CN2011/074100 CN2011074100W WO2012155328A1 WO 2012155328 A1 WO2012155328 A1 WO 2012155328A1 CN 2011074100 W CN2011074100 W CN 2011074100W WO 2012155328 A1 WO2012155328 A1 WO 2012155328A1
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endostatin
sirna
liposome
peg
pharmaceutical composition
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French (fr)
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徐根兴
殷妍
郭佳佳
张昌栋
吴稚伟
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JIANGSU RESEARCH CENTER FOR GENE PHARMACEUTICAL ENGINEERING AND TECHNOLOGY
Nanjing University
Nanjing Tech University
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JIANGSU RESEARCH CENTER FOR GENE PHARMACEUTICAL ENGINEERING AND TECHNOLOGY
Nanjing University
Nanjing Tech University
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Priority to PCT/CN2011/074100 priority Critical patent/WO2012155328A1/zh
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    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • composition comprising endostatin and RNA interference molecules and application thereof
  • the invention belongs to the field of biomedicine and relates to a composition and application of endostatin as a delivery system and a chemically synthesized RNA interference molecule.
  • Endostatin is produced by primary tumor cells and inhibits tumor blood vessel growth (0' Reily et al 1997, Cel l 88 : 277 ). It is a fragment of collagen XVI II composed of 183 amino acids. With a high positive charge, it is a rare protein with a high positive charge in recombinant proteins. At present, there are 183 amino acids, 184 amino acids, 193 amino acids with 6 histidines at the N-terminus, and PEG-modified endostatin have been clinically studied or already on the market. However, there is no corresponding report on the delivery of chemically synthesized RNA interference molecules to the corresponding target tissues by endostatin as a delivery system.
  • Endostatin is currently known to have the following biological functions: (1) Inhibition of angiogenesis by inhibiting proliferation and growth of vascular endothelial cells. Endostatin can specifically inhibit the proliferation of vascular endothelial cells induced by fibroblast growth factor (bFGF), inhibit the migration of endothelial cells, induce apoptosis of endothelial cells, but for non-endothelial cells, such as smooth muscle cells, 3T3 fibrils There is no inhibition of cells and the like. (2) It inhibits the growth of blood vessels and does not contribute to resting blood vessels. (3) inhibit tumor growth and metastasis. A number of experiments have shown that recombinant endostatin or endostatin genes can be used for tumor therapy. It has been reported that endostatin inhibits the growth and metastasis of more than 60 different solid tumors. (4) Endostatin has a certain role in inhibiting tumor cells in vitro.
  • bFGF fibroblast growth factor
  • endostatin itself has a certain therapeutic effect on tumors, clinical use of 7.5 mg/m 2 endostatin alone has no obvious effect. Need to be combined with chemotherapy drugs.
  • RNA interference has attracted much attention since its discovery in 1998.
  • Fire et al (Nature, 1998, 391: 806-811) first discovered double strand RNA (dsRNA) in the nematode to silence gene expression.
  • siRNA synthetic small interfering RNA
  • siRNA can mediate RNA interference in cells, recognize specific mRNA complementary to its sequence, and silence the expression of related genes.
  • the action is specific and efficient, and is a powerful method for specifically inhibiting gene expression.
  • siRNA has become a potential therapeutic tool for many disease-related genes and has become a hot topic in clinical application research. Twelve of the siRNA drugs have entered the I-111 clinical study internationally.
  • aiRNA Asymmetric double-stranded interfering RNA
  • aiRNA Asymmetric double-stranded interfering RNA
  • siRNA has many advantages and may replace siRNA for innovative drug research in the future.
  • siRNA-mediated gene silencing is more efficient, durable, structurally stable, and can reduce the off-target effects of the sense strand.
  • AsiRNA drugs are currently not reported in clinical studies internationally.
  • MicroRNA is a non-coding small RNA of about 22-28 nt in length, and mi RNA passes through the target mRNA 3 ' UTR (Untranslated Regions, the untranslated region, which is a non-coding fragment at both ends of the mRNA molecule).
  • UTR Untranslated Regions, the untranslated region, which is a non-coding fragment at both ends of the mRNA molecule.
  • Complete or incomplete complementary binding results in degradation of target mRNA or translational inhibition, thereby regulating expression of target genes, affecting cell proliferation, differentiation and apoptosis.
  • Their regulation of post-transcriptional gene expression is regulated by miRNA-mediated specific gene silencing leading to degradation of target mRNA and inhibition of protein synthesis. miRNAs have important regulatory roles in cell proliferation, differentiation and apoptosis. Gene expression regulation studies are often performed using microRNA mimics or inhibitors.
  • miRNA mimics are miRNAs that mimic in vivo organisms and are synthesized by chemical synthesis to enhance the function of endogenous miRNAs. miRNA mimics can further enhance the silencing of endogenous miRNAs and reduce the amount of targeted protein expression in cells.
  • MicroRNA inhibitors directly inhibit the function of miRNAs. For example, the Danish pharmaceutical company Santafis Pharma announced that its new treatment for hepatitis C, SPC3649 (locked nucleic acid LNA-antimiRTM-122), has entered the first phase of human clinical trials, the world's first miRNA drug for human clinical trials. Direct inhibition of the function of hepatitis C microRNA-122.
  • RNA interference molecules whether chemically modified or unmodified, two strand-symmetric small nucleic acid interference (siRNA), chemically modified or unmodified two strands paired with asymmetric small nucleic acid interference (asiRNA, aiRNA) ), or a mimetic or inhibitor of microRNA (miRNA).
  • siRNA two strand-symmetric small nucleic acid interference
  • aiRNA asymmetric small nucleic acid interference
  • miRNA mimetic or inhibitor of microRNA
  • the object of the present invention is to provide a high dose, a poor stability, a large toxic side effect or a high manufacturing cost when the siRNA, a siRNA, and miRNA gene drugs characterized by high negative charge in the prior art are administered intravenously.
  • a pharmaceutical composition comprising an endostatin recombinant protein carrying a charge of +5 to +15 and a chemically synthesized RNA interference molecule carrying a charge of -15 to -55, which are combined by positive and negative charges to form a charge value at -20
  • Compositions between +20 preferably form a composition having a charge value between -10 and +10.
  • the pharmaceutical composition further comprises a cationic liposome, a neutral liposome or a PEG-modified long-acting liposome for encapsulating the endostatin recombinant protein and the RNA interference molecule to modulate the amount of positive and negative charges.
  • the composition having a positive or negative electric charge value can also be directly used without being dissolved in the physiological saline solution for injection without liposome encapsulation.
  • the endostatin recombinant protein is a recombinant protein purified and expressed by Escherichia coli, yeast, mammalian or human cells, and a viral vector, and may be all endostatin products currently commercially available, or may be in accordance with the art.
  • the endostatin recombinant protein preferably does not carry a 6 histidine tag of a human-structured endostatin recombinant protein consisting of 183-184 amino acids, N-terminal or / and C-terminal
  • a human-structured endostatin fusion protein consisting of 190-200 amino acids carrying a 6-histidine tag, or a long-acting endothelin formed by PEG modification or albumin modification of the above two types of endostatin recombinant proteins Or a synthetic truncated endostatin (including the major amino acid composition of the 183 amino acid sequence).
  • All types of endostatin recombinant proteins are commonly characterized by carrying a high positive charge from +5 to +15 and have a specific affinity for vascular endothelial cells of active lesions.
  • RNA interference molecule is selected from a chemically modified or unmodified two strand symmetric small nucleic acid interference molecule (siRNA), and the chemically modified or unmodified two strand pairing asymmetric small nucleic acid interference molecule (asiRNA, aiRNA), Or chemically modified or unmodified miRNA.
  • siRNA chemically modified or unmodified two strand symmetric small nucleic acid interference molecule
  • asiRNA aiRNA
  • miRNA chemically modified or unmodified miRNA
  • the two strand-symmetric small nucleic acid interference molecules are double-stranded paired RNAs of the length of the sense strand and the antisense strands, which are 19-25 nt in length, and the two strands of the two strands contain 2_4
  • the UU base or the deoxyribonucleic acid dTdT is single-stranded;
  • the two strands pair asymmetric amino acid interference molecules are chemically synthesized, starting from the target gene symmetric small nucleic acid interference molecule siRNA
  • siRNA By dividing the 5' or 3' end of the siRNA sense strand or the antisense strand by l_5nt bases, the lengths of the two strands are inconsistent, and there are 2-4 UU bases at each 3' end of the strand or dTdT of deoxyribonucleic acid Single-stranded suspension, 14-21 nt double-stranded asymmetric small nucleic acid interference molecule asiRNA
  • the miRNAs include miRNA mimics and miRNA inhibitors.
  • miRNA mimics are miRNAs that mimic in vivo sources and are synthesized by chemical synthesis to enhance the function of endogenous miRNAs.
  • miRNA inhibitors are chemically modified inhibitors specific to target miRNAs in cells that specifically silence a single gene or simultaneously silence multiple related but not identical genes.
  • the chemical modification refers to a thio modification, a methylation modification, a phosphorylation modification, a cholesterol modification, or a fluoro modification.
  • siRNA, or asiRNA, or miRNA involved in the present invention are chemically synthesized RNA interference molecules whether or not chemically modified, and the common feature is that the high negative charge is from -15 to -55, and the expression of the target gene can be regulated.
  • the components of the pharmaceutical composition are separately packaged for use as they are, or directly mixed and packaged together.
  • composition for the preparation of a medicament for treating malignant tumor, hepatitis or AIDS.
  • the components of the pharmaceutical composition are separately packaged for use as they are, or directly mixed and packaged together.
  • the endostatin recombinant protein used in the invention and the RNA interference molecule inhibiting gene expression are combined by positive and negative charges, and can be directly mixed according to the ratio of positive and negative charges, and can be directly mixed according to the positive or negative charge ratio.
  • the charge ratio is mixed and used.
  • the ratio of positive and negative charges can be calculated by electrophoresis and charge measurement, and the proper ratio means that the final composition has a charge value between -20 and +20 after mixing.
  • the composition may also be encapsulated with cationic liposomes, neutral liposomes, or PEGylated long-acting liposomes to adjust the ratio of positive and negative charges to a more suitable range of charge values (ie, charge values between -10 and +10).
  • composition is not precipitated in the physiological saline for injection, does not precipitate, does not affect the activity, is more stable, and is more likely to enter the cells, and can deliver chemically synthesized RNA by subcutaneous injection, local injection, intravenous injection, intravenous infusion.
  • Interfering molecules The target organ regulates the expression of the gene of interest.
  • the recombinant human endostatin provided by the present invention has a high positive charge property, and is combined with a chemically synthesized RNA interference molecule carrying a high negative charge by positive and negative charges to form a composition with or without liposome encapsulation. It does not precipitate in the physiological saline for injection, does not precipitate, does not affect activity, is more stable, and is more likely to enter cells.
  • the pharmaceutical composition greatly reduces the dosage and charge value of endostatin and chemically synthesized RNA interference molecules, and since the delivery of endostatin has the specific property of targeting focal vascular endothelial cells, the composition is in vein
  • the target tissue can be targeted and stably distributed within 1 hour or 24 hours after injection, so that the therapeutic effect is better, the activity is more stable, the effective action time is longer, and the side effects are smaller.
  • the pharmaceutical composition of the present invention has both a targeting effect and a gene regulation function, and plays an active role in the treatment of malignant tumor, hepatitis or AIDS, and can be used for preparing a medicament for treating malignant tumor, hepatitis or AIDS.
  • FIG. 1 Schematic diagram of Bcl2-siRNA, endostatin, PEG liposome (complex lipid) forming a Bcl2_siRNA interference composition that can be administered intravenously.
  • a separate bottle of Bcl2-siRNA, endostatin, and PEG liposomes is mixed together at the time of use.
  • Figure 2. Correspondence diagram between the charge of endostatin and the pH of the solvent. The abscissa is the solvent pH and the ordinate is the endostatin charge.
  • FIG. 4 Zeta potential map of a combination of endostatin and VEGF-a siRNA and PEG liposome-chitosan. Zeta potential is +13.7 mV
  • Figure 5 Electropherogram of the formation of a composition of endostatin and Bcl2-siRNA in the presence of different liposomes.
  • FIG. 1 Electropherogram of endostatin with different gene siRNA, asiRNA, miRNA forming a composition with or without liposome encapsulation.
  • Electropherogram of the composition of VEGF-a siRNA and endostatin in the absence of liposome encapsulation, from left to right, VEGF-a siRNA and endostatin are 1:0, 1:5, 1: 10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60 mass ratio electrophoretic pattern of the composition formed;
  • Electropherogram of a composition of anti-Has-122 miRNA inhibitor and PEG endostatin in the absence of liposome encapsulation, from left to right, respectively, anti-Has-122 miRNA inhibitor and PEG endothelin An electropherogram of a composition formed by mass ratios of 3:0, 3:5, 3:10, 3:15, 3:20, 3:25, 3:30, 3:40, 3:50.
  • Electropherogram of the composition of Rabllb-siRNA and Endostatin in the absence of liposome encapsulation The lanes from left to right are Rabllb-siRNA and endostatin at 1:0, 1:5, respectively. Electropherogram of the composition formed by 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50 mass ratio.
  • Figure 7 Particle size of the composition formed by endostatin and chemically synthesized RNA interference molecules.
  • the control group did not receive any transfection, and only different concentrations of cisplatin were added.
  • the B C 12-a siRNA-endostatin group was transfected with B C 12-a siRNA using the endostatin-PEG liposome delivery system followed by the addition of different concentrations of cisplatin.
  • Cytotoxicity of different liposome, endostatin and B C 12-a siRNA forming compositions wherein 1-4 represents a composition formed by Lipo 2000 liposome and B C 12-a siRNA, endostatin-PEG lipid
  • Guang 6 represents: 1 : Lipo 2000 transfected B C 12-a siRNA group; 2 : PEG liposome-endostatin complex transfected Bcl2-a siRNA group; 3: Lipo 2000 transfected Bcl2_a siRNA + low dose cisplatin (1 Micrograms group; 4: PEG liposome-endostatin complex transfected with B C 12-a siRNA + low dose cisplatin group; 5: low dose cisplatin (1 ⁇ g) alone group; 6: doubling dose of cisplatin ( 2 ⁇ g) Individual group.
  • Figure 11 Inhibitory curves of endostatin and chemically modified B C 12-a siRNA forming compositions against tumor volume of H22 liver cancer-bearing mice.
  • the saline group was a blank control for intravenous injection of normal saline; the CTX (15 mg/kg) group and the CTX (30 mg/kg) group were positive controls for intraperitoneal injection of the chemotherapy drug cyclophosphamide; PEG liposome-endostatin-no
  • the relevant siRNA group was a negative control for the formation of a complex of PEG liposome-endostatin with unrelated siRNA in the tail vein; PEG liposome-endostatin-Bcl2-a siRNA group was injected intravenously with PEG liposome-endostatin Complex formed with B C 12-a siRNA; PEG liposome-endostatin_Bcl2-a siRNA+CTX group was a combination group using CTX (15 mg/kg) on the previous group.
  • Figure 12 Inhibition curve of endothelin and chemically synthesized RNA interference molecule forming composition against tumor volume of H22 liver cancer mice.
  • the saline group was a blank control for intravenous saline injection; the CTX (30 mg/kg) group was a positive control for intraperitoneal injection of the chemotherapy drug cyclophosphamide; the PEG liposome group was injected with PEG liposome, PEG liposome.
  • - Endostatin-VEGF-a siRNA group was injected intravenously with PEG liposome-endostatin and VEGF-a siRNA complex.
  • FIG 14. Survival curves of H22 tumor-bearing mice injected with endostatin and chemically synthesized VEGF RNA interference molecules. The samples and injections of each group are the same as in Figure 12.
  • Figure 16 Distribution of endostatin and chemically modified and fluorescently labeled B C 12-a siRNA in various organs of human hepatocarcinoma nude mice and targeting tumor effects. From left to right, the heart, liver, spleen, Lung, kidney, tumor.
  • FIG. 17 Distribution of Endostatin and chemically modified and fluorescently labeled CCR5_a siRNA in various organs of normal nude mice. a: 1 hour and 24 hours of sample injection for quantitative analysis of CCR5_as iRNA residual amount in each organ; b: 1 hour after the injection of the sample, the animal was removed and the viscera was taken for biopsy, from left to right, heart, liver, spleen, Lung, kidney; c: Samples were sacrificed 24 hours after the animals were sacrificed and the viscera was taken for biopsy. From left to right, the heart, liver, spleen, lungs and kidneys were followed. Figure 18. Effect of endostatin and chemically synthesized RNA interference molecule forming composition on human lung cancer A549 xenografts in nude mice. Concrete
  • Fig. 1 The process of forming endothelin and siRNA by positive and negative charge in the presence of liposomes to form a complex is represented by a pattern diagram (Fig. 1), which is divided into three bottles of siRNA, endostatin and liposome.
  • Fig. 1 The advantage is that the quality control of each component is relatively easy, the storage is convenient, but it is troublesome to use. It is also possible to premix the three bottles of the ingredients in a certain proportion of the charge ratio described in the following examples, and combine them in one bottle, which is convenient to use, but the quality control of the mixed components is difficult.
  • Figure 1 shows the siRNA of the tumor anti-apoptosis gene (B-cell lymphoma/le kemia-2, Bcl2, formerly known as B-cell lymphoma/leukemia 2 gene, Bcl2), which can be replaced with asiRNA of this gene or other genes.
  • the liposome exemplified in Fig. 1 may be a cationic liposome, a neutral liposome, or a PEG-modified long-acting liposome, or a liposome, and the endostatin is injected with physiological saline for injection.
  • the mixture is mixed with the RNA interference molecule composition and intravenously instilled.
  • the endostatin recombinant protein used in this embodiment is produced by Jiangsu Zhongzhong Pharmaceutical Group Co., Ltd. Zhongkai Biopharmaceutical Factory, hereinafter referred to as endostatin, consisting of 184 amino acids, the amino acid sequence is: SEQ ID N0. 1, isoelectric The point is 9.3.
  • the mixture is mixed at a mass ratio of 1:10:5 and diluted 15 times.
  • the Zeta is measured by Zeta Sizer 3000 dynamic laser scatterometer. Potential.
  • the zeta potential of PEG liposome-endostatin-B C 12_siRNA is shown in Figure 3. 5 mV ⁇ The average zeta potential of the PEG liposome-endostatin-Bcl2-siRNA was 2. 6mV.
  • VEGF vascular endothelial growth factor
  • VEGF-a siRNA, endostatin, PEG liposome, chitosan, ⁇ -cyclodextrin were mixed at a mass ratio of 1:10:5:15:5, diluted 20-fold to 5 ml with DEPC water, and used 0 The 22 ⁇ m filter was filtered and sealed. A mixture in which PEG liposome, chitosan, and ⁇ -cyclodextrin are formed at 5:15:5 is called chitosan liposome, the same below.
  • the zeta potential of the endostatin-VEGF-a siRNA-chitosan liposome composition was measured, and the average zeta potential was 13.7 mV. The results are shown in Fig. 4.
  • the method determines the charge profile of a composition consisting of an endostatin recombinant protein and an interfering molecule in the presence or absence of liposome encapsulation.
  • RNA loading buffer 100 V, electrophoresis for 30 min.
  • the gel is placed under an ultraviolet lamp to be photographed, and the result is shown in Fig. 5. It can be seen from Fig. 5 that the negatively charged various concentrations of Bcl2-siRNA are mixed with endostatin, and after being encapsulated by liposome and encapsulated by liposome, the electrophoresis strip has hysteresis and appears from negative to negative A certain gradient of positive charge indicates that Bcl2-siRNA forms a positively charged combination with endostatin and liposomes.
  • VEGF-aiRNA powder (sequence and charge value were the same as in Example 2) was dissolved in DEPC water to prepare a 20 nM stock solution.
  • the agarose gel configuration method is the same as above.
  • VEGF-a siRNA and endostatin (sequence and charge value are the same as in Example 2) with a mass ratio of 1:0, 1:5, 1:10, 1:20, 1:30, 1:40, 1: 50, 1:60 were mixed in an RNase-free EP tube, allowed to stand for 15 min, and added to the agarose gel well. Electrophoresis was carried out for 30 min at a constant pressure of 100 V. Take a picture and observe the band. The result is shown in Figure 6a.
  • VEGF-a siRNA sequence, charge value are the same as in Example 2
  • endostatin sequence, charge value are the same as in Example 2
  • PEG liposome mixed at a mass ratio of 1:10:5, respectively, with chitosan :1: 0, 1: 5, 1: 10, 1: 15, 1: 20, 1: 30 mass ratio mixing, and then ⁇ _cyclodextrin with a mass ratio of 1:2.5, 1: 5, in The RNase-free fistula was mixed and allowed to stand for 15 min. Add the agarose gel pores in proportion to the mass ratio from small to large. At 70V constant pressure, electrophoresis for 30min. Photographs were taken to observe the amount of charge carried by the strips and the analysis composition at different ratios. The results are shown in Figure 6b.
  • the electrophoresis of the same method can be used to observe the charge of a composition composed of an endostatin recombinant protein and an RNA interference molecule without liposome encapsulation.
  • Hepatitis-associated Has-122 miRNA inhibitor anti-Has-122 (sequence 5 'ACAAACACC AUUGUC ACACUCCA-3 (SEQ ID N0.2) ) 4.5 ⁇ l, PEG-modified long-acting endostatin (1 mg/ml, Jiangsu Wuzhong Pharmaceutical Group Co., Ltd.
  • Has-122 miRNA inhibitor anti-Has-122 and PEG endostatin are respectively in mass ratio of 3:0, 3:5, 3:10, 3:15, 3: 20, 3: 25, 3: 30, 3: 40, 3: 50 mixed, room temperature for 20min.
  • the above sample was added to RNA loading buffer, and subjected to agarose gel electrophoresis, 100 V, and the gel was placed under ultraviolet light for imaging at 10 min, 15 min, 20 min, and 25 min, respectively, and photographed.
  • the electropherogram is shown in Figure 6c.
  • Different ratios of anti-Has-122 and PEG The composition formed by endostatin exhibits different charge values in electrophoresis, and a composition with a weak positive charge is selected for zeta potential measurement, and the measured potential value is usually within 10 mV, so that it is convenient to find a suitable combination by electrophoresis.
  • the ratio of anti-Has-122 and PEG endostatin corresponding to the charge value of the substance.
  • siRNA receptor-associated gene CCR5 was obtained as asiRNA (sequence: sense strand 5'GUCAAGUCCAAUCUAUGdTdT 3% antisense strand 3 'dTdTCAC AGUUC AGGUUAGAUAC 5'), tumor ion channel-associated Rabllb siRNA (sequence: sense strand 5 'UGUCAGACAGACGCGAAAAdTdT3', antisense strand 3'dTdTACAGUCUGUCUGCGCUUUU 5'), siRNA for the drug resistance gene MDR1 related to tumor chemotherapy drug resistance (sequence: sense chain 5'AAAAUGUUGUCUGGACAAGCAdTdT3', antisense strand 3 'dTdTUUUUACAACAGACCUGUUCGU 5') 1.5 ⁇ 1 and N-terminal endostatin with 6 histidine (lmg/ml, trade name Endu, produced by Shandong Xiansheng Maidjin Bio-Pharmac
  • Bcl2-a siRNA with a charge value of -38 (sequence: sense strand: 5' GAGGCUGGGAUGCCUUUdTdT 3', antisense strand: 3' dTdTGCCUCCGACCCUACGGAAA 5', concentration 20nM) and endostatin (lmg/ml, sequence, charge value
  • PEG liposome (1 mg/ml) was gently mixed at a mass ratio of 1:10:5, and left at room temperature for 15 minutes.
  • the above mixture was diluted 15 times with 0.05 M acetic acid-sodium acetate buffer.
  • the particle size was measured using a Mastersizer FOO particle size analyzer. 4nm ⁇
  • the particle size distribution of the PEG liposome-endostatin-B C 12-a siRNA is relatively uniform, the average particle size is 142.4 nm.
  • the Zeta potential was measured to be +3 mV.
  • VEGF-a siRNA (sequence, charge value are the same as in Example 2), endostatin (1 mg/ml, sequence, charge value are the same as in Example 2), PEG lipid, chitosan (CS), ⁇ -cyclodextrin Fine ( ⁇ -CD) mixed with a mass ratio of 1: 10 : 5 : 15 : 5, that is, 30 ⁇ 1 VEGF-aiRNA was gently mixed with 80 ⁇ 1 endostatin, allowed to stand at room temperature for 5 min, and added with PEG lipid.
  • the present invention used the PEG liposome-endostatin system to transfect the B C 12-a siRNA fragment with the charge value and sequence as in Example 4.
  • the B C 12-a siRNA powder was dissolved in DEPC water to prepare a 20 nM stock solution.
  • human cervical cancer cell HeLaB2 cells purchased from the Institute of Oncology, Chinese Academy of Medical Sciences, Chinese Academy of Medical Sciences
  • DMEM cell culture medium containing calf serum, penicillin and streptomycin.
  • the density of the cells reaches 50%, the original medium is discarded and cultured in DMEM without calf serum and penicillin or streptomycin.
  • the IC50 of cisplatin is significantly reduced, that is, cells transfected with PEG liposome-endostatin-B C 12-a siRNA can inhibit cancer cell proliferation only by lower concentration of cisplatin .
  • Human cervical cancer cell line HeLaB2 with high expression of Bcl2 (purchased from the Chinese Academy of Medical Sciences, Peking Union Medical College, Cancer Research) The cells were inoculated into a 96-well plate and cultured at 37 ° C in an incubator containing 5% CO 2 . When the density of the cells reached 50%, transfection was carried out. The experiment was divided into 4 groups with at least three parallel control wells in each group. Group 1 per well was added 0. 25 ⁇ ILipof ectAMINETM2000 (Lipo 2000) liposome (Life Techonolobies, product number: 11668-019, trade name Inivitrogen, the same below) +0.
  • 25 ⁇ 1 Bcl2_a siRNA (concentration, charge value The sequence is the same as in Example 4, the same below); Group 2 is added 0. 25 ⁇ 1 Bcl2-a siRNA + 0. 5 ⁇ 1 Endostatin (concentration, charge value, sequence are the same as in Example 4, the same below) + 0. 25 ⁇ 1 PEG liposome (concentration as in Example 4, the same below), the zeta potential of the PEG liposome-endostatin-B C 12-a siRNA was determined to be +3 mV; group 3 was added 0 per well. 25 ⁇ 1 Bcl2_a siRNA +0. 5 ⁇ 1 endostatin + 0. 25 ⁇ 1 chitosan liposome; Group 4 is a blank control without any treatment.
  • the culture plate was placed in a 37 V, 5% (0 2 incubator) for 24 h, and the medium was aspirated.
  • the CCK-8 kit instructions 100 ⁇ l of DMEM medium and 10 ⁇ l of CCK were added per well.
  • -8 reagent placed in a 37 ° C incubator for further 1 hour, and 0D450 detection with a microplate reader.
  • the culture plate was placed in a 37 V, 5% C0 2 incubator for 24 h, and then the medium was aspirated.
  • the CCK-8 kit instructions 100 ⁇ l of DMEM medium and 10 ⁇ l of CCK-8 were added per well.
  • the reagent was placed in a 37 ° C incubator for further 1 hour, and the 0D450 was detected by a microplate reader, and the cell viability was calculated as in Example 5.
  • the relationship between the concentration of PEG liposome-endostatin-B C 12-a siRNA complex and cytotoxicity is shown in Figure 6%.
  • the liposomes as PEG - endostatin -B C increased 12-asiRNA complex concentration, cell viability decreased, i.e., increased toxicity to the cells, but not the whole toxicity to cells, when When the concentration of PEG liposome-endostatin-Bcl2-a siRNA complex reached 32 times the normal transfection concentration, the cell viability was about 77%, which was not toxic to cells.
  • Bcl2_a siRNA group was transfected with Lipof ectAMINETM2000 (Lipo 2000) liposome (charge value, sequence is the same as in Example 4); 2: PEG liposome-endostatin complex transfection B C 12-a siRNA group; 3: Lipo 2000 Transfection of B C 12-a siRNA + low dose cisplatin (1 ⁇ g) group; 4: PEG liposome-endothelial complex transfection of B C 12-a siRNA + low dose cisplatin (1 ⁇ g) group; 5 : low dose cis Platinum (1 ⁇ g) alone; 6: doubling dose of cisplatin (2 ⁇ g); 7, blank control group.
  • HelaB2 cells in logarithmic growth phase were inoculated into 96-well plates one day before transfection. When the cell density grew to 50% coverage, the original medium was discarded, and the serum-free and penicillin-free, streptomycin were used. The DMEM medium was washed once, and 100 ⁇ l of DMEM medium containing no calf serum and penicillin or streptomycin was added. Group 1 and Group 3 were transfected as follows: 0.25 ⁇ l of Bcl2-a siRNA (20 nM) was diluted with 25 ⁇ l of serum-free Opti-MEM and gently incubated for 5 minutes at room temperature.
  • Groups 2 and 4 replaced the 0.25 ⁇ l Lipo 2000 liposome in the above transfection method with 0.25 ⁇ l PEG liposome + 0.5 ⁇ l endostatin complex. Other transfection methods are the same as above.
  • the zeta potential of Group 2 PEG liposome-endostatin-Bcl2-a siRNA was determined to be +5 mV.
  • the blank control group was not transfected. After 24 h of transfection, groups 3, 4, and 5 were added with 1 ⁇ g of cisplatin, and group 6 was added with 2 ⁇ g of cisplatin. After 72 h of transfection, CCK-8 assay was performed as described in Example 5.
  • the 0D value of the blank control group was set to 100%, and the relative values of the 0D values of the other groups and the 0D values of the blank control group could reflect the relative levels of the number of cells in each group.
  • the in vitro inhibitory effect of PEG liposome-endostatin-B C 12-a siRNA complex in combination with cisplatin on HelaB2 cells is shown in Figure 10. It can be seen from Figure 10 that the PEG liposome-endostatin complex can also transfect B C 12-a siRNA into cells compared to the commercial transfected liposome Lipo 2000, thereby inhibiting cell growth. Moreover, PEG liposome-endostatin complex transfection with B C 12-a siRNA can inhibit cell proliferation better when combined with low cisplatin, and its inhibition rate on cell growth is higher than that of cisplatin alone. Group ( Figure 10).
  • the interference molecule used in this example was a cholesterol-modified B C 12-a siRNA with a charge value of -35 (sense strand: 5 ' chol-GAGGCUGGGAUGCCUUUdTdT3, antisense strand: 3 'dTdTGCCUCCGACCCUACGGAAA5 ', chol indicates cholesterol modification).
  • the irrelevant siRNA sequence is (sense strand: 5 ' UUCUCCGAACGUGUCACGUdTdT 3 ', antisense strand: 3 'dTdTAAGAGGCUUGCACAGUGCA 5 ').
  • Mouse liver cancer H22 liver cancer ascites cells (Laboratory Animal Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences) After resuscitation at 37 °C, each mouse was intraperitoneally injected with 0.3 ml, and the second generation was inoculated after 7 days. The second generation of ascites was adjusted, and the cell concentration was adjusted to 5 ⁇ 10 6 /ml with physiological saline. Each mouse was injected subcutaneously into the right side of the forelimb with 0.2 mL, about 1 ⁇ 10 6 tumor cells. The mice were fed ad libitum and fed normally.
  • mice were randomly divided into six groups: saline group, chemotherapy drug cyclophosphamide CTX (15 mg/kg) group, chemotherapy drug cyclophosphamide CTX (30 mg/kg) group, PEG liposome-endostatin-no siRNA group, PEG liposome-endostatin_Bcl2-a siRNA group (Zeta potential measurement +3mV), PEG liposome-endostatin _Bcl2-a siRNA Combined chemotherapy drug cyclophosphamide CTX (15mg/kg) group.
  • the administration was started the next day after the inoculation, and the dose of B C 12-a siRNA was 1 mg/kg, and the mass ratio of B C 12-a siRNA to endostatin and PEG liposome was 1:10:5.
  • the amide CTX (15 mg/kg) group was administered with continuous tail vein for 7 days (0.4 ml/mouse), CTX (15 mg/kg) group and CTX (30 mg/kg) group at the 2nd, 4th, 6th, and 8th after inoculation.
  • Tianjing is administered by intraperitoneal injection.
  • the length and width of the tumor were measured with a vernier caliper on the 5th, 10th, 15th and 20th day of administration, and the volume of the tumor was calculated by the following formula: a*b 2 *0.5 (a is long and b is wide) .
  • the tumor volume curve of each group is shown in Fig. 11. It can be seen from Figure 11 that PEG liposome-endostatin-B C 12-a siRNA can inhibit tumor growth well compared with saline group and unrelated siRNA group, and combined with chemotherapy drug cyclophosphamide CTX, antitumor effect Further increase.
  • the sense chain 5' cholesterol-modified VEGF symmetric and asymmetric RNA interference molecule used in this example the VEGF-a siRNA with a charge value of -38 (19+2/21+2, sense strand 5' cho 1 - GUGAAUGCAGACCAAAGAAdTdT 3', antisense strand 3' dTdTUACACUUACGUCUGGUUUCUU 5' ) and VEGF-siRNA with a charge value of -40 (21+2/21+2, sequence: sense strand 5' AUGUGAAUGCAGACCAAAGAAdTdT3 ', antisense strand 3' dTdTUACACUUACGUCUGGUUUCUU 5 ' ).
  • mice were randomly divided into 5 groups after vaccination: saline group (NS), chemotherapy drug cyclophosphamide CTX group, PEG liposome group, PEG liposome-endostatin _VEGF-a siRNA21/23 (Zeta potential measurement was +13mV). PEG liposome-endostatin-VEGF-siRNA23/23 group (Zeta potential measurement was +15 mV.).
  • the administration was started the next day after inoculation.
  • the doses of siRNA and as i RNA were 1 mg/kg, NS (0.4 ml/mouse), PEG liposome group (0.3 ml/mouse), and continuous tail vein administration for 14 days.
  • CTX (30 mg/kg) was administered intraperitoneally on days 2, 4, 6, 8, 10, and 12 after inoculation.
  • the length and width of the tumor were measured with a vernier caliper, and the volume of the tumor was calculated by the following formula: a*b3 ⁇ 40.5 (a is long and b is wide).
  • the tumor volume of the liposome-VEGF-a siRNA group was significantly smaller than that of the other groups, and it had a good tumor suppressing effect.
  • the tumor volume curve of each group is shown in Fig. 12.
  • mice Male Kunming mice were inoculated, grouped, specifically administered and dosed in the same manner as the Bcl2 fraction in Example 8, using PEG liposome, B C 12-a siRNA, endostatin and PEG liposome-endostatin- The charge values of B C 12-a siRNA were also the same as in Example 8. During the whole treatment, the mice were free to eat and drink water, and the living conditions and survival of the mice were recorded. The number of days was counted from the next day when the tumor cells were inoculated. By the 60th day, the survival time of 60 days or more was also calculated according to 60 days.
  • the median survival time of each group was as follows: saline group: 29 days; chemotherapy drug cyclophosphamide CTX (15 mg/kg) group: 39 days; chemotherapy drug cyclophosphamide CTX (30 mg/kg) group: 17 days; lipid - endostatin-unrelated siRNA group: 34 days; liposome-endostatin-Bcl2-a siRNA group: 41 days; liposome-endostatin-B C 12-a siRNA combined with chemotherapy drug cyclophosphamide CTX (15 mg/kg) Group: 58 days.
  • the survival curves of the mice in each group are shown in Fig. 13.
  • liposome-endostatin-B C 12-a siRNA can significantly prolong the survival of tumor-bearing mice, and when combined with low-dose chemotherapy drugs, when compared with chemotherapy drugs alone The survival period has been extended.
  • Male Kunming mice were inoculated, grouped, specifically administered and dosed in the VEGF fraction of Example 8, PEG liposomes, RNA interference molecules, endostatin and PEG liposome-endostatin-B C
  • the charge values of 12-VEGF-a siRNA21/23, PEG liposome-endostatin_Bcl2-VEGF-siRNA23/23 were also the same as in Example 8.
  • mice were free to eat and drink water, and the living conditions and survival of the mice were recorded. The number of days was counted from the next day when the tumor cells were inoculated. By the 60th day, the survival time of 60 days or more was also calculated according to 60 days.
  • the median survival time of each group was as follows: saline group: 35 days; CTX (30 mg/kg) group: 18 days; PEG liposome group: 20 days; PEG liposome-endostatin-VEGF-a siRNA21/23 Group: 38 days; PEG liposome-endostatin_VEGF_siRNA23/23 group: 32 days.
  • the survival curves of the mice in each group are shown in Fig. 14. From the median survival time and survival curve of each group, PEG liposome-VEGF-a siRNA21/23 could significantly prolong the survival of tumor-bearing mice.
  • the sample was prepared before administration, and PEG liposome-endostatin complex 2ml was taken. (The mass ratio of endostatin to PEG liposome was 2:1) and the fluorescently labeled BCL2-asiRNA-Cy5 20nmol was shaken and incubated for 20 min at room temperature (the PEG liposome-endostatin_Bcl2-a siRNA- The zeta potential of Cy5 was determined to be +7 mV).
  • BALB/C nude mice SPF grade, Shanghai Slack Laboratory Animals Co., Ltd.
  • male weighing 18_20g
  • transplanted tumor is S ⁇ C-7721 liver cancer.
  • Human hepatoma cells S ⁇ C-7721 purchased from Shanghai Chinese Academy of Sciences cell bank) at a concentration of 5 ⁇ 10 6 (0.2 mL/only) were injected subcutaneously into the neck and back of BLBA/c nude mice. Animal experiments were started when the tumor grew to 40-50 mm 3 . 2 ⁇ PEG ⁇ The prepared test drug, the tail vein injection (0.
  • CCR5_a siRNA a chemically modified and Cy5 fluorescently labeled CCR5_a siRNA was used (sequence: sense strand: 5' Choi - (mG) (mU) (mC) AAGUCCAAUCU (FA) (FU) (FG) dT-s-dT- Cy5 3, , Choi is cholesterol modification, m is methylation modification, F is fluoro modification, s is thio modification, antisense strand: 3' dTdTCACAGUUCAGGUUAGAUAC 5' ), by PEG liposome-endo endostatin
  • the delivery system was injected into the tail vein of BALB/c nude mice and observed for distribution in nude mice.
  • PEG liposome-endo endostatin complex 2ml (where Endo endostatin)
  • the mass ratio of PEG liposome is 2:1, the preparation method is the same as PEG liposome-endostatin complex), and the fluorescently labeled chemically modified CCR5- asiRNA - Cy5 20nmol mixed shaker, incubation at room temperature for 20 min, Zeta potential determination It is +10mV.
  • BALB/C nude mice SPF grade, Shanghai Slack Laboratory Animals Co., Ltd.
  • male weighing 18_20g, taking the prepared test drug, normal nude mice tail vein injection (0.
  • CCR5_a siRNA-Cy5 was distributed in all major organs of BLBA/c nude mice at different time points.
  • CCR5-a siRNA-Cy5 was abundantly accumulated by 24 h fluorescence quantification. In the kidney tissue, this product is mainly for renal excretion.
  • RNA interference molecule used in this example is a chemically modified B C 12-a siRNA with a charge value of -35, and the sequence is: sense strand: 5' chol- (mG) (mA) (mG) GCUGGGAUGCC (mU) (mU (mU) dT-s-dT 3', Choi is cholesterol modified, m is methylated, s is thio modified, antisense strand: 3' dTdT GCCUCCGACCCUACGGAAA 5' , endostatin-PEG lipid used The same as Example 2. Forty-two ICR mice were selected according to the weight of the animals according to the randomized grouping method of the group, 20 in each group, half male and half female.
  • This acute toxicity study selected B C 12-a siRNA at a dose of 100 mg/kg as the dose for the acute toxicity study (equivalent to 100 times the effective dose of 10 mg/kg in mice), endostatin-PEG lipid
  • the combined dose was 50 mg/kg (equivalent to 10 times the pharmacodynamically effective dose of 5 mg/kg of endostatin mice); and the negative control group was given 0.1% sterile DEPC water. 0. 4ml / 20g body weight.
  • the test results were as follows: Compared with the negative control group, no significant abnormalities were observed in the clinical symptoms of the animals in the test group, and no significant abnormalities were observed in the weight gain.
  • the minimum lethal dose (LD50) of the test sample B C 12-a siRNA administered to the ICR mice was greater than 100 mg/kg.
  • B C 12-a siRNA, endostatin used in this example, and Example 11, BALB/C nude mice (SPF grade), male, 18-20 g were purchased from Shanghai Slack Laboratory Animals Co., Ltd. Take well-grown A549 human lung cancer solid tumor (provided by Shanghai Pharmaceutical Industry Research Institute), cut into small pieces of about 3mm size under sterile conditions, inoculate a piece of mitral each mouse with right sac, and randomly divide The five groups were: saline group (blank control), chemotherapy drug cyclophosphamide CTX (30 mg/kg) group (positive control), PEG liposome-endostatin-B C 12-a siRNA high dose group, PEG liposome-endostatin-Bcl2-a siRNA in the low dose group of PEG liposome-endostatin-Bcl2_a siRNA.
  • the ratios of the groups were the same, except that the amount injected was 0.4, 0.2, 0. lml, so the zeta potential of the three groups was +3 mV.
  • the tumors were regrouped according to the tumor size, and the animals with too large and too small tumors were eliminated.
  • the average volume of each group of tumors was basically the same, and the administration started.
  • the high-dose group was administered with Bcl2- as iRNA 2 mg/kg, endostatin 15 mg/kg, and PEG liposome 5 mg/kg.
  • the medium dose group was administered 1/2 of the high dose group and the low dose group was 1/4 of the high dose group.
  • the saline group and PEG liposome-endostatin-B C 12-a siRNA were injected into the tail vein for 14 days in the high, medium and low dose groups.
  • the CTX (30 mg/kg) group was given intraperitoneal injection for 7 days.
  • the animals were sacrificed 29 days after inoculation, and the tumor pieces were dissected and photographed.
  • PEG liposome-endostatin-B C 12-a siRNA had a certain anti-tumor effect, and the anti-tumor effect increased with the dose, and the anti-tumor effect of the high-dose group.
  • the tumor segments after dissection in each group are shown in Figure 18.
  • the charge values of all liposome-endostatin recombinant protein-RNA interference molecules involved in Example 12 of the present invention were measured by Zeta potential method using a Zeta Sizer 3000 dynamic laser scatterometer.

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Abstract

本发明公开了一种组合物及其应用，其中所述组合物包含作为递送系统的内皮抑素和化学合成的RNA干扰分子。所述组合物包含携带+5至+15电荷的内皮抑素重组蛋白和携带-15至-55电荷的化学合成的RNA干扰分子，二者的正负电荷结合使所述组合物具有-20到+20的电荷值。

Description

说明书
包含内皮抑素和 RNA干扰分子的组合物及其应用 领域
本发明属于生物医药领域， 涉及内皮抑素作为递送系统和化学合成的 RNA干扰分子组成 的组合物及应用。 背景
内皮抑素（endostatin)最早由原发性肿瘤细胞产生， 能抑制肿瘤血管生长（0' Rei ly 等 1997， Cel l 88 : 277 ), 是胶原蛋白 XVI I I的一个片段， 由 183个氨基酸组成， 带高正电荷， 是重组蛋白中极少见的带高正电荷的一种蛋白质。 目前已经有 183个氨基酸、 184个氨基酸、 N端带 6个组氨酸的 193个氨基酸和 PEG修饰的内皮抑素均已经进行了临床研究或已经上市。 但是内皮抑素作为递送系统递送化学合成的 RNA干扰分子到相应的靶组织没有相应的报道。
目前已知内皮抑素有如下生物学功能： （1 )通过抑制血管内皮细胞的增殖和生长来抑制血 管生成。内皮抑素能特异性地抑制血管内皮细胞在成纤维细胞生长因子（bFGF)诱导下的增殖， 抑制内皮细胞的迁移， 诱导内皮细胞凋亡， 但对非内皮细胞， 如平滑肌细胞、 3T3成纤维细胞 等均无抑制作用。 （2 )对生长的血管产生抑制作用， 而对静止的血管组织不起作用。 （3 )抑制 肿瘤的生长和转移。 多项实验表明可以利用重组内皮抑素蛋白或内皮抑素基因进行肿瘤治疗， 已经有报道表明， 内皮抑素对 60种以上的不同实体肿瘤的生长和转移能产生抑制作用。 （4) 内皮抑素有一定的体外抑制肿瘤细胞的作用。
内皮抑素虽然本身对肿瘤具有一定的治疗效果， 但是临床单独使用 7. 5mg/m2内皮抑素没 有明显的疗效。 需要同化疗药物联合使用。
RNA干扰（RNA interference, RNAi)自 1998年发现以来备受瞩目。 1998年， Fire等（Nature, 1998, 391 : 806-811 ) 在线虫体内首次发现双链 RNA (double strand RNA， dsRNA)能使基因表达 沉默。 2002年报道合成的小干扰 RNA ( smal l interfering RNA, siRNA) 可以在培养的哺乳动 物细胞中实现序列特异性的基因敲除。 自首次成功地运用 siRNA的基因沉默作用对小鼠丙型肝 炎进行治疗以来， 研究者开始尝试将 siRNA应用于各种疾病的治疗， 包括艾滋病和癌症的基因 治疗等。
siRNA的作用原理为 siRNA在细胞内能介导 RNA干扰效应，识别与其序列互补的特异性 mRNA， 沉默相关基因的表达。 作用具有特异性和高效性， 是特异性抑制基因表达的强效方法。 siRNA 已成为许多疾病相关基因潜在的治疗手段， 已经是临床应用研究的热点。 siRNA药物在国际上 已经有 12项进入了 I-111期临床研究。
2008年 12月 Sun等 (Nature 2001, 411 : 494 - 498. ) 提出正义链和反义链长度不一致 的不对称双链小干扰 RNA ( asymmetric interfering RNA, asiRNA, aiRNA) 可以更加有效地使 哺乳细胞中相应的靶基因沉默。 认为 asiRNA有较多的优点将来可能取代 siRNA进行创新药物 的研究。 与 siRNA相比， asiRNA介导的基因沉默方式更加有效， 持久， 结构稳定， 并且可以 降低正义链的脱靶效应。 asiRNA药物目前在国际上没有进入临床研究的报道。
微小 RNA (microRNA， miRNA)是一种长度约为 22-28 nt的非编码小分子 RNA， mi RNA通过 与靶 mRNA 3 ' UTR (Untranslated Regions,即非翻译区， 是 mRNA分子两端的非编码片段）完全 或不完全的互补结合， 导致靶 mRNA降解或翻译抑制， 从而调控靶基因的表达， 影响细胞增殖、 分化和凋亡。它们通过 miRNA介导的特异性的基因沉默导致靶 mRNA降解及抑制蛋白质的合成， 从而调控转录后基因表达水平。 miRNA对细胞的增殖、 分化和凋亡有重要的调节作用。 通常采 用微小 RNA模拟物 （miRNA mimics ) 或抑制剂来进行基因表达调控研究。 miRNA mimics是模 拟生物体内源的 miRNAs，运用化学合成的方法合成，能增强内源性 miRNA的功能。 miRNA mimics 能进一步增强内源 miRNA的沉默作用， 降低细胞内靶向蛋白表达量。微小 RNA抑制剂可以直接 抑制 miRNAs的功能。 例如， 丹麦制药公司 Santafis Pharma宣布其开发的丙型肝炎新型治疗 手段 SPC3649 (锁核酸 LNA-antimiRTM-122)开始进入人体第一阶段临床试验， 这是世界上首个 进行人体临床试验的 miRNA药物， 直接抑制丙型肝炎微小 RNA-122的功能。
上述三类化学合成的 RNA干扰分子，无论是化学修饰或不修饰的两条链对称的小核酸干扰 ( siRNA), 化学修饰或不修饰的两条链配对不对称的小核酸干扰 （asiRNA, aiRNA )、 还是微小 RNA (miRNA)的模拟物或抑制剂。 其共同特征为携带高负电荷 _15 至 -55。 在进行静脉全身给药 时仍面临有剂量高、 或稳定性不长、 或毒副作用比较大、 或制造成本高等问题。 其中高负电荷 是主要问题所在。 麵!^
本发明的目的在于针对现有技术中以高负电荷为特征的 siRNA、 asiRNA, miRNA基因药物 在进行静脉全身给药时存在的剂量高、稳定性差、毒副作用比较大或制造成本高等问题， 提供 一种内皮抑素作为递送系统和化学合成的 RNA干扰分子组成的组合物及应用。
一种药用组合物， 包含携带 +5至 +15电荷的内皮抑素重组蛋白和携带 -15至 -55电荷的化 学合成的 RNA干扰分子， 二者通过正负电荷结合形成电荷值在 -20到 +20之间的组合物， 优选 形成电荷值在 -10到 +10之间的组合物。
所述的药用组合物还包含用于包裹所述的内皮抑素重组蛋白与 RNA干扰分子以调节正负 电荷量的阳离子脂质体、 中性脂质体或 PEG修饰长效脂质体。正负电荷值符合要求的组合物也 可以不用脂质体包裹用注射用生理盐水溶解直接使用。
所述的内皮抑素重组蛋白为通过大肠杆菌、酵母菌、 哺乳动物或人细胞、病毒载体表达纯 化后的重组蛋白，可以为目前市售的所有内皮抑素产品， 也可以为按照本领域的常规实验手段 与方法制备的内皮抑素；所述的内皮抑素重组蛋白优选不携带 6组氨酸标签的由 183-184个氨 基酸组成的人源结构的内皮抑素重组蛋白， N端或 /和 C端携带 6组氨酸标签的由 190-200个 氨基酸组成的人源结构改构的内皮抑素融合蛋白，或上述两类内皮抑素重组蛋白通过 PEG修饰 或白蛋白修饰后形成的长效内皮抑素； 或人工合成的截短型内皮抑素（包括了 183个氨基酸序 列中的主要氨基酸组成）。所有类型的内皮抑素重组蛋白共同特征为携带高正电荷从 +5至 +15， 并且与活跃病灶的血管内皮细胞具有特殊的亲合性。
所述的 RNA干扰分子选自化学修饰或不修饰的两条链对称的小核酸干扰分子（siRNA)，化 学修饰或不修饰的两条链配对不对称的小核酸干扰分子 （asiRNA, aiRNA), 或者化学修饰或不 修饰的 miRNA。
所述的两条链对称的小核酸干扰分子（siRNA)为正义链与反义链长度一致、 能抑制目的 基因表达， 长度为 19_25nt的双链配对的 RNA, 其两条链 3 ' 端含有 2_4个 UU碱基或脱氧核 糖核酸 dTdT呈单链悬挂； 所述的两条链配对不对称的小核酸干扰分子 （asiRNA, aiRNA) 是经 化学合成得到，从靶基因对称性小核酸干扰分子 siRNA出发，通过将 siRNA正义链或反义链 5 ' 或 3 ' 端裁减 l_5nt碱基， 形成两条链碱基长度不一致，每条链 3 ' 端有 2-4个 UU碱基或脱氧 核糖核酸 dTdT呈单链悬挂， 具有 14-21nt 的能诱发 RNA干扰的双链不对称小核酸干扰分子 asiRNAo
所述的 miRNA包括 miRNA模拟物 （miRNA mimics ) 和 miRNA抑制剂。 miRNA mimics是模拟 生物体内源的 miRNAs , 运用化学合成的方法合成， 能增强内源性 miRNA的功能。 miRNA抑制剂 是化学修饰的专门针对细胞中特异的靶 miRNA的抑制剂， 能够特异性地沉默单一基因， 也可以 同时沉默多个相关但不相同的基因。
所述的化学修饰指硫代修饰、 甲基化修饰、 磷酸化修饰、 胆固醇修饰或氟代修饰。
本发明所涉及的 siRNA、 或 asiRNA、 或 miRNA无论是否经过化学修饰， 均属于化学合成的 RNA干扰分子， 共同特征为带高负电荷从 -15至 -55， 均能调控目的基因的表达。
所述的药用组合物中各组分分开封装临用时混合， 或直接混合后封装在一起。
所述的药用组合物在制备治疗恶性肿瘤、 肝炎或艾滋病药物中的应用。
所述的药用组合物中各组分分开封装临用时混合， 或直接混合后封装在一起。
本发明采用的内皮抑素重组蛋白同抑制基因表达的 RNA干扰分子通过正负电荷结合，既可 以直接按正负电荷比例计算出合适的配比直接混合一起，也可以分别瓶装临用时按照正负电荷 比例混合后使用。其中正负电荷比例可通过电泳、 电荷测定计算出合适的配比， 合适的配比指 混合后使最终组合物的电荷值在 -20到 +20之间。 该组合物还可以用阳离子脂质体、 中性脂质 体、或 PEG化长效脂质体包裹以调节正负电荷的比例达到更合适的电荷值范围（即电荷值在 -10 到 +10 ) 内， 并且使该组合物在注射用生理盐水中不析出、 不沉淀、 不影响活性、 更稳定， 更 容易进入细胞， 能皮下注射、 局部注射、 静脉注射、 静脉滴注递送化学合成的 RNA干扰分子到 靶器官调控目的基因的表达。
有益效果：本发明提供的重组人内皮抑素具有高正电荷特性，在有或无脂质体包裹情况下， 与携带高负电荷的化学合成的 RNA干扰分子通过正负电荷结合形成组合物。在注射用生理盐水 中不析出、 不沉淀、 不影响活性、 更稳定， 更容易进入细胞。 该药用组合物大大降低了内皮抑 素和化学合成的 RNA干扰分子的使用剂量和电荷值，并且由于起递送作用的内皮抑素具有靶向 病灶血管内皮细胞的特殊性质， 该组合物在静脉注射后 1小时内或 24小时内能靶向目标组织 并稳定分布， 使治疗效果更好， 活性更稳定， 有效作用时间更长， 毒副作用更小。 本发明药用 组合物兼具了靶向性和基因调控两种作用， 在治疗恶性肿瘤、 肝炎或艾滋病中发挥积极作用， 可用于制备治疗恶性肿瘤、 肝炎或艾滋病药物。 附图说明
图 1、 Bcl2-siRNA、 内皮抑素、 PEG脂质体（复合脂）形成能静脉给药的 Bcl2_siRNA干扰组合 物的模式图。 其中 Bcl2-siRNA、 内皮抑素、 PEG脂质体各单独一瓶， 临用时混合在一起。 图 2、 内皮抑素的电荷与溶剂 pH的对应关系图， 横坐标为溶剂 pH值， 纵坐标为内皮抑素电荷 值。
图 3、 内皮抑素与 Bcl2-siRNA及 PEG脂质体结合形成的组合物的 Zeta电位图。 Zeta电位为 +2.6mV0
图 4、 内皮抑素与 VEGF-asiRNA及 PEG脂质体-壳聚糖结合形成的组合物的 Zeta电位图。 Zeta 电位为 +13.7 mV
图 5、 内皮抑素与 Bcl2-siRNA在不同脂质体包裹情况下形成组合物的电泳图。
各条带对应的样品如下： 1、 Bcl2-siRNA (20nM) 1.5μ 1； 2、 Blc2_siRNA: 内皮抑素： PEG脂质体质量比 1:10:5; 3、Bcl2-siRNA ：内皮抑素： PEG脂质体：壳聚糖质量比 1:10:5:2.5； 4、 Bcl2-siRNA: 内皮抑素： PEG脂质体： 壳聚糖质量比 1： 10： 5： 5; 5、 Bcl2_siRNA: 内皮抑素： PEG脂质体： 壳聚糖质量比 1:10:5:10; 6、 Bcl2-siRNA: 内皮抑素： PEG脂质体： 壳聚糖质量 比 1: 10:5: 15; 7、Bcl2- siRNA:内皮抑素： PEG脂质体：壳聚糖质量比 1:10:5:20； 8、 Bcl2- siRNA: 内皮抑素： PEG脂质体： 壳聚糖质量比 1:10:5:25。
图 6、 内皮抑素与不同基因的 siRNA、 asiRNA, miRNA在有或没有脂质体包裹情况下形成组合 物的电泳图。
a: 无脂质体包裹情况下 VEGF-asiRNA与内皮抑素形成的组合物的电泳图， 从左到右各泳 道分别为 VEGF-asiRNA与内皮抑素以 1:0、 1:5、 1:10、 1:15、 1:20、 1:30、 1:40、 1:50、 1:60 质量比形成的组合物的电泳图；
b: 有脂质体包裹情况下 VEGF-asiRNA与内皮抑素形成的组合物的电泳图， 从左到右各泳 道分别为 VEGF-asiRNA、 内皮抑素、 PEG 脂质体、 壳聚糖、 β_环糊精以 1:10:5:0:2.5、 1:10:5:5:2.5、 1:10:5:10:2.5 1:10:5:15:2.5 1:10:5:20:2.5、 1:10:5:30:2.5、 1:10:5:0: 5、 1:10:5:5:5、 1:10:5:10:5、 1:10:5:15:5、 1:10:5:20:5、 1 : 10 : 5 : 30 : 5质量比形成的组合 物的电泳图。
c: 无脂质体包裹情况下 anti-Has-122 miRNA抑制剂与 PEG内皮抑素形成的组合物的电泳 图， 从左到右各泳道分别为 anti-Has-122 miRNA抑制剂与 PEG内皮抑素以 3： 0、 3:5、 3:10、 3:15、 3:20、 3:25、 3:30、 3:40、 3:50质量比形成的组合物的电泳图。
d: 无脂质体包裹情况下 CCR5_asiRNA与恩度内皮抑素形成的组合物的电泳图， 从左到右 各泳道分别为 CCR5_asiRNA与内皮抑素以 1:0、 1:5、 1:10、 1:15、 1:20、 1:25、 1:30、 1:40、 1:50质量比形成的组合物的电泳图。
e: 无脂质体包裹情况下 Rabllb-siRNA与恩度内皮抑素形成的组合物的电泳图， 从左到 右各泳道分别为 Rabllb-siRNA与内皮抑素以 1:0、 1:5、 1:10、 1:15、 1:20、 1:25、 1:30、 1:40、 1:50质量比形成的组合物的电泳图。
f: 无脂质体包裹情况下 MDRl-siRNA与恩度内皮抑素形成的组合物的电泳图， 从左到右各 泳道分别为 MDRl-siRNA与内皮抑素以 1:0、 1:5、 1:10、 1:15、 1:20、 1:25、 1:30、 1:40、 1:50 质量比形成的组合物的电泳图。
图 7、 内皮抑素与化学合成的 RNA干扰分子形成组合物的粒径。
a, BC12-asiRNA与内皮抑素及 PEG脂质体的组合物的粒径图，
b, VEGF-asiRNA与内皮抑素及壳聚糖脂质体的组合物的粒径图。
图 8、 内皮抑素与 BC12-asiRNA形成组合物对顺铂 IC50的影响。
其中对照组不进任何转染， 只加入不同浓度顺铂。 BC12-asiRNA-内皮抑素组为利用内皮抑 素 -PEG脂质体递送系统转染 BC12-asiRNA后再加入不同浓度顺铂。
图 9、 内皮抑素与 BC12-asiRNA形成组合物的细胞毒性。
a.不同脂质体、内皮抑素与 BC12-asiRNA形成组合物的细胞毒性；其中 1_4分别代表 Lipo 2000脂质体与 BC12-asiRNA形成的组合物、 内皮抑素 -PEG脂质体与 BC12-asiRNA形成的组合 物、内皮抑素-壳聚糖脂质体与 BC12-asiRNA形成的组合物及不进行任何处理的空白细胞对照。
b. PEG脂质体-内皮抑素 -BC12-asiRNA复合物浓度与细胞毒性的关系； 其中 1_8分别为 1: 不进行任何处理 2： 0.125 μ 1 PEG脂质体 +0.25 μ 1内皮抑素 +0.125 μ 1 Bcl2- asiRNA复合物； 3： 0.25 μ 1 PEG脂质体 +0.5 μ 1内皮抑素 +0.25 μ 1 Bcl2- asiRNA复合物； 4： 0.5 μ 1 PEG脂 质体 +1 μ 1内皮抑素 +0.5 μ 1 Bcl2-asiRNA复合物； 5、 1 μ 1 PEG脂质体复合物 +2 μ 1内皮抑素 + 1 μ 1 Bcl2-asiRNA; 6、 2μ 1 PEG月旨质体 +4 μ 1内皮抑素 +2 μ 1 Bcl2_asiRNA复合物； 7、 4μ 1 PEG脂质体 +8 μ ΐ 内皮抑素 +4μ 1 Bcl2-asiRNA复合物； 8、 8μ 1 PEG脂质体 +16 μ ΐ 内皮抑 素 +8μ 1 Bcl2-asiRNA复合物。
图 10、 PEG脂质体-内皮抑素 -BC12-asiRNA复合物与顺铂联合使用对 HelaB2细胞体外抑制作 用试验结果图。
广 6分别代表： 1 : Lipo 2000转染 BC12-asiRNA组； 2 : PEG脂质体-内皮抑素复合物转染 Bcl2-asiRNA组； 3: Lipo 2000转染 Bcl2_asiRNA+低剂量顺铂 （1微克）组； 4: PEG脂质体 - 内皮抑素复合物转染 BC12-asiRNA+低剂量顺铂组； 5: 低剂量顺铂 （1微克）单独组； 6: 增倍 剂量顺铂 （2微克） 单独组。
图 11、 内皮抑素与经化学修饰的 BC12-asiRNA形成组合物对荷 H22肝癌小鼠肿瘤体积的抑制 曲线。
其中生理盐水组为静脉注射生理盐水的空白对照； CTX ( 15mg/kg) 组及 CTX ( 30mg/kg) 组为腹腔注射化疗药物环磷酰胺的阳性对照； PEG脂质体 -内皮抑素-不相关 siRNA组为尾静脉 注射 PEG 脂质体 -内皮抑素与不相关 siRNA 形成复合物的阴性对照； PEG 脂质体-内皮抑素 -Bcl2-asiRNA组尾静脉注射 PEG脂质体 -内皮抑素与 BC12-asiRNA形成的复合物； PEG脂质体- 内皮抑素 _Bcl2-asiRNA+CTX组为在上一组基础上使用 CTX ( 15mg/kg) 的联合组。
图 12、 内皮抑素与化学合成的 RNA干扰分子形成组合物对荷 H22肝癌小鼠肿瘤体积的抑制曲 线。
其中生理盐水组为静脉注射生理盐水的空白对照； CTX ( 30mg/kg)组为腹腔注射化疗药物 环磷酰胺的阳性对照； PEG 脂质体组为尾静脉注射 PEG 脂质体， PEG 脂质体-内皮抑素 -VEGF-asiRNA组尾静脉注射 PEG脂质体 -内皮抑素与 VEGF-asiRNA形成的复合物。
图 13、 注射内皮抑素-经化学修饰的 BC12-asiRNA形成组合物的 H22荷瘤小鼠的生存曲线。 各 组样品及注射情况同图 11。
图 14、注射内皮抑素与化学合成的 VEGF的 RNA干扰分子形成组合物的 H22荷瘤小鼠的生存曲 线。 各组样品及注射情况同图 12。
图 15、 内皮抑素与经化学修饰且进行荧光标记的 BC12-asiRNA 在荷人肝癌裸鼠各器官的 Bcl2-asiRNA残留量。
图 16、 内皮抑素与经化学修饰且进行荧光标记的 BC12-asiRNA在荷人肝癌裸鼠各器官的分布 及靶向肿瘤作用， 图中从左向右依次为心、 肝、 脾、 肺、 肾、 肿瘤。
图 17、 恩度内皮抑素与经化学修饰且进行荧光标记的 CCR5_asiRNA在正常裸鼠各器官的分布。 a: 样品注射 1小时和 24小时进行活体成像定量观察各器官 CCR5_as iRNA残留量柱状图； b: 样品注射 1小时处死动物取出内脏进行活体成像图， 从左向右依次为心、 肝、 脾、 肺、 肾； c: 样品注射 24小时处死动物取出内脏进行活体成像图， 从左向右依次为心、 肝、 脾、 肺、 肾。 图 18. 内皮抑素与化学合成的 RNA干扰分子形成组合物对人体肺癌 A549裸鼠移植瘤的疗效。 具体实
在本发明中所使用的术语， 除非有另外说明，一般具有本领域普通技术人员通常理解的含 义。
下面结合具体的制备实施例和应用实施例，并参照数据进一步详细地描述本发明。应理解， 这些实施例只是为了举例说明本发明， 而非以任何方式限制本发明的范围。
在以下的实施例中， 未详细描述的各种过程和方法是本领域中公知的常规方法。所用到的 引物， 均在首次出现时标明， 其后所用相同引物， 均以首次标明的内容相同。
实施例 1
将内皮抑素与 siRNA在有脂质体存在情况下通过正负电荷结合形成复合物的过程用模式 图 （图 1 ) 表示， 这种将 siRNA、 内皮抑素、 脂质体分三瓶分装的优点是每一种成分的质量控 制比较容易， 储存比较方便， 但是使用时比较麻烦。也可以将三瓶的成分按照以下实施例描述 的一定的电荷比例配比预先混合在一起， 组合于 1瓶， 这样使用方便， 但是混合组分的质量控 制比较难。 图 1所示的是肿瘤抗凋亡基因 (B- cell lymphoma/le kemia-2, Bcl2， 原称 B 细 胞淋巴瘤 /白血病 2基因， Bcl2 )的 siRNA, 可以更换为该基因或其它基因的 asiRNA或 miRNA， 图 1所例举的脂质体可以是阳离子脂质体、 中性脂质体、 或 PEG修饰长效脂质体、 也可以不用 脂质体， 用注射用生理盐水将内皮抑素与 RNA干扰分子组合物溶解混合后静脉滴注。
实施例 2
本实施例使用的内皮抑素重组蛋白由江苏吴中医药集团有限公司中凯生物制药厂生产出 品， 以下简称内皮抑素， 由 184个氨基酸组成， 氨基酸序列为： SEQ ID N0. 1， 等电点为 9. 3。
PH5. 0时， 内皮抑素电荷为 +12. 89; pH5. 5时， 内皮抑素电荷为 +10. 84; pH6时， 内皮抑素电 荷为 +8. 65 (图 2)。用 pH5. 5、 电荷值为 +10. 84的内皮抑素，与电荷值为 -40的 Bcl2_siRNA (序 列为：正义链 5' CGGAGGCUGGGAUGCCUUUdTdT 3' ，反义链 3' dTdTGCCUCCGACCCUACGGAAA 5 ' )， 通过正负电荷计算结合比例， 将 Bcl2-siRNA、 内皮抑素、 PEG脂质体 （聚乙二醇 2000-二硬酯 酰磷脂酰乙醇胺 （PEG-PE) 与二油基磷脂酰丝氨酸 （D0PS) 的混合物， PEG-PE与 D0PS质量比 为 1 : 1， 终浓度为 lmg/ml， 下同） 以质量比 1： 10： 5的比例混合， 并稀释 15倍， 用 Zeta Sizer 3000动态激光散射仪测定其 Zeta电位。 PEG脂质体-内皮抑素 -BC12_siRNA的 Zeta电位见图 3。 由图 3可见， PEG脂质体 -内皮抑素- Bcl2-siRNA的平均 Zeta电位为 2. 6mV。
同样方法， 用溶剂 pH5. 5、 电荷值为 +10. 84的内皮抑素， 与电荷值为 -38的血管内皮生 长因子 ( vascular endothelial growth factor , VEGF ) 不对称 asiRNA (序歹 lj为： 正义链 5' GUGAAUGCAGACCAAAGAA dTdT 3' , 反义链 3 ' dTdTUACACUUACGUCUGGUUUCUU 5 ' ) , 通过正 负电荷计算结合比例。 将 VEGF -asiRNA、 内皮抑素、 PEG脂质体、 壳聚糖、 β _环糊精分别以 1 : 10 : 5： 15： 5的质量比混合， 用 DEPC水稀释 20倍至 5ml， 并用 0. 22 μ m的滤膜过滤， 封口。 其中 PEG脂质体、 壳聚糖、 β -环糊精以 5: 15： 5形成的混合物称为壳聚糖脂质体， 下同。 检 测该内皮抑素 -VEGF-asiRNA-壳聚糖脂质体组合物的 zeta 电位， 平均 Zeta 电位为 13. 7 mV, 结果见图 4。 实施例 3
除了按照实施例 2的方法在有脂质体包裹情况下进行检测 zeta电位， 以确定内皮抑素重 组蛋白与 RNA干扰分子通过正负电荷结合后的组合物的电荷值外，还可以通过电泳的方法判断 有脂质体包裹或没有脂质体包裹情况下内皮抑素重组蛋白与 ■干扰分子组成的组合物的电 荷情况。配制 TBE溶液， Tris 10.8g，硼酸 5.5g， EDTA 0.58g，加入灭菌的 DEPC水定容至 1000ml。 称取 0.6g 琼脂糖， 溶于 60ml TBE溶液中， 加热两分钟， 冷却后加入少量 EB， 倒入制胶槽， 插入梳子， 制得琼脂糖凝胶。将 Bcl2-siRNA 1.5μ 1 (序列、 电荷值均同实施例 2,浓度为 20ηΜ) 与内皮抑素 (序列、 电荷值均同实施例 2)、 PEG脂质体轻轻混勾， 使其质量比为 1:10:5， 再与 不同比例的壳聚糖混合，使 Bcl2-siRNA、与壳聚糖之间的质量比分别为 1:0、 1:2.5、 1:5、 1:10、 1： 15、 1： 20、 1:25。 将上述样品室温静置 15min后加入 RNA loading buffer, 上样进行琼 脂糖凝胶电泳， 100V， 电泳 30min。 电泳结束后将胶置于紫外灯下显像， 拍照， 结果见图 5。 由图 5可见， 带负电荷的各种不同浓度的 Bcl2-siRNA与内皮抑素混合后， 不经脂质体包裹及 经过脂质体包裹后， 电泳条带有滞后现象， 并呈现从负到正电荷一定的梯度，说明 Bcl2-siRNA 与内皮抑素及脂质体形成带正电荷的一系列组合物。
将合成的 VEGF-aiRNA粉末 （序列、 电荷值均同实施例 2) 用 DEPC水溶解， 配成 20nM的 储备溶液。 琼脂糖凝胶配置方法同上。 将 VEGF-asiRNA和内皮抑素（序列、 电荷值均同实施例 2) 分别以质量比为 1:0、 1:5、 1:10、 1:20、 1:30、 1:40、 1:50、 1 :60在无 RNA酶的 EP管内 混合， 静置 15min， 加入琼脂糖凝胶孔中。 以 100V恒压， 电泳 30min。 拍照， 观察条带， 结果 见图 6a。
VEGF-asiRNA (序列、 电荷值均同实施例 2)、 内皮抑素（序列、 电荷值均同实施例 2)、 PEG 脂质体以 1:10:5 质量比混合， 分别与壳聚糖以：1: 0、 1： 5、 1： 10、 1： 15、 1： 20、 1： 30 的质量比混合， 再分别与 β_环糊精以 1: 2.5、 1： 5的质量比， 在无 RNA酶的 ΕΡ管内混合， 静置 15min。 按照质量比从小到大的比例， 加入琼脂糖凝胶孔中。 以 70V恒压， 电泳 30min。 拍照， 观察条带和分析组合物不同比例所带的电荷量， 结果见图 6b。
同样方法的电泳可以用于观察没有脂质体包裹情况下内皮抑素重组蛋白与 RNA干扰分子 组成的组合物的电荷情况。 取肝炎相关的 Has-122 miRNA抑制剂 anti-Has-122 (序列为 5 'ACAAACACC AUUGUC ACACUCCA-3 (SEQ ID N0.2) )4.5μ1, PEG修饰的长效内皮抑素 (lmg/ml, 江苏吴中医药集团有限公司中凯生物制药厂生产出品， 采用单甲氧基聚乙二醇丙 醛与内皮抑素在还原剂氰基硼酸氢钠条件下反应形成的 PEG内皮抑素， 纯度 98%以上， 以下 简称 PEG内皮抑素）， Has-122 miRNA抑制剂 anti-Has-122与 PEG内皮抑素两者分别按照质量 比 3: 0、 3： 5、 3： 10、 3： 15、 3： 20、 3： 25、 3： 30、 3： 40、 3： 50混合， 室温放置 20min。 将上述样品加入 RNA loading buffer, 上样进行琼脂糖凝胶电泳， 100V， 分别于 10min、 15min、 20min、 25min将胶置于紫外灯下显像， 拍照。 电泳图见图 6c。不同配比的 anti-Has-122和 PEG 内皮抑素形成的组合物在电泳中呈现不同电荷值， 选择其中带微弱正电荷的组合物进行 Zeta 电位测定， 测得的电位值通常在 10 mV内， 可见通过电泳能方便的找到合适的组合物电荷值 所对应的 anti-Has-122和 PEG内皮抑素的配比。
同样， 分别取艾滋病受体相关基因 CCR5 的 asiRNA (序列为： 正义链 5'GUCAAGUCCAAUCUAUGdTdT 3% 反义链 3 ' dTdTCAC AGUUC AGGUUAGAUAC 5' ) , 肿 瘤离子通道相关的 Rabllb的 siRNA (序列为：正义链 5'UGUCAGACAGACGCGAAAAdTdT3'， 反义链 3'dTdTACAGUCUGUCUGCGCUUUU 5')， 肿瘤化疗药物耐药相关的耐药基因 MDR1 的 siRNA, (序列为： 正义链 5'AAAAUGUUGUCUGGACAAGCAdTdT3'， 反义链 3 'dTdTUUUUACAACAGACCUGUUCGU 5')各 1.5μ1与 N端带 6个组氨酸的内皮抑素（ lmg/ml， 商品名为恩度， 由山东先声麦得津生物制药有限公司生产出品， 南京先声药业有限公司经销， 由 193个氨基酸组成， 以下简称恩度内皮抑素） 分别按照质量比 1 : 0、 1： 5、 1： 10、 1： 15、 1 : 20、 1 : 25、 1 : 30、 1： 40、 1： 50混合，室温放置 20min。将上述样品加入 RNA loading buffer, 上样进行琼脂糖凝胶电泳， 100V， 分别于 10min、 15min、 20min、 25min将胶置于紫外灯下显 像， 拍照。 恩度内皮抑素与 CCR5_asiRNA、 Rabl lb-siRNA, MDRl-siRNA正负电荷结合的电泳图 分别见图 6d、 6e、 6f。 不同配比的 siRNA、 asiRNA, miRNA和恩度内皮抑素形成的组合物在电 泳中呈现不同电荷值， 选择其中带微弱正电荷的组合物进行 Zeta电位测定， 测得的电位值在 10 mV 内， 因此通过电泳能方便的找到合适的组合物电荷值所对应的 siRNA、 asiRNA, miRNA 和恩度内皮抑素的配比。 由图 6a_f可见， 带不同负电荷的 RNA干扰分子与带不同正电荷的内 皮抑素重组蛋白混合后，不管是否经脂质体包裹， 电泳条带均有滞后现象，并呈现一定的梯度， 说明不同■干扰分子与不同浓度的内皮抑素形成带不同量正电荷的一系列组合物,均改变了 siRNA asiRNA、 miRNA的高负电荷特征。
实施例 4
将电荷值为 -38的 Bcl2-asiRNA (序列为： 正义链： 5' GAGGCUGGGAUGCCUUUdTdT 3' ， 反 义链： 3' dTdTGCCUCCGACCCUACGGAAA 5' ， 浓度为 20nM)与内皮抑素 （lmg/ml， 序列、 电荷值 均同实施例 2)、 PEG脂质体 ( lmg/ml ) 以质量比 1： 10： 5轻轻混勾， 室温放置 15min。 用 0. 05M 醋酸 -醋酸钠缓冲液将以上混合物稀释 15倍。用 MastersizerfOOO粒度仪测定其粒径。粒径见 图 7a， 由图 7a可见， PEG脂质体-内皮抑素 -BC12-asiRNA的粒径分布比较均勾， 平均粒径为 142. 4nm。 Zeta电位测定为 +3mV。
将 VEGF-asiRNA (序列、 电荷值均同实施例 2)、 内皮抑素 （lmg/ml， 序列、 电荷值均同实 施例 2)、 PEG脂质、 壳聚糖 (CS)、 β _环糊精 ( β -CD) 以 1 : 10 : 5 : 15 : 5的质量比混合， 即 30 μ 1 VEGF-aiRNA与 80 μ 1内皮抑素轻轻吹打混合， 室温下静置 5min， 加入 PEG脂质 40 μ 1， 混合后室温下静置 10min， 再加入 CS 60 l、 β -CD 40 μ 1 , 均勾混合后， 用灭菌的 DEPC水稀 释 20倍至 5ml。 将上述壳聚糖脂质体-内皮抑素 -VEGF-asiRNA组合物用 0. 22 μ m的滤膜过滤， 封口。 Mastersizer2000粒度仪检测 PEG/壳聚糖脂质体 _VEGF_aiRNA组合物的粒径为 219. 6皿。 见图 7b。 Zeta电位测定为 +14mV。
实施例 5
本发明使用 PEG脂质体 -内皮抑素系统转染 BC12-asiRNA片段的电荷值、序列同实施例 4， BC12-asiRNA粉末用 DEPC水溶解， 配成 20nM的储备溶液。 转染操作前一天， 用含小牛血清、 青霉素、 链霉素的 DMEM细胞培养基将高表达 Bcl2的人宫颈癌细胞 HeLaB2细胞 （购于中国医 学科学院中国协和大学肿瘤研究所） 接种至 96孔板中， 于 37 V， 含 5% (：02的培养箱进行培 养， 当细胞的密度达到 50%时， 弃去原培养基， 用不含小牛血清和青霉素、 链霉素的 DMEM培 养基洗一次， 并加入 100 μ 1不含小牛血清和青霉素、链霉素的 DMEM培养基。用 25 μ 1不含血 清培养基的 Opti-MEM 稀释 50pmol Bcl2_asiRNA， 轻轻混勾并室温放置 5 分钟。 用 25 μ 1 Opti-MEM稀释 0. 5 μ 1内皮抑素 （电荷值、 序列同实施例 2) 及 0. 25 μ 1 PEG脂质体， 轻轻混 勾并室温放置 5分钟， 将上述两溶液轻轻混勾得到 PEG脂质体 -内皮抑素 -BC12-asiRNA混合 液， Zeta电位测定为 +3mV， 室温孵育 20分钟后， 取 50 μ 1 Bcl2-asiRNA -内皮抑素 -PEG脂质 体混合液加入含有细胞及培养液的培养板相应孔中， 轻轻混勾。将培养板置于 37°C， 含 5% C02 的培养箱进行培养。 培养 4-6小时后， 将孔中含有 PEG脂质体 -内皮抑素 -BC12-asiRNA混合 液的培养基移去， 并更换新鲜的含小牛血清、 青霉素和链霉素的 DMEM培养基 100 μ 1， 作为实 验组。 对照组不进行转染。 转染 24h后， 实验组、 对照组各孔均加入不同浓度梯度的顺铂， 使 顺铂的终浓度分别为 0. 5、 1、 2、 4、 8 g/ml。 每个浓度至少做 3个平行孔。 将培养板置于培 养箱中继续培养 48小时后， 吸去培养基， 按照 CCK-8试剂盒 (购于上海同仁化学研究所)说明 书， 每孔加 100 μ 1单独 DMEM培养液和 10 μ 1 CCK-8试剂， 置于 37°C培养箱中继续培养 1小 时， 并用酶标仪进行 0D450的检测。 按照以下公式进行细胞活力的计算： 细胞话力 ΐ¼〗= A" x 100% 其中， As为实验组 0D值 （含细胞、 PEG脂质体 -内皮抑素 -BC12-asiRNA)， Ab为空白组 的 0D值 （不含细胞和 PEG脂质体 -内皮抑素 -BC12-asiRNA)， Ac为不经过转染的对照组的 0D 值 （含细胞， 不含 PEG脂质体-内皮抑素 -Bcl2-asiRNA)， 最终求出细胞活力为 50%时的顺铂 浓度， 即为 IC50。 对照组顺铂的 IC50 为 ( 2. 19 ±0. 08 ) g/ml , 实验组顺铂的 IC50 为
( L 09 ±0. 02 ) g/ml。 CCK8 检测不同浓度顺铂对转染及不转染 PEG 脂质体-内皮抑素 -Bcl2-asiRNA的 HelaB2细胞增殖影响的试验结果见图 8，由图 8可见 HelaB2转染 PEG脂质体
-内皮抑素 -Bcl2-asiRNA 后， 顺铂的 IC50 明显降低， 即细胞转染 PEG 脂质体-内皮抑素 -BC12-asiRNA后仅需较低浓度的顺铂即可抑制癌细胞增殖。
实施例 6
将高表达 Bcl2的人宫颈癌细胞 HeLaB2细胞（购于中国医学科学院中国协和大学肿瘤研究 所） 接种至 96孔板中， 于 37°C， 含 5% C02的培养箱进行培养， 当细胞的密度达到 50%， 进行 转染。 实验分为 4 组， 每组至少有三个平行对照孔。 组 1 每孔加入 0. 25 μ ILipof ectAMINE™2000 (Lipo 2000)脂质体 (Life Techonolobies, 产品货号： 11668-019, 商 标为 Inivitrogen, 下同） +0. 25 μ 1 Bcl2_asiRNA (浓度、 电荷值、 序列同实施例 4， 下同）； 组 2每孔加入 0. 25 μ 1 Bcl2-asiRNA+0. 5 μ 1 内皮抑素 （浓度、 电荷值、 序列均同实施例 4， 下同） +0. 25 μ 1 PEG脂质体（浓度同实施例 4， 下同）， 该 PEG脂质体 -内皮抑素 -BC12-asiRNA 的 Zeta电位测定为 +3mV; 组 3每孔加入 0. 25 μ 1 Bcl2_asiRNA +0. 5 μ 1内皮抑素 +0. 25 μ 1壳 聚糖脂质体； 组 4为不进行任何处理的空白对照。 培养板放入 37 V， 5% (：02的培养箱中继续 培养 24h后，吸去培养基，按照 CCK-8试剂盒说明书，每孔加 100 μ 1单独 DMEM培养液和 10 μ 1 CCK-8试剂， 置于 37°C培养箱中继续培养 1小时， 并用酶标仪进行 0D450的检测。 按实施例 5 中的方法进行细胞活力的计算， 可得出 PEG脂质体 -内皮抑素 -BC12-asiRNA复合物、 壳聚糖 脂质体-内皮抑素 -BC12-asiRNA复合物对细胞的毒性， 实验结果见图 9a， 由图可见在正常的转 染浓度下 PEG脂质体-内皮抑素 -BC12-asiRNA复合物、 壳聚糖脂质体-内皮抑素 -BC12-asiRNA 复合物对细胞均几乎没有任何毒性， 且对细胞的影响小于商业化转染试剂 Lipo 2000。
在上述浓度 PEG脂质体-内皮抑素 -BC12-asiRNA复合物对细胞几乎无毒性的基础上， 进一 步增加 PEG脂质体-内皮抑素 -BC12-asiRNA复合物的浓度观察细胞毒性。细胞接种至 96孔板， 分别加入以下浓度的 PEG脂质体-内皮抑素 -BC12-asiRNA复合物。 1 : 不进行任何处理； 2: 加 入 0. 125 μ 1 PEG脂质体 +0. 25 μ 1内皮抑素 +0. 125 μ 1 Bcl2- asiRNA复合物； 3： 加入 0. 25 μ 1 PEG脂质体 +0. 5 μ 1内皮抑素 +0. 25 μ 1 Bcl2- asiRNA复合物； 4： 加入 0. 5 μ 1 PEG脂质体 +1 μ 1 内皮抑素 +0. 5 μ 1 Bcl2-asiRNA 复合物； 5、 加入 Ι μ ΐ PEG 脂质体 +2 μ 1 内皮抑素 +1 μ 1 Bcl2-asiRNA复合物； 6: 加入 2 μ 1 PEG脂质体复合物 +4 μ 1内皮抑素 +2 μ 1 Bcl2_asiRNA; 7： 加入 4 μ 1 PEG脂质体 +8 μ 1内皮抑素 +4 μ 1 Bcl2-asiRNA复合物； 8: 加入 8 μ 1 PEG脂质体 +16 μ 1内皮抑素 +8 μ 1 BC12-asiRNA复合物。 培养板放入 37 V , 5% C02的培养箱中继续培养 24h 后， 吸去培养基， 按照 CCK-8试剂盒说明书， 每孔加 100 μ 1单独 DMEM培养液和 10 μ 1 CCK-8 试剂， 置于 37°C培养箱中继续培养 1小时， 并用酶标仪进行 0D450的检测， 按实施例 5中的 方法进行细胞活力的计算。 PEG脂质体-内皮抑素 -BC12-asiRNA复合物浓度与细胞毒性的关系 如图％。 由图可见， 随着 PEG脂质体-内皮抑素 -BC12-asiRNA复合物浓度的增加， 细胞存活率 下降， 即对细胞的毒性增加， 但整体来看对细胞的毒性不大， 当 PEG 脂质体-内皮抑素 -Bcl2-asiRNA复合物浓度达到正常转染浓度的 32倍时， 细胞活力约为 77%， 对细胞的毒性不 大。
实施例 7
实验分为以下 7组： 1 :用 Lipof ectAMINE™2000 (Lipo 2000)脂质体转染 Bcl2_asiRNA组（电 荷值、 序列同实施例 4) ; 2 : PEG脂质体-内皮抑素复合物转染 BC12-asiRNA组； 3: Lipo 2000 转染 BC12-asiRNA+低剂量顺铂（1微克）组； 4: PEG脂质体 -内皮抑复合物转染 BC12-asiRNA+ 低剂量顺铂 （1微克） 组； 5 : 低剂量顺铂 （1微克） 单独组； 6: 增倍剂量顺铂 （2微克） 组； 7、 空白对照组。 转染前一天将处于对数生长期的 HelaB2细胞接种至 96孔板， 待到细胞密度 生长至 50%覆盖率时， 弃去原培养基， 用不含小牛血清和青霉素、 链霉素的 DMEM培养基洗一 次， 并加入 100 μ 1不含小牛血清和青霉素、链霉素的 DMEM培养基。 组 1和组 3按如下方法进 行转染： 用 25 μ 1不含血清培养基的 Opti-MEM稀释 0. 25 μ lBcl2-asiRNA (20nM)， 轻轻混勾 并室温放置 5分钟。 用 25 μ 1 Opti-MEM稀释 Lipo 2000脂质体， 轻轻混勾并室温放置 5分钟， 将上述两溶液轻轻混勾， 室温孵育 20分钟后， 把混合液加入到 96孔板中， 轻轻混勾。 将培养 板置于 37°C， 含 5% C02的培养箱进行培养。 培养 4-6小时后， 将孔中含有 PEG脂质体-内皮抑 素 - BC12-asiRNA 混合液的培养基移去， 并更换新鲜的含小牛血清、 青霉素和链霉素的 DMEM 培养基 100 μ 1。 组 2和组 4将上述转染方法中的 0. 25 μ 1 Lipo 2000脂质体换为 0. 25 μ 1 PEG 脂质体 +0. 5 μ 1 内皮抑素复合物。 其他转染方法同上。 组 2 的 PEG 脂质体 -内皮抑素 -Bcl2-asiRNA的 Zeta电位测定为 +5mV。 空白对照组不进行转染。 转染 24h后， 组 3、 4、 5分 别加入 1微克顺铂， 组 6加入 2微克顺铂。 转染 72h后， 按实施例 5中所述方法进行 CCK-8 检测。 设定空白对照组 0D值为 100%， 其余各组的 0D值与空白对照组 0D值的相对值即可反映 各组细胞数的相对水平。 PEG脂质体-内皮抑素 -BC12-asiRNA复合物与顺铂联合使用对 HelaB2 细胞体外抑制作用试验结果见图 10。 由图 10可见与商业化转染脂质体 Lipo 2000相比， PEG 脂质体-内皮抑素复合物同样可以转染 BC12-asiRNA进入细胞， 从而对细胞生长产生抑制。 且 PEG脂质体-内皮抑素复合物转染 BC12-asiRNA与低倍顺铂联合使用时能更好地抑制细胞增殖， 其对细胞生长的抑制率高于单独增倍剂量的顺铂组 （图 10)。
注： 本实施例所使用的内皮抑素同实施例 2。
实施例 8
本实施例所用 ■ 干扰分子为电荷值为 -35 的胆固醇修饰的 BC12-asiRNA (正义链： 5 ' chol-GAGGCUGGGAUGCCUUUdTdT3， ， 反义链： 3 ' dTdTGCCUCCGACCCUACGGAAA5 ' ， chol表示 胆固醇修饰 ）。 不相关 siRNA序列为 （正义链： 5 ' UUCUCCGAACGUGUCACGUdTdT 3 ' ， 反义链： 3 ' dTdTAAGAGGCUUGCACAGUGCA 5 ' )。 雄性昆明种小鼠， 约 8周龄， 体重 20 ± lg， 购自南京江 宁青龙山实验动物中心， 用颗粒词料喂养在 21 ± 2°C的环境中， 使其自由取食和饮水， 实行 12 小时的白天和黑夜循环。小鼠肝癌 H22肝癌腹水细胞（中科院上海药物研究所实验动物中心 ) 37 °C复苏后每只小鼠腹腔注射 0. 3ml， 7天后接种第二代。 取第二代腹水， 用生理盐水调整细胞 浓度为 5 X 106个 /ml， 每只小鼠前肢右侧腋下皮下注射 0. 2 mL， 约 1 X 106个瘤细胞。 小鼠自由 进食， 正常喂养。 小鼠接种后随机分为六组： 生理盐水组、 化疗药物环磷酰胺 CTX ( 15mg/kg) 组、 化疗药物环磷酰胺 CTX ( 30mg/kg) 组、 PEG脂质体 -内皮抑素-不相关 siRNA组、 PEG脂质 体-内皮抑素 _Bcl2-asiRNA 组 （Zeta 电位测定为 +3mV)、 PEG脂质体-内皮抑素 _Bcl2-asiRNA 联合化疗药物环磷酰胺 CTX (15mg/kg)组。 接种后次日开始给药， BC12-asiRNA的给药剂量为 lmg/kg, BC12-asiRNA与内皮抑素、 PEG脂质体的质量比为 1: 10:5。 生理盐水、 PEG脂质体- 内皮抑素-不相关 siRNA组、 PEG脂质体-内皮抑素 -BC12-asiRNA组和 PEG脂质体-内皮抑素 -Bcl2-siRNA联合化疗药物环磷酰胺 CTX (15mg/kg)组连续尾静脉给药 7天（0.4ml/只）， CTX (15mg/kg) 组及 CTX (30mg/kg) 组在接种后的第 2、 4、 6 、 8、 天经腹腔注射给药。 在给药 的第 5、第 10、第 15和第 20天用游标卡尺测量肿瘤的长和宽， 并通过以下的公式计算肿瘤的 体积： a*b2*0.5 (a为长， b为宽）。 各组肿瘤体积曲线如图 11。 由图 11可见与生理盐水组及 不相关 siRNA组相比， PEG脂质体-内皮抑素 -BC12-asiRNA能很好地抑制肿瘤生长， 联合化疗 药物环磷酰胺 CTX后， 抑瘤效果进一步增加。
同上肿瘤模型， 本实施例所用正义链 5' 胆固醇修饰的 VEGF对称和不对称的 RNA干扰分 子，电荷值为 -38的 VEGF-asiRNA (19+2/21+2，正义链 5' cho 1 -GUGAAUGCAGACCAAAGAAdTdT 3' ， 反义链 3' dTdTUACACUUACGUCUGGUUUCUU 5' )和电荷值为 -40的 VEGF-siRNA (21+2/21+2， 序 列为：正义链 5' AUGUGAAUGCAGACCAAAGAAdTdT3 ' ，反义链为 3' dTdTUACACUUACGUCUGGUUUCUU 5' )。 小鼠接种后随机分为 5组： 生理盐水组 （NS)、 化疗药物环磷酰胺 CTX组、 PEG脂质体 组、 PEG脂质体-内皮抑素 _VEGF-asiRNA21/23 (Zeta电位测定为 +13mV)。、 PEG脂质体-内皮抑 素 -VEGF-siRNA23/23组， （ Zeta电位测定为 +15mV。）。 接种后次日开始给药， siRNA、 as i RNA 的给药剂量为 lmg/kg， NS (0.4 ml/只）、 PEG脂质体组 （0.3 ml/只）， 连续尾静脉给药 14天， CTX (30mg/kg)在接种后的第 2、 4、 6 、 8、 10、 12天经腹腔注射给药。 在给药的第 5、 第 10、 第 15天用游标卡尺测量肿瘤的长和宽，并通过以下的公式计算肿瘤的体积： a*b¾0.5 (a为长， b为宽）。 脂质体 -VEGF-asiRNA组小鼠的肿瘤体积明显小于其他组， 具有良好的抑瘤效果。 各 组肿瘤体积曲线如图 12。
注： 本实施例所使用的内皮抑素同实施例 2。
实施例 9
雄性昆明种小鼠接种、分组、具体给药方式及剂量均同实施例 8中的 Bcl2部分，所用 PEG 脂质体、 BC12-asiRNA、 内皮抑素以及 PEG脂质体-内皮抑素 -BC12-asiRNA的电荷值也均与实 施例 8中相同。 整个治疗过程中小鼠自由进食饮水， 并观察记录小鼠的生活状态和生存情况， 从接种肿瘤细胞次日开始计算天数， 到第 60天为止， 60天以上生存时间也按照 60天计算。 各组的中位生存时间如下： 生理盐水组： 29天； 化疗药物环磷酰胺 CTX (15mg/kg)组： 39天； 化疗药物环磷酰胺 CTX (30mg/kg) 组： 17天； 脂质体 -内皮抑素-不相关 siRNA组： 34天； 脂 质体-内皮抑素 -Bcl2-asiRNA组： 41天； 脂质体-内皮抑素 -BC12-asiRNA联合化疗药物环磷酰 胺 CTX (15mg/kg) 组： 58天。 各组小鼠的生存曲线如图 13。 由各组中位生存时间及生存曲线 可见脂质体-内皮抑素 -BC12-asiRNA能明显延长荷瘤小鼠的生存期， 且与低剂量化疗药物联合 时比化疗药单独使用时的生存期有所延长。 雄性昆明种小鼠接种、 分组、 具体给药方式及剂量均实施例 8中的 VEGF部分， 所用 PEG 脂质体、各 RNA干扰分子、 内皮抑素以及 PEG脂质体-内皮抑素 -BC12- VEGF-asiRNA21/23、 PEG 脂质体-内皮抑素 _Bcl2- VEGF-siRNA23/23的电荷值也均与实施例 8中相同。 整个治疗过程中 小鼠自由进食饮水， 并观察记录小鼠的生活状态和生存情况， 从接种肿瘤细胞次日开始计算天 数， 到第 60天为止， 60天以上生存时间也按照 60天计算。 各组的中位生存时间如下： 生理 盐水组： 35 天； CTX ( 30mg/kg) 组： 18 天； PEG 脂质体组： 20 天； PEG 脂质体-内皮抑素 -VEGF-asiRNA21/23组： 38天； PEG脂质体-内皮抑素 _VEGF_siRNA23/23组： 32天。 各组小鼠 的生存曲线如图 14。由各组中位生存时间及生存曲线可见 PEG脂质体 -VEGF-asiRNA21/23能明 显延长荷瘤小鼠的生存期。
实施例 10
经化学修饰及 Cy5荧光标记的 BC12-asiRNA (电荷值为 -35， 序列为： 正义链： 5' Choi - (mG) (mA) (mG) GCUGGGAUGCC (FU) (FU) (FU) dT-s-dT-Cy5 3' ， Choi 为胆固醇修饰， m为甲基化 修饰， F为氟代修饰， s为硫代修饰， 反义链： 3' dTdTGCCUCCGACCCUACGGAAA 5' )， 通过内 皮抑素 -PEG脂质体递送系统将其尾静脉注射入 BALB/c荷瘤裸鼠体内， 并观察其在裸鼠体内的 分布情况， 样品给药前现配， 取 PEG脂质体-内皮抑素复合物 2ml (其中内皮抑素与 PEG脂质 体的质量比为 2 : 1 )与荧光标记的 BCL2-asiRNA-Cy5 20nmol混合摇勾，室温孵化 20min (该 PEG 脂质体-内皮抑素 _Bcl2-asiRNA-Cy5的 Zeta电位测定为 +7mV)。 BALB/C裸鼠 （SPF级， 上海斯 莱克实验动物有限责任公司）， 雄性， 体重 18_20g， 移植性肿瘤为 S匪 C-7721肝癌。 将浓度为 5 X 106个（0. 2mL/只）的人肝癌细胞 S匪 C-7721细胞（购于上海中国科学院细胞库）注入 BLBA/c 裸鼠的颈背部皮下。 待肿瘤长到 40-50mm3时开始动物实验。 取配制好的受试药物， 尾静脉注 射（0. 3ml/只）荷瘤裸鼠， 在不同时间点进行活体成像观察， 在 24h处死动物取内脏进行荧光 定量； 将剩余的 0. 2mlPEG脂质体-内皮抑素 -BC12-asiRNA-Cy5混合物稀释成六个浓度用活体 成像进行定量，做浓度与荧光强度的标准曲线，最后根据活体成像仪指示的数据根据标准曲线 计算主要脏器心、肝、脾、肺、肾的 BC12-asiRNA残留量，结果见图 15^16。所用仪器为 in-vivo imaging system (Maestro, Cambridge Research & Instrument ) 禾口 光分光光度计 (LS55, 美国 Perkin Elmer), 实验结果见图 16， 结果显示在不同时间点 Bcl-2-asiRNA_Cy5在 BLBA/c 荷瘤裸鼠的各主要脏器中均有分布， 通过 24h的荧光定量看到 BCl-2-asiRNA-Cy5大量聚集在 肿瘤组织中， 有明显的肿瘤靶向作用。 注： 本实验中使用的内皮抑素同实施例 2。
本实施例又采用化学修饰及 Cy5荧光标记的 CCR5_asiRNA (序列为： 正义链： 5' Choi - (mG) (mU) (mC) AAGUCCAAUCU (FA) (FU) (FG) dT- s- dT- Cy5 3， ， Choi 为胆固醇修饰， m为甲基化 修饰， F 为氟代修饰， s 为硫代修饰，， 反义链： 3' dTdTCACAGUUCAGGUUAGAUAC 5' )， 通过 PEG脂质体 -恩度内皮抑素将其递送系统尾静脉注射入 BALB/c裸鼠体内， 并观察其在裸鼠体内 的分布情况。 样品给药前现配， 取 PEG脂质体-恩度内皮抑素复合物 2ml (其中恩度内皮抑素 与 PEG脂质体的质量比为 2 : 1， 制备方法同 PEG脂质体 -内皮抑素复合物） 与荧光标记化学修 饰的 CCR5- asiRNA - Cy5 20nmol混合摇勾， 室温孵化 20min， Zeta电位测定为 +10mV。 BALB/C 裸鼠 （SPF级， 上海斯莱克实验动物有限责任公司）， 雄性， 体重 18_20g， 取配制好的受试药 物， 正常裸鼠尾静脉注射 （0. 3ml/20g)， 在 1、 3、 5、 24h时进行活体成像观察， 在 lh及 24h (各 3 只） 后处死取内脏进行活体成像观察， 将剩余的 0. 2ml PEG 脂质体-恩度内皮抑素 -CCR5-asiRNA-Cy5 混合物稀释成六个浓度用活体成像进行定量， 做浓度与荧光强度的标准曲 线，最后根据活体成像仪指示的数据定量计算主要脏器心、肝、脾、肺、肾的 CCR5-asiRNA-Cy5 残留剂量。所用仪器同上，实验结果见图 17，结果显示在不同时间点 CCR5_asiRNA-Cy5在 BLBA/c 裸鼠的各主要脏器中均有分布，通过 24h的荧光定量看到 CCR5-asiRNA-Cy5大量聚集在肾组织 中， 说明本品主要为肾排泄。
实施例 11
本实施例使用的 RNA干扰分子为电荷值为 -35的经化学修饰的 BC12-asiRNA ，序列为： 正 义链： 5' chol- (mG) (mA) (mG) GCUGGGAUGCC (mU) (mU) (mU) dT-s-dT 3' ， Choi 为胆固醇修饰， m为甲基化修饰， s为硫代修饰， 反义链： 3' dTdT GCCUCCGACCCUACGGAAA 5' ， 使用的内皮抑 素 -PEG脂质体同实施例 2。 试验选用 ICR小鼠 40只， 根据动物体重按区组随机化分组法分为 两组， 每组 20只， 雌雄各半。本次急性毒性研究选择 BC12-asiRNA的剂量为 100mg/kg作为急 性毒性研究的给药剂量（相当于小鼠药效学有效剂量 10mg/kg的 100倍）， 内皮抑素 -PEG脂质 体剂量为 50mg/kg (相当于内皮抑素小鼠药效学有效剂量 5mg/kg的 10倍）进行联合使用； 同 时设阴性对照组给予 0. 1% 的灭菌 DEPC水， 给药体积均按 0. 4ml/20g体重。 试验结果如下： 与阴性对照组相比，供试品给药组动物临床症状观察未发现明显异常情况，体重增加亦未出现 明显的异常情况。观察期结束后，所有存活实验小鼠解剖后大体肉眼观察结果显示与阴性对照 组相比， 供试品给药组小鼠各主要脏器组织均未见明显异常病理变化。 因此， ICR小鼠一次性 静脉推注给予供试品 BC12-asiRNA的最小致死剂量（LD50)大于 100mg/kg。
实施例 12
本实施例所用 BC12-asiRNA、 内皮抑素同实施例 11， BALB/C裸鼠（SPF级）， 雄性， 18_20g 购于上海斯莱克实验动物有限责任公司。 取生长良好的 A549人源肺癌实体瘤 （上海医药工业 研究院提供）， 无菌条件下切割成约 3mm大小的均勾小块， 用套管针每只小鼠右腋皮下接种一 块， 随机分为 5组， 分别为： 生理盐水组 （空白对照）、 化疗药物环磷酰胺 CTX ( 30mg/kg) 组 (阳性对照）、 PEG 脂质体-内皮抑素 -BC12-asiRNA 高剂量组、 PEG 脂质体-内皮抑素 -Bcl2-asiRNA中剂量组、 PEG脂质体-内皮抑素 -Bcl2_asiRNA低剂量组。 各组配比相同， 只是 所注射的量分别为 0. 4、 0. 2、 0. lml , 因此三组的 Zeta电位测定均为 +3mV。 接种后 13日根据 肿瘤大小重新分组， 淘汰肿瘤过大和过小的动物， 每组肿瘤平均体积基本一致， 开始给药。 高 剂量组的给药剂量为 Bcl2-asiRNA 2mg/kg、 内皮抑素 15mg/kg、 PEG脂质体 5mg/kg， 给药体积 为 0. 4ml， 中剂量组的给药剂量是高剂量组的 1/2， 低剂量组的剂量是高剂量组的 1/4。 生理 盐水组及 PEG脂质体-内皮抑素 -BC12-asiRNA高、 中、 低剂量组均连续尾静脉注射 14天， CTX ( 30mg/kg)组连续腹腔注射 7天。 接种后 29天处死动物， 解剖取瘤块， 拍照。 与生理盐水组 相比， PEG脂质体-内皮抑素 -BC12-asiRNA各组均有一定的抑瘤效果， 且抑瘤效果随剂量增大 而升高， 高剂量组的抑瘤效果最为明显， 各组解剖后的瘤块如图 18。
本发明实施例 12中所涉及的所有脂质体 -内皮抑素重组蛋白 -RNA干扰分子的电荷值均 采用 Zeta Sizer 3000动态激光散射仪测定其 Zeta电位法测得。
本发明各实施例中所使用的恩度内皮抑素的生产方法详见中国专利申请 200510040941. 9 中具体实施方式部分 1PEG-ES的制备部分。本发明各实施例中所使用的内皮抑素的生产方法详 见中国专利申请 97107112. 8实施例。 本发明各实施例中所使用的 PEG内皮抑素的生产方法详 见中国专利申请 200510040941. 9中具体实施方式部分的制备部分。

Claims

权利要求书
、 一种药用组合物， 其特征在于所述的组合物包含携带 +5至 +15电荷的内皮抑素重组蛋白和 携带 -15至 -55电荷的化学合成的 RNA干扰分子， 二者通过正负电荷结合形成电荷值在 -20 至 IJ+20之间的组合物， 优选形成电荷值在 -10到 +10之间的组合物。
、 根据权利要求 1所述的药用组合物， 其特征在于所述的药用组合物还包含用于包裹所述的 内皮抑素重组蛋白与 RNA干扰分子以调节正负电荷量的阳离子脂质体、中性脂质体、或 PEG 修饰长效脂质体。
、 根据权利要求 1所述的药用组合物， 其特征在于所述的内皮抑素重组蛋白选自不携带 6组 氨酸标签的由 183-184个氨基酸组成的人源结构的内皮抑素重组蛋白， N端或 /和 C端携带 6组氨酸标签的由 190-200个氨基酸组成的人源结构改构的内皮抑素融合蛋白， 或上述两 类内皮抑素重组蛋白通过 PEG修饰或白蛋白修饰后形成的长效内皮抑素； 或人工合成的截 短型内皮抑素。
、 根据权利要求 1所述的药用组合物， 其特征在于所述的 RNA干扰分子选自化学修饰或不修 饰的两条链对称的小核酸干扰分子， 化学修饰或不修饰的两条链配对不对称的小核酸干扰 分子， 或者化学修饰或不修饰的 miRNA。
、 根据权利要求 4所述的药用组合物， 其特征在于所述的两条链对称的小核酸干扰分子为正 义链与反义链长度一致的能抑制目的基因表达的长度为 19_25nt的双链配对 RNA, 其每条 链 3 ' 端含有 2-4个 UU碱基或脱氧核糖核酸 dTdT呈单链悬挂。
、 根据权利要求 4所述的药用组合物， 其特征在于所述的两条链配对不对称的小核酸干扰分 子是从靶基因对称性小核酸干扰分子 siRNA出发，通过将 siRNA正义链或反义链 5 ' 或 3 ' 端裁减 l_5nt碱基， 形成两条链碱基长度不一致，每条链 3 ' 端有 2-4个 UU碱基或脱氧核 糖核酸 dTdT呈单链悬挂， 具有 14-21nt的能诱发 RNA干扰的双链不对称小核酸干扰分子 asiRNAo
、 根据权利要求 6中任一项所述的药用组合物， 其特征在于所述的化学修饰指硫代修饰、 甲基化修饰、 磷酸化修饰、 胆固醇修饰和氟代修饰。
、 根据权利要求广 4中任一项所述的药用组合物， 其特征在于所述的药用组合物中各组分分 开封装临用时混合， 或直接混合后封装在一起。
、 权利要求广 4中任一项所述的药用组合物在制备治疗恶性肿瘤、 肝炎或艾滋病药物中的应 用。 、根据权利要求 9所述的应用，其特征在于所述的药用组合物中各组分分开封装临用时混合， 或直接混合后封装在一起， 用于皮下注射、 局部注射、 静脉注射或静脉滴注。
PCT/CN2011/074100 2011-05-16 2011-05-16 包含内皮抑素和rna干扰分子的组合物及其应用 Ceased WO2012155328A1 (zh)

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Citations (1)

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CN101111154A (zh) * 2004-12-09 2008-01-23 表达遗传学公司 联合免疫基因疗法和化学疗法用于治疗癌症和过度增生性疾病

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101111154A (zh) * 2004-12-09 2008-01-23 表达遗传学公司 联合免疫基因疗法和化学疗法用于治疗癌症和过度增生性疾病

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MA, CHUNHONG ET AL.: "Inhibition effect of recombinant endostatin combined with anti-sense A targeting PreS2 on hepatoma cells in vivo", CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY, vol. 24, no. 2, February 2004 (2004-02-01), pages 145 *
WANG DONG ET AL.: "Synergetic role of pSilence Ape1 and endostatin in antiangiogenesis of WANG, Dong et al. Synergetic role of pSilence Apel and endostatin in antiangiogenesis of osteosarcoma in animal model", CHINESE JOURNAL OF EXPERIMENTAL SURGERY, vol. 23, no. 8, August 2006 (2006-08-01), pages 990 - 992 *

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