WO2017193460A1 - 一种基于金纳米簇联合NGF siRNA治疗胰腺癌的方法 - Google Patents

一种基于金纳米簇联合NGF siRNA治疗胰腺癌的方法 Download PDF

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WO2017193460A1
WO2017193460A1 PCT/CN2016/087364 CN2016087364W WO2017193460A1 WO 2017193460 A1 WO2017193460 A1 WO 2017193460A1 CN 2016087364 W CN2016087364 W CN 2016087364W WO 2017193460 A1 WO2017193460 A1 WO 2017193460A1
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sirna
ngf
gold
gold nanocluster
nanocluster
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French (fr)
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蒋兴宇
雷祎凤
谢阳州昀
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国家纳米科学中心
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/242—Gold; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention relates to a gold nanocluster, in particular to a NGF siRNA combined with a gold nanocluster, a pharmaceutical composition and a kit containing the NGF siRNA combined with a gold nanocluster, and a preparation method and application of the NGF siRNA combined with a gold nanocluster.
  • GMC Gold nanoclusters
  • the electronic structure similar to semiconductors also makes it have special fluorescent properties, so it is used in biomedical applications for bioimaging and biomolecules (DNA, protein, Enzyme) detection and the like.
  • Gold nanoclusters have been reported, for example, as gold nanoclusters modified with TAT polypeptides, which can be used for photodynamic therapy of fluorescence imaging, gene delivery and near-infrared light excitation; gold nanoclusters contain polyacrylic acid and calcium phosphate to provide enhancement Fluorescent properties and can be used for chemotherapy; gold nanoclusters combined with cisplatin prodrugs and folic acid, can be used for fluorescence imaging and targeted chemotherapy for breast cancer; gold nanoclusters contain chitosan and form a stable complex with suicide genes, available For optical imaging without the use of additional dyes, and confirmed by in vitro cell experiments to inhibit cervical cancer cells; gold nanoclusters contain BSA and DOX, which can be used as therapeutic agents for fluorescent probes and cervical cancer; and, gold nano Clusters combined with Herceptin, in vitro cell experiments to verify its use for imaging and treatment of breast cancer;
  • Pancreatic cancer is one of the most deadly cancers in humans, with a 5-year survival rate of less than 5% after diagnosis.
  • the growth and development of pancreatic cancer is closely related to the neuromicroenvironment in which it is located.
  • Clinical results suggest that the most common phenomenon in pancreatic cancer tumors is a high increase in nerve density within the tumor.
  • Pancreatic tumors can secrete neurotrophic factors such as nerve growth factor (NGF).
  • NGF nerve growth factor
  • NGF nerve growth factor
  • NGF nerve growth factor
  • NGF nerve growth factor
  • siRNA is an effective means of genetic intervention. It has been reported that the use of subcutaneous tumor models and injection of siRNA near tumors have demonstrated that NGF siRNA can be used to inhibit breast cancer growth. However, free siRNA is easily degraded by nucleases in serum, so further development is needed. More qualitatively NGF siRNA-based pharmaceutical compositions and formulations for the prevention and/or treatment of cancer.
  • NGF siRNA combined with gold nanoclusters with mild conditions and better stability
  • a method for preparing the NGF siRNA combined with gold nanoclusters A pharmaceutical composition, kit for NGF siRNA in combination with gold nanoclusters, and anti-tumor application of the NGF siRNA in combination with gold nanoclusters.
  • a first aspect of the present invention provides an NGF siRNA in combination with a gold nanocluster comprising a gold nanocluster and an NGF siRNA, and the gold nanocluster and the NGF siRNA The combination is electrostatic adsorption.
  • NGF siRNA itself can be used to silence the NGF gene in pancreatic tumors.
  • free NGF siRNA is easily degraded by nucleases in serum and is easily cleared by renal organs, resulting in lower silencing efficiency.
  • free NGF siRNA is difficult to enter into cells and tumors to play a role in gene silencing. Therefore, most of the current reports on siRNA use a local introduction strategy such as subcutaneous injection, intratumoral injection, and the like.
  • the present invention employs gold nanoclusters as an efficient delivery platform for delivery of NGF siRNA, which has not been reported so far.
  • the gold nanocluster can provide effective protection for NGF siRNA, thereby improving the stability of NGF siRNA and the circulation time in vivo and tumor targeting, and then increasing the NGF siRNA in the tumor.
  • the efficiency of delivery in cells and tumor tissues, to achieve effective NGF gene silencing, in order to inhibit the occurrence and development of cancer (such as pancreatic cancer) caused by over-expression of NGF opens up a promising treatment for pancreatic cancer New approach.
  • the NGF siRNA according to the first aspect of the present invention is combined with a gold nanocluster, wherein the mass ratio of the NGF siRNA to the gold nanoclusters in the NGF siRNA combined with the gold nanoclusters is from 1.5:1 to 3.5:1, preferably 2:1. ⁇ 3:1, more preferably 3:1.
  • the NGF siRNA according to the first aspect of the invention is combined with a gold nanocluster, wherein the gold nanoclusters have an average diameter of 1.63 to 3.57 nm, preferably 2.2 to 3.1 nm, for example 2.66 ⁇ 0.45 nm;
  • the surface of the gold nanoclusters is preferably positively charged; and/or
  • the potential of the gold nanoclusters is preferably 19 to 21 mV, preferably 19.1 to 20.7 mV, for example, 19.9 ⁇ 0.8 mV.
  • siRNA refers to a class of RNA molecules that are processed from transcripts that form siRNA precursors. Mature siRNAs typically have 18 to 26 nucleotides (nt) and do not exclude siRNA molecules with other numbers of nucleotides. siRNA is typically detected by Northern blotting.
  • siRNA can be generated by mimicking the miRNA production machinery, and such siRNA can be processed from precursor RNA (Precursor RNA, Pre-RNA).
  • precursor RNA Precursor RNA, Pre-RNA
  • the precursor RNA can be folded into a stable stem-loop (hairpin) structure, which is typically 50-100 bp in length.
  • the precursor RNA can be folded into a stable stem-loop structure, and the stem of the stem-loop structure comprises two sequences that are substantially complementary on both sides.
  • the precursor RNA can be natural or synthetic.
  • the precursor RNA can be cleaved to generate siRNA that is substantially complementary to at least a portion of the sequence encoding the mRNA of the gene.
  • substantially complementary means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure).
  • two "substantially complementary" nucleotide sequences are complementary to each other by 70% of the nucleotides, preferably at least 80% of the nucleotides are complementary, more preferably at least 90% of the nucleosides
  • the acids are complementary, further preferably at least 95% of the nucleotides are complementary, and may even be 96%, 97%, 98%, 99%, 100% complementary.
  • stem loop structure also referred to as “hairpin” structure, refers to a nucleotide molecule which can form a secondary structure comprising a double-stranded region (stem portion) from which the double-stranded region Two regions of the nucleotide molecule (on the same molecule) are formed, and the two regions are separated on both sides of the double-stranded portion. It also includes at least one "loop” structure, including non-complementary nucleotide molecules, ie, single-stranded regions. Even if the two regions of the nucleotide molecule are not completely complementary, the double-stranded portion of the nucleotide can remain in a double-stranded state.
  • insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself.
  • the two regions are still substantially complementary and interact in a foreseeable manner to form a double-stranded region of the stem-loop structure.
  • Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid forms a stem-loop structure.
  • siRNAs of the invention generally refers to NGF siRNA, which is generally used for inhibiting the expression of nerve growth factor (NGF).
  • NGF nerve growth factor
  • siRNAs of the invention may also include siRNA variants and derivatives.
  • siRNA derivatives in a broad sense may also include siRNA variants.
  • One skilled in the art can modify NGF siRNA using a general method including, but not limited to, methylation modification, hydrocarbyl modification, glycosylation modification (eg 2-methoxy-glycosyl modification, hydrocarbyl-glycosyl) Modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification) and the like.
  • NGF siRNA according to the first aspect of the invention in combination with a gold nanocluster, wherein the NGF
  • the sequence of the siRNA is:
  • siRNA sense 5'-CCACAGACAUCAAGGGCAA dTdT-3' SEQ ID NO: 1
  • siRNA antisense 3'-dTdT GGUGUCUGUAGUUCCCGUU-5' SEQ ID NO: 2.
  • the siRNA is purchased and/or synthesized from ribobio.
  • the siRNA sequences involved in the present invention were determined from the NGF database (accession number NM_002506) of the National Center for Biotechnology Information.
  • a second aspect of the invention provides a preparation method for preparing the NGF siRNA combined with a gold nanocluster according to the first aspect of the invention, the preparation method comprising the steps of:
  • the reducing agent is preferably a glutathione (GSH) and a polypeptide molecule.
  • the polypeptide molecule is preferably CRRRRRRRRR (CR 9 ).
  • the molar ratio of HAuCl 4 to glutathione is 1:0.5 to 3, preferably 1:1.5, and the molar ratio of HAuCl 4 to CRRRRRRRRR is 1:0.5 to 1.5, preferably 1:0.75.
  • a reducing agent is added to the HAuCl 4 solution at room temperature, preferably at 24 to 26 ° C, more preferably at 25 ° C; and/or
  • the temperature at which the temperature is heated is 70 °C.
  • the time of constant temperature stirring is preferably from 12 to 36 h, more preferably 24 h.
  • the mass ratio of the NGF siRNA to the gold nanoclusters is from 0.5 to 100:1, preferably from 1.5 to 5:1, more preferably from 2 to 3.5:1, and most preferably 3:1;
  • the normal temperature is 22 to 28 ° C, preferably 24 to 26 ° C, for example, 25 ° C;
  • the agitation is vortex agitation; and/or
  • the agitation time is from 15 to 90 minutes, preferably from 15 to 45 minutes, and most preferably from 30 minutes.
  • a third aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the NGF siRNA of the first aspect of the invention in combination with a gold nanocluster or a method according to the second aspect of the invention
  • the prepared NGF siRNA is combined with gold nanoclusters.
  • the dosage form of the pharmaceutical composition is preferably an injection, more preferably an intravenous injection.
  • a fourth aspect of the invention provides a kit comprising the first aspect of the invention NGF siRNA in combination with gold nanoclusters or NGF siRNA prepared according to the method of the second aspect of the invention in combination with gold nanoclusters.
  • a fifth aspect of the invention provides an application of the NGF siRNA in combination with a gold nanocluster according to the first aspect of the invention, the NGF siRNA in combination with the method of the second aspect of the invention, or the third nanoparticle of the invention
  • the cancer is preferably a cancer caused by overexpression of NGF, and may be, for example, pancreatic cancer; and/or the drug is preferably an injection, more preferably an intravenous injection.
  • a sixth aspect of the invention provides a method of preventing and/or treating cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of the NGF siRNA in combination with the gold nanocluster of the first aspect of the invention or according to the second aspect of the invention
  • the NGF siRNA prepared by the method is combined with a gold nanocluster or a pharmaceutical composition of the third aspect of the invention.
  • the invention also provides a preferred method of treatment comprising one or more of the following steps: (1) determining whether the disease species is a cancer caused by overexpression of NGF; (2) directly employing the NGF siRNA of the invention Combining gold nanoclusters or preparing NGF siRNAs in combination with gold nanoclusters according to the method of the present invention; (3) administering a therapeutically effective amount of NGF siRNA to gold nanoclusters to a subject in need thereof; (4) detecting the content of NGF siRNA in serum of the subject (5) detecting the content of NGF siRNA in the target cells; (6) detecting the expression level of NGF mRNA and/or NGF protein in the target cells; (7) detecting the therapeutic effect of the disease.
  • a seventh aspect of the present invention provides an NGF siRNA-conjugated gold nanocluster for preventing and/or treating cancer, the NGF siRNA in combination with a gold nanocluster as the NGF siRNA in combination with the gold nanocluster of the first aspect of the invention or according to the present invention
  • the NGF siRNA prepared by the method of the second aspect is combined with a gold nanocluster.
  • the NGF siRNA of the present invention in combination with a gold nanocluster may have, but is not limited to, the following beneficial effects:
  • the GNC-siRNA of the present invention can improve the stability of siRNA and prolong the circulation time in vivo. It was confirmed by in vitro experiments that the stability of GNC-siRNA in serum was higher than that of free siRNA; moreover, the fluorescence intensity of GNC-siRNA and free siRNA after different blood circulation time in mice was compared, and the results also indicated that GNC - siRNA is more stable in serum.
  • the GNC-siRNA of the present invention can improve the targeting of pancreatic tumors and improve the silencing effect of NGF.
  • In vitro and in vivo experiments show that GNC-siRNA is significantly higher in pancreatic cancer cells and pancreatic tumors than in free siRNA, and can significantly reduce pancreatic cancer cells and NGF mRNA expression and protein expression in pancreatic tumor tissues.
  • the GNC-siRNA of the present invention can enhance the anti-tumor therapeutic effect of pancreatic cancer. It can be seen from in vivo experiments that GNC-siRNA can significantly inhibit the growth, development and metastasis of pancreatic cancer tumors in mice.
  • the preparation method of the GNC-siRNA of the present invention is simple, and the reaction conditions are very mild (may be normal temperature, normal pressure, aqueous phase, near pH neutrality), and the activity of the siRNA is greatly protected. The adverse effects of previous chemical modification methods on siRNA activity were also avoided.
  • the GNC-siRNA of the present invention has high efficiency of delivery to NGF siRNA, and can reach 3000 mg siRNA/g gold nanoclusters (mass ratio of siRNA/GNC is about 3:1), which is equivalent to 226 ⁇ mol siRNA/g gold nanoclusters.
  • Gold nanoclusters deliver siRNA more efficiently than known gold nanoparticle delivery siRNAs.
  • Figure 2 is a photograph of the gold nanocluster solution of the first embodiment
  • Figure 3 is a TEM image of the gold nanocluster of the first embodiment
  • Figure 5 is a zeta potential of the NGF siRNA in combination with the gold nanoclusters of the first to tenth embodiments
  • 6 is a surface XPS spectrum of a gold nanocluster and an NGF siRNA combined with a gold nanocluster in the first embodiment
  • Figure 7 is a gel electrophoresis pattern of polyacrylamide in the first test example
  • Figure 8 is a graph showing the amount of pancreatic cancer cell uptake in the second test example.
  • Figure 9 is a chart showing the level of NGF mRNA expression in pancreatic cancer cells by PCR in the third test example.
  • Figure 10 is a graph showing the level of NGF protein in pancreatic cancer cells by Western blotting in the third test example
  • Figure 11 is a graph showing changes in fluorescence intensity of free siRNA and GNC-siRNA in mouse blood over time using Cy-5 fluorescent molecule-labeled siRNA in the fourth test example;
  • Figure 12 is a graph showing the aggregation of siRNA, Cy-siRNA and GNC-siRNA in pancreatic tumor tissues in mice in the fifth test example using Cy-5 fluorescent molecules.
  • the white circle indicates a subcutaneous pancreatic tumor.
  • Figure 13 is an in vitro picture of the aggregation of siRNA, free siRNA and GNC-siRNA in pancreatic tumor tissue with Cy-5 fluorescent molecule labeled in the fifth test example;
  • Figure 14 is a diagram showing the construction of the orthotopic pancreatic tumor model in the sixth test case, the administration time, and the sacrifice of the mouse. Schematic diagram of time
  • Figure 15 is a graph showing changes in body weight of mice during administration in the sixth test example.
  • Figure 16 is a graph showing the growth results of bioluminescence-labeled orthotopic pancreatic tumors in the sixth test example.
  • Figure 17 is a graph showing the statistical results of the luminescence intensity of the orthotopic pancreatic tumor in the sixth test example.
  • Figure 18 is an in vitro photograph of an orthotopic pancreatic tumor collected after the end of the experiment in the sixth test example
  • Figure 19 is a graph showing the results of weighing of the orthotopic pancreatic tumors in the sixth test example.
  • Figure 20 is a graph showing the results of bioluminescence-labeled orthotopic pancreatic tumor metastasis in the mesentery in the sixth test example.
  • Fig. 21 is a graph showing the statistical results of the luminescence intensity of pancreatic tumors transferred to the mesentery in the sixth test example.
  • Figure 22 is a chart showing the level of NGF mRNA expression in pancreatic tumor tissues by PCR in the seventh test example
  • Figure 23 is a graph showing the level of NGF protein in pancreatic tumor tissues by Western blotting in the seventh test example.
  • This example is intended to illustrate the NGF siRNA combined with gold nanoclusters (GNC-siRNA) of the present invention and a preparation method thereof.
  • the GNC-siRNA of the present invention was prepared by the following procedure (technical route is shown in Figure 1):
  • reducing agents GSH and CR 9 to HAuCl 4 solution at 25 ° C, wherein HAuCl 4 is 100 mM, GSH is 150 mM, CR 9 is 75 mM, which corresponds to a molar ratio of HAuCl 4 to GSH of 1:1.5, HAuCl 4
  • the molar ratio to CR 9 was 1:0.75, then heated to 70 ° C and stirred at a constant temperature for 24 h to obtain a gold nanocluster solution.
  • the color of the gold nanocluster solution is light yellow-green (as shown in Figure 2.
  • the figure 2 may display an incorrect color, which does not limit the invention).
  • the size and surface morphology of the gold nanoclusters were observed by TEM.
  • the TEM image is shown in Fig. 3.
  • the average diameter was 2.66 ⁇ 0.45 nm, and the dispersion was good.
  • the surface of the gold nanoclusters was positively charged using a zeta potentiometer (as shown in Figure 4), and the zeta potential was 19.9 ⁇ 0.8 mV.
  • NGF siRNA was added to the gold nanocluster solution, and the mixture was stirred at 25 ° C for 30 min to obtain the GNC-siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • negatively charged NGF siRNA was adsorbed on the surface of positively charged gold nanoclusters by electrostatic adsorption.
  • the zeta potential of this GNC-siRNA is shown in Figure 5.
  • Figure 6 is a surface XPS map of GNC (from step (1)) and GNC-siRNA (from step (2)) as determined by XPS spectrometer. It can be seen from the map that GNC has no peak of P, indicating that phosphorus is not contained, and a peak of P appears in GNC-siRNA, indicating that GNC-siRNA contains a phosphorus element (P element is derived from a nucleic acid), that is, NGN siRNA is electrostatically adsorbed on the surface of GNC.
  • P element is derived from a nucleic acid
  • This example is intended to illustrate the GNC-siRNA of the present invention and a method for its preparation.
  • the GNC-siRNAs of the second to tenth examples were prepared in the same manner as in the first embodiment except that the mass ratio of the NGF siRNA to the gold nanoclusters in the step (2) was 0.5:1, 1:1, respectively. , 1.5:1, 2:1, 2.5:1, 3.5:1, 4:1, 5:1, 100:1.
  • the zeta potential of the above GNC-siRNA was measured using a zeta potentiometer, and the results are shown in Fig. 5.
  • the gold nanocluster solution of the step (2) is used as a mass ratio of 0:1.
  • the zeta potential of the solution does not change, indicating that the siRNA is saturated. Therefore, GNC-siRNA has a siRNA loading of up to 3000 mg siRNA/g gold nanoclusters, which corresponds to 226 ⁇ mol siRNA/g gold nanoclusters.
  • the mass ratio of the NGF siRNA to the gold nanocluster is 3:1, 0.5:1, 1:1, 1.5:1, 2:1, respectively. 2.5:1, 3:1, 3:1, 3:1, 3:1.
  • This example is intended to illustrate the GNC-siRNA of the present invention and a method for its preparation.
  • the GNC-siRNA of the present invention was prepared by the following procedure (technical route is shown in Figure 1):
  • the reducing agents GSH and CR 9 were added to the HAuCl 4 solution at 24 ° C, wherein HAuCl 4 was 100 mM, GSH was 50 mM, and CR 9 was 50 mM, which corresponds to a molar ratio of HAuCl 4 to GSH of 1:0.5, HAuCl 4 .
  • the molar ratio to CR 9 was 1:0.5.
  • the mixture was heated to 70 ° C and stirred at a constant temperature for 12 hours to obtain a gold nanocluster solution.
  • the color of the gold nanocluster solution is pale yellowish green.
  • the size and surface topography of the gold nanoclusters were observed by TEM, and the average diameter was 2.41 nm, and the dispersibility was good.
  • the surface of the gold nanoclusters was positively charged using a zeta potentiometer, and the zeta potential was 20.3 mV.
  • NGF siRNA was added to the gold nanocluster solution, and vortexed at 24 ° C for 15 min to obtain the GNC-siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • the XPS pattern of the GNC-siRNA of the present example has a P peak, indicating that the NGC surface is electrostatically adsorbed with NGF siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • This example is intended to illustrate the GNC-siRNA of the present invention and a method for its preparation.
  • the GNC-siRNA of the present invention was prepared by the following procedure (technical route is shown in Figure 1):
  • reducing agents GSH and CR 9 to HAuCl 4 solution at 26 ° C, wherein HAuCl 4 is 100 mM, GSH is 300 mM, CR 9 is 150 mM, which corresponds to a molar ratio of HAuCl 4 to GSH of 1:3, HAuCl 4
  • the molar ratio to CR 9 was 1:1.5.
  • the mixture was heated to 70 ° C and stirred at a constant temperature for 36 hours to obtain a gold nanocluster solution.
  • the color of the gold nanocluster solution is pale yellowish green.
  • the size and surface morphology of the gold nanoclusters were observed by TEM, and the average diameter was 2.89 nm, and the dispersibility was good.
  • the surface of the gold nanoclusters was positively charged using a zeta potentiometer, and the zeta potential was 20.1 mV.
  • NGF siRNA was added to the gold nanocluster solution, and the mixture was stirred at 26 ° C for 45 min to obtain the GNC-siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • the XPS pattern of the GNC-siRNA of the present example has a P peak, indicating that the NGC surface is electrostatically adsorbed with NGF siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • This example is intended to illustrate the GNC-siRNA of the present invention and a method for its preparation.
  • the GNC-siRNA of the present invention was prepared by the following procedure (technical route is shown in Figure 1):
  • reducing agents GSH and CR 9 to HAuCl 4 solution at 25 ° C, wherein HAuCl 4 is 100 mM, GSH is 250 mM, CR 9 is 100 mM, which corresponds to a molar ratio of HAuCl 4 to GSH of 1:2.5, HAuCl 4
  • the molar ratio to CR 9 is 1:1.
  • the mixture was heated to 70 ° C and stirred at a constant temperature for 24 hours to obtain a gold nanocluster solution.
  • the color of the gold nanocluster solution is pale yellowish green.
  • the size and surface morphology of the gold nanoclusters were observed by TEM, and the average diameter was 2.75 nm, and the dispersibility was good.
  • the surface of the gold nanoclusters was positively charged using a zeta potentiometer, and the zeta potential was 19.6 mV.
  • NGF siRNA was added to the gold nanocluster solution, and vortexed at 25 ° C for 60 min to obtain the GNC-siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • the XPS pattern of the GNC-siRNA of the present example has a P peak, indicating that the NGC surface is electrostatically adsorbed with NGF siRNA.
  • the mass ratio of NGF siRNA to gold nanoclusters was 3:1.
  • the free NGF siRNA and the GNC-siRNA of the first example were cultured in a medium containing 10% serum (v/v), and the initial concentration of the siRNA was 100 nM, and after 0 min, 15 min, 30 min, 45 min, and 60 min incubation, extraction was performed. 5 ⁇ l of each solution was added to a polyacrylamide gel electrophoresis tank, and the gel was run at 160 V for 45 min, and the nucleic acid was stained and UV-developed with GelRed to evaluate the position and distribution of the siRNA nucleic acid. The results are shown in Figure 7, indicating that the GNC-siRNA of the present invention is more stable in serum than the free siRNA.
  • This test example was used to verify the uptake of different siRNAs in pancreatic cancer cells.
  • Pancreatic cancer cell Panc-1 cells were cultured in a culture dish to 80% cell fusion degree, and free siRNA and GNC-siRNA of the first example were separately added to the culture medium so that the initial concentration of siRNA was 100 nM. After 1 h of culture, the cells were washed, the cells were fixed with 4% paraformaldehyde, and the cells were washed. Then, the uptake of Cy5-siRNA by Panc-1 cancer cells was observed by confocal laser microscopy. The results are shown in Fig. 8, indicating that the uptake of the GNC-siRNA of the present invention in pancreatic cancer cells is significantly higher than that of the free siRNA.
  • Pancreatic cancer cell Panc-1 cells were cultured in a culture dish to 70% to 80% confluency, and free siRNA and GNC-siRNA of the first example were separately added to the culture medium to make the initial concentration of siRNA. All were 100 nM, and the untreated group, GNC-nsRNA (gold nanoclusters combined with nonsense siRNA, wherein the initial concentration of siRNA was 100 nM) was used as a control. After 48 hours of culture, the cells were washed. The expression level of NGF mRNA in the cells was characterized by PCR, and the expression level of NGF protein in the cells was characterized by Western blotting. The results are shown in Figures 9 and 10, respectively, indicating that the GNC-siRNA of the present invention significantly reduced NGF mRNA expression and NGF protein expression in pancreatic cancer cells relative to free siRNA.
  • the mouse tail vein was separately injected with the free siRNA and the GNC-siRNA of the first example, so that the siRNA injection amount was the same, and each mouse was injected with 30 ⁇ g of Cy5-siRNA. After the tail vein injection for 0h, 1h, 2h, 3h, 6h, the blood of the mice was collected about 150 ⁇ l.
  • This test is used to verify the tumor targeting of different siRNAs in mice.
  • free siRNA and GNC-siRNA of the first example were injected into the tail vein to make the same amount of siRNA injection.
  • Each mouse was injected with 30 ⁇ g of Cy5-siRNA to physiologically.
  • the saline injection group served as a control.
  • the CRI Maestro 2 multi-spectral small animal in vivo imaging system was used to analyze the distribution of free siRNA and the GNC-siRNA of the first example in mice, using the yellow filter of the CRI Maestro 2 imaging system.
  • the light sheet collects fluorescent emission signals from 650 nm to 800 nm. The result is shown in FIG.
  • mice After 24 hours of siRNA injection, the mice were dissected and the tumor tissues were taken out and imaged in vitro using the above imaging system to observe the fluorescence distribution of free siRNA and GNC-siRNA at the tumor site. The result is shown in FIG.
  • mice 1 ⁇ 10 6 luciferase-labeled pancreatic cancer cells (Panc-1-luc) were implanted in the pancreatic head of the pancreas to construct an orthotopic pancreatic cancer tumor model. After about 2 weeks, an in situ pancreatic tumor is formed.
  • the mice were injected with free siRNA and the GNC-siRNA of the first example in the tail vein, and the amount of siRNA injected was 30 ⁇ g siRNA per mouse, and the saline injection group and the GNC injection group (the same amount of GNC injection).
  • the GNC-nsRNA injection group (the same amount of GNC and siRNA injection) was used as a control, and it was injected every two days for a total of 7 injections.
  • Figure 15 shows changes in body weight of mice during the experiment. The results showed that the body weight of the mice did not change significantly during the administration, indicating that different modes of administration did not cause significant toxicity to the mice.
  • Figure 16 is a graph showing the growth results of bioluminescent labelled orthotopic pancreatic tumors.
  • Figure 17 is a statistical result of the luminescence intensity of an orthotopic pancreatic tumor.
  • Figure 18 is an in vitro photograph of an orthotopic pancreatic tumor collected after the end of the experiment.
  • Figure 19 is a statistical result of weighing of an orthotopic pancreatic tumor. The above results all indicate that the GNC-siRNA of the present invention can significantly inhibit the growth of mouse orthotopic pancreatic tumors.
  • Figure 20 is a graph showing the results of metastasis of an orthotopic pancreatic tumor labeled with bioluminescence in the mesentery.
  • Figure 21 is a statistical result of the luminescence intensity of pancreatic tumors metastasized on the mesentery. The above results indicate that the GNC-siRNA of the present invention can significantly inhibit the metastasis of pancreatic tumors.
  • This test was used to verify the NGF gene silencing effect of different siRNAs in pancreatic tumors in mice.
  • the tumor tissue in the sixth test case was taken, and the expression level of NGF mRNA in the cells was characterized by PCR, and the expression level of NGF protein in the cells was characterized by Western blotting.
  • the results are shown in Figures 22 and 23, respectively, indicating that the GNC-siRNA of the present invention significantly reduced NGF mRNA expression and NGF protein expression in pancreatic tumor tissues relative to free siRNA.
  • the invention also provides a kit comprising the NGF siRNA in combination with the gold nanoclusters of the first aspect of the invention or the NGF siRNA in combination with the gold nanoclusters prepared according to the method of the second aspect of the invention.
  • reagents required for PCR amplification, vector construction, and the like including but not limited to amplification buffers, primers, template DNA, enzymes, and the like, can also be included in the kit.
  • instructions for use and/or use/analysis software may also be included in the kit.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and the NGF siRNA of the first aspect of the invention in combination with a gold nanocluster or an NGF prepared according to the method of the second aspect of the invention siRNA combined with gold nanoclusters.
  • the NGF siRNA in combination with the gold nanoclusters can be an effective amount or a therapeutically effective amount in the pharmaceutical composition.
  • an effective amount refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a "pharmaceutically acceptable” ingredient is suitable for use in humans and/or animals (eg, mammals and birds) without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, has reasonable benefits / risk ratio substance.
  • “Pharmaceutically acceptable carrier” means a carrier for administration, and may include various excipients, diluents and the like.
  • the pharmaceutical composition of the present invention may contain a safe and effective amount of the NGF siRNA of the present invention in combination with a gold nanocluster as an active ingredient and a pharmaceutically acceptable carrier.
  • Such carriers can include, but are not limited to, physiological saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched with the administration mode, and the dosage form of the pharmaceutical composition of the present invention can be prepared as an injection, an oral preparation (tablet, capsule, oral liquid), a transdermal agent, a diluent, and the like as needed.
  • physiological saline or an aqueous solution containing glucose and other excipients is usually prepared in a conventional manner.
  • the pharmaceutical composition is more suitably manufactured under sterile conditions. According to the above experimental results, since the NGF siRNA of the present invention in combination with the gold nanoclusters has good stability in serum, it is suitable for preparation as an injection, especially an intravenous injection.
  • the effective amount of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, The route of medicine, etc. Generally, in most cases, when the active ingredient of the present invention can be used in an amount of about 0.01 mg to 50 mg/kg of animal body per day (more A good dose of 1 mg to 5 mg/kg of animal body weight) can give a satisfactory effect.
  • the active ingredient of the present invention can be used in an amount of about 0.01 mg to 50 mg/kg of animal body per day (more A good dose of 1 mg to 5 mg/kg of animal body weight) can give a satisfactory effect.
  • the dosage is: 30 ⁇ g/mouse ( ⁇ 20 g), ie ⁇ 1.5 mg/kg; typically, the tail vein injection of siRNA ranges from 1 ⁇ g to 400 ⁇ g/mouse, ie
  • the amount administered is 0.05-20 mg/kg, and so on. For example, several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the pharmaceutically acceptable carrier of the present invention includes, but is not limited to, water, physiological saline, liposome, lipid, protein, protein-antibody conjugate, peptide substance, cellulose, nanogel, or combination.
  • the choice of carrier should generally be matched to the mode of administration, as is well known to those of ordinary skill in the art.
  • the invention also provides the use of the pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of cancer.
  • the cancer is a cancer caused by overexpression of NGF, and may be, for example, pancreatic cancer.
  • the invention also provides a method of preventing and/or treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the NGF siRNA in combination with the gold nanoclusters of the first aspect of the invention or the method according to the second aspect of the invention
  • the prepared NGF siRNA is combined with a gold nanocluster or a pharmaceutical composition of the third aspect of the invention.
  • the NGF siRNA of the present invention in combination with a gold nanocluster or a pharmaceutical composition can pass through the gastrointestinal tract, nasal cavity, trachea, lung, non-lesional vein or epidermis, intradermal, subcutaneous, intracardiac, intramuscular, bone marrow, abdominal cavity, hard It is administered by means of extramembranous, buccal, sublingual, ocular, rectal, vaginal, urethral, or ear canal.
  • Preferred modes of administration or modes of administration include oral, respiratory, injection, transdermal, mucosal, or intraluminal administration.
  • the oral administration means includes swallowing, incorporation, and the like.
  • the method of administration of the respiratory tract includes an inhalation method such as ultrasonic atomization inhalation, oxygen atomization inhalation, hand pressure atomization inhalation, and the like.
  • the administration mode of injection includes arterial injection, intravenous injection, intramuscular injection, intracardiac injection, intradermal injection, and the like.
  • the transdermal or transdermal administration methods include iontophoresis, electroporation, and the like.
  • the mucosal administration forms include nasal mucosa administration, oral mucosal administration, ocular mucosal administration, rectal mucosal administration, uterine administration, and vaginal mucosal administration.
  • the method of administration of the lumen includes rectal administration, vaginal administration, urethral administration, nasal administration, ear canal administration, and the like.
  • NGF siRNA in combination with gold nanoclusters has good stability in serum, and thus is particularly suitable for administration by arterial injection or intravenous injection.

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Abstract

一种NGF siRNA联合金纳米簇及其制备方法，含有该NGF siRNA联合金纳米簇的药物组合物、试剂盒，以及该NGF siRNA联合金纳米簇在制备用于预防和/或治疗癌症的药物中的应用。所述NGF siRNA联合金纳米簇包含金纳米簇和NGF siRNA，并且所述金纳米簇与所述NGF siRNA之间以静电吸附作用相结合。

Description

一种基于金纳米簇联合NGF siRNA治疗胰腺癌的方法
相关申请的交叉引用
本申请要求2016年05月13日提交的第CN201610319982.X号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明涉及一种金纳米簇，具体涉及一种NGF siRNA联合金纳米簇，含有该NGF siRNA联合金纳米簇的药物组合物和试剂盒，以及该NGF siRNA联合金纳米簇的制备方法和应用。
背景技术
金纳米簇(GNC)具有显著的库伦阻塞效应和特殊的磁性质，与半导体类似的电子结构也使它具有特殊的荧光性能，因而在生物医药领域用于生物成像和生物分子(DNA、蛋白质、酶)检测等。已报道的金纳米簇应用例如为：金纳米簇修饰有TAT多肽，可用于荧光成像、基因递送和近红外光激发的光动力治疗；金纳米簇包载有聚丙烯酸和磷酸钙，以提供增强的荧光性质并可用于化疗；金纳米簇结合顺铂前药和叶酸，可用于荧光成像以及乳腺癌的靶向化疗；金纳米簇包载有壳聚糖并与自杀基因形成稳定复合物，可用于光学成像而无需使用额外的染料，且通过体外细胞实验验证其可抑制宫颈癌细胞；金纳米簇包载有BSA和DOX，可用作荧光探针和宫颈癌的治疗药物；以及，金纳米簇结合赫赛汀(Herceptin)，以体外细胞实验验证其可用于乳腺癌的成像和治疗；等。
胰腺癌是人类最致命的癌症之一，其确诊后5年生存率不到5％。胰腺癌的生长和发展与其所处的神经微环境密切相关。临床结果表明，胰腺癌肿瘤中最常见的现象是肿瘤内的神经密度的高度增加。胰腺肿瘤能分泌神经营养因子如神经生长因子(NGF)等。NGF是调节肿瘤中神经生长的关键生长因子，NGF及其受体在胰腺肿瘤和胰腺癌细胞中过度表达，促进了胰腺肿瘤的生长、侵袭和转移。这些现象表明，沉默胰腺肿瘤中的NGF基因，是治疗胰腺癌的一种潜在的有效途径。
siRNA是一种有效的基因干预手段。目前已报道采用皮下肿瘤模型和在肿瘤附近注射siRNA的方式验证了NGF siRNA可用于抑制乳腺癌的生长。然而，游离siRNA容易被血清中的核酸酶降解，因此，还需要进一步研发稳 定性更佳的用于预防和/或治疗癌症的基于NGF siRNA的药物组合物及制剂。
发明内容
因此，本发明的目的在于克服现有技术中的缺陷，提供一种制备条件温和、稳定性更好的NGF siRNA联合金纳米簇，以及用于制备该NGF siRNA联合金纳米簇的方法，含有该NGF siRNA联合金纳米簇的药物组合物、试剂盒，和该NGF siRNA联合金纳米簇的抗肿瘤应用。
为实现上述目的，本发明的第一方面提供了一种NGF siRNA联合金纳米簇，所述NGF siRNA联合金纳米簇包含金纳米簇和NGF siRNA，并且所述金纳米簇与所述NGF siRNA之间以静电吸附作用相结合。
NGF siRNA本身可用于沉默胰腺肿瘤中的NGF基因。但是，游离NGF siRNA很容易被血清中的核酸酶降解，并且容易被肾器官清除，导致其沉默效率较低。而且，由于其较大的尺寸和表面负电荷，游离NGF siRNA很难进入细胞和肿瘤中发挥基因沉默的作用。因此，目前较多关于siRNA的报道采用的是局部导入策略，例如皮下注射、瘤内注射等。尽管有将粒径较大的金纳米颗粒结合EGFR siRNA或GM3S siRNA的实验，但其也均为经皮肤给药，且均用于治疗皮肤相关疾病或症状(分别用于抑制皮肤肿瘤和加速糖尿病小鼠的皮肤伤口愈合)。为保持siRNA的稳定性，现有做法通常是对其进行化学修饰(如糖基修饰等)，或者采用脂质体包埋、高分子纳米颗粒富集(例如壳聚糖、环糊精、PEI等)、聚合物胶束包埋等方式，具体可参见文献Gavrilov 2012,Therapeutic siRNA:Principles,challenges,and strategies。
与现有技术明显不同的是，本发明采用金纳米簇作为递送NGF siRNA的高效递送平台，目前尚未见这样的报道。在此过程中发现，通过采用本发明的技术方案，金纳米簇可以为NGF siRNA提供有效保护，从而提高NGF siRNA的稳定性和在体内的循环时间以及肿瘤靶向性，继而提高NGF siRNA在肿瘤细胞和肿瘤组织中的递送效率，实现有效的NGF基因沉默，以此来抑制因NGF过度表达而引发的癌症(如胰腺癌)的发生和发展，为胰腺癌的治疗开辟了一种有前途的新途径。
根据本发明第一方面的NGF siRNA联合金纳米簇，其中，在所述NGF siRNA联合金纳米簇中，NGF siRNA与金纳米簇的质量比为1.5:1～3.5:1，优选为2:1～3:1，更优选为3:1。
根据本发明第一方面的NGF siRNA联合金纳米簇，其中，所述金纳米簇的平均直径为1.63～3.57nm，优选为2.2～3.1nm，例如为2.66±0.45nm；
所述金纳米簇的表面优选带正电荷；和/或
所述金纳米簇的电位优选为19～21mV，优选为19.1～20.7mV，例如为19.9±0.8mV。
一般而言，siRNA是指一类RNA分子，从可形成siRNA前体的转录物加工而来。成熟的siRNA通常具有18～26个核苷酸(nt)，也不排除具有其他数目核苷酸的siRNA分子。siRNA通常可被Northern印迹检测到。
siRNA可以通过模拟miRNA产生机制来生成，这样的siRNA就可从前体RNA(Precursor RNA，Pre-RNA)加工而来。该前体RNA可折叠成一种稳定的茎环(发夹)结构，所述茎环结构的长度一般为50-100bp。所述前体RNA可折叠成稳定的茎环结构，茎环结构的茎部两侧包含基本上互补的两条序列。所述前体RNA可以是天然的或是人工合成的。
前体RNA可被剪切生成siRNA，所述siRNA可与编码基因的mRNA的至少一部分序列基本上互补。本文所使用的“基本上互补”是指核苷酸的序列是足够互补的，可以以一种可预见的方式发生相互作用，如形成二级结构(如茎环结构)。通常，两条“基本上互补”的核苷酸序列互相之间有70％的核苷酸是互补的，优选至少有80％的核苷酸是互补的，更优选至少有90％的核苷酸是互补的，进一步优选至少有95％的核苷酸是互补的，甚至可以为96％、97％、98％、99％、100％互补。
上述“茎环”结构也被称作“发夹”结构，是指一种核苷酸分子，其可形成一种包括双链区域(茎部)的二级结构，所述双链区域由该核苷酸分子的两个区域(位于同一分子上)形成，两个区域分列双链部分的两侧。其还包括至少一个“环”结构，包括非互补的核苷酸分子，即单链区域。即使该核苷酸分子的两个区域不是完全互补的，核苷酸的双链部分也可保持双链状态。例如，插入、缺失、取代等可导致一个小区域的不互补或该小区域自身形成茎环结构或其他形式的二级结构。然而，该两个区域仍可基本上互补，并在可预见的方式中发生相互作用，形成茎环结构的双链区域。茎环结构是本领域技术人员所熟知的，通常在获得了一条具有一级结构的核苷酸序列的核酸后，本领域技术人员能够确定该核酸是否形成茎环结构。
本发明的技术方案中述及的siRNA一般是指NGF siRNA，其通常用于抑制神经生长因子(NGF)的表达。本发明的siRNA还可包括siRNA变体和衍生物。此外，广义上的siRNA衍生物也可以包括siRNA变体。本领域技术人员可以使用通用的方法对NGF siRNA进行修饰，修饰方式包括但不限于：甲基化修饰、烃基修饰、糖基化修饰(如2-甲氧基-糖基修饰、烃基-糖基修饰、糖环修饰等)、核酸化修饰、肽段修饰、脂类修饰、卤素修饰、核酸修饰(如“TT”修饰)等。
根据本发明第一方面的NGF siRNA联合金纳米簇，其中，所述NGF  siRNA的序列为：
siRNA sense 5’-CCACAGACAUCAAGGGCAA dTdT-3’(SEQ ID NO:1)，siRNA antisense 3’-dTdT GGUGUCUGUAGUUCCCGUU-5’(SEQ ID NO:2)。
优选地，所述siRNA购自和/或合成自锐博生物科技有限公司(ribobio)。本发明涉及的siRNA序列确定自国家生物技术信息中心(National Center for Biotechnology Information)的NGF数据库(accession number NM_002506)。
本发明的第二方面提供了一种制备方法，其用于制备本发明第一方面所述的NGF siRNA联合金纳米簇，该制备方法包括以下步骤：
(1)向HAuCl4溶液加入还原剂，加热并恒温搅拌，得到金纳米簇溶液；
(2)向所述金纳米簇溶液加入NGF siRNA并常温搅拌，得到所述NGF siRNA联合金纳米簇。
所述还原剂优选为谷胱甘肽(GSH)和多肽分子。所述多肽分子优选为CRRRRRRRRR(CR9)。
根据本发明第二方面的制备方法，其中，在步骤(1)中：
HAuCl4与谷胱甘肽的摩尔比为1:0.5～3，优选为1:1.5，HAuCl4与CRRRRRRRRR的摩尔比为1:0.5～1.5，优选为1:0.75。
优选地，在室温下，优选在24～26℃下，更优选在25℃下，向HAuCl4溶液加入还原剂；和/或
优选地，加热恒温的温度为70℃。恒温搅拌的时间优选为12～36h，更优选为24h。
根据本发明第二方面的制备方法，其中，在步骤(2)中：
所述NGF siRNA与金纳米簇的质量比为0.5～100:1，优选为1.5～5:1，更优选为2～3.5:1，最优选为3:1；
所述常温为22～28℃，优选为24～26℃，例如为25℃；
所述搅拌为涡旋搅拌；和/或
所述搅拌的时间为15～90分钟，优选为15～45分钟，最优选为30分钟。
本发明的第三方面提供了一种药物组合物，所述药物组合物包含药学上可接受的载体，以及本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇。所述药物组合物的剂型优选为注射剂，更优选为静脉注射剂。
本发明的第四方面提供了一种试剂盒，所述试剂盒包括本发明第一方面 的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇。
本发明的第五方面提供了一种应用，该应用为本发明第一方面的NGF siRNA联合金纳米簇、按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇或本发明第三方面的药物组合物在制备用于预防和/或治疗癌症的药物中的应用。所述癌症优选为NGF过度表达而引发的癌症，例如可以为胰腺癌；和/或所述药物优选为注射剂，更优选为静脉注射剂。
本发明的第六方面提供了一种预防和/或治疗癌症的方法，该方法包括向需要的对象给予治疗有效量的本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇或本发明第三方面的药物组合物。
本发明还提供了一种优选的治疗方法，该方法包括以下步骤中的一个或多个：(1)确定疾病种类是否属于NGF过度表达而引发的癌症；(2)直接采用本发明的NGF siRNA联合金纳米簇或按照本发明的方法制备NGF siRNA联合金纳米簇；(3)向需要的对象给予治疗有效量的NGF siRNA联合金纳米簇；(4)检测对象体内的血清中NGF siRNA的含量；(5)检测靶细胞中NGF siRNA的含量；(6)检测靶细胞中NGF mRNA和/或NGF蛋白的表达水平；(7)检测疾病治疗效果。
本发明的第七方面提供了一种用于预防和/或治疗癌症的NGF siRNA联合金纳米簇，该NGF siRNA联合金纳米簇为本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇。
与现有技术相比，本发明的NGF siRNA联合金纳米簇(以下简称为GNC-siRNA)可以具有但不限于以下有益效果：
1、本发明的GNC-siRNA可提高siRNA的稳定性，延长体内循环时间。通过体外实验验证可知，GNC-siRNA在血清中的稳定性比游离siRNA的稳定性更高；而且，对比GNC-siRNA和游离siRNA在小鼠体内不同血液循环时间后的荧光强度，结果也表明GNC-siRNA在血清中的稳定性更强。
2、本发明的GNC-siRNA可改善胰腺肿瘤的靶向性，提高NGF的沉默效应。通过体外和体内实验可得知，GNC-siRNA在胰腺癌细胞和胰腺肿瘤中的摄取量均明显高于游离siRNA的摄取量，并且可显著降低胰腺癌细胞和 胰腺肿瘤组织中的NGF mRNA表达和蛋白表达。
3、本发明的GNC-siRNA可提高胰腺癌的抗肿瘤治疗效果。通过体内实验可得知，GNC-siRNA能显著抑制小鼠中胰腺癌肿瘤的生长、发展和转移。
4、本发明的GNC-siRNA的制备方法简单，反应条件十分温和(可以为常温，常压，水相，pH中性附近)，极大地保护了siRNA的活性。也避免了以往的化学修饰方法对siRNA活性可能造成的不良影响。
5、本发明的GNC-siRNA对NGF siRNA的递送效率高，可达到3000mg siRNA/g金纳米簇(siRNA/GNC的质量比约为3:1)，相当于226μmol siRNA/g金纳米簇。相对于已知的金纳米颗粒递送siRNA而言，金纳米簇递送siRNA的效率更高。
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。
附图的简要说明
图1为本发明的NGF siRNA联合金纳米簇的合成技术路线图；
图2为第一实施例的金纳米簇溶液的照片；
图3为第一实施例的金纳米簇的TEM图；
图4为第一实施例的金纳米簇的zeta电位；
图5为第一至第十实施例的NGF siRNA联合金纳米簇的zeta电位；
图6为第一实施例中的金纳米簇和NGF siRNA联合金纳米簇的表面XPS图谱；
图7为第一试验例中的聚丙烯酰胺凝胶电泳图；
图8为第二试验例中的胰腺癌细胞摄取量图；
图9为第三试验例中的PCR表征胰腺癌细胞中的NGF mRNA水平图表；
图10为第三试验例中的蛋白印迹法表征胰腺癌细胞中的NGF蛋白水平图；
图11为第四试验例中的用Cy-5荧光分子标记siRNA，游离siRNA和GNC-siRNA在小鼠血液中随时间的荧光强度的变化图；
图12为第五试验例中的用Cy-5荧光分子标记siRNA，游离siRNA和GNC-siRNA在小鼠体内的胰腺肿瘤组织中的聚集。其中白色圆圈表示皮下胰腺肿瘤。
图13为第五试验例中的用Cy-5荧光分子标记siRNA，游离siRNA和GNC-siRNA在胰腺肿瘤组织中聚集的体外图片；
图14为第六试验例中的原位胰腺肿瘤模型的构建、给药时间和小鼠牺 牲时间示意图；
图15为第六试验例中的给药过程过小鼠的体重变化图；
图16为第六试验例中的生物发光法标记的原位胰腺肿瘤的生长结果图；
图17为第六试验例中的原位胰腺肿瘤的发光强度的统计结果图；
图18为第六试验例中的实验结束后收集的原位胰腺肿瘤的体外照片；
图19为第六试验例中的原位胰腺肿瘤的称重统计结果图；
图20为第六试验例中的生物发光法标记的原位胰腺肿瘤在肠系膜的转移结果图；
图21为第六试验例中的肠系膜上转移的胰腺肿瘤的发光强度的统计结果图。
图22为第七试验例中的PCR表征胰腺肿瘤组织中的NGF mRNA水平图表；
图23为第七试验例中的蛋白印迹法表征胰腺肿瘤组织中的NGF蛋白水平图。
实施发明的最佳方式
下面通过具体的实施例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细具体地说明之用，而不应理解为用于以任何形式限制本发明。
本部分对本发明试验中所使用到的材料以及试验方法进行一般性的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。本领域技术人员清楚，在上下文中，如果未特别说明，本发明所用材料和操作方法是本领域公知的。
第一实施例
本实施例用于说明本发明的NGF siRNA联合金纳米簇(GNC-siRNA)及其制备方法。
通过以下步骤制备本发明的GNC-siRNA(技术路线如图1所示)：
(1)在25℃下向HAuCl4溶液加入还原剂GSH和CR9，其中HAuCl4为100mM，GSH为150mM，CR9为75mM，相当于HAuCl4与GSH的摩尔比为1:1.5，HAuCl4与CR9的摩尔比为1:0.75，然后加热至70℃并恒温搅拌24h，得到金纳米簇溶液。
金纳米簇溶液的颜色呈浅黄绿色(如图2所示。因专利公布的原因，图 2可能显示不正确的颜色，这不能限制本发明)。使用TEM观察金纳米簇的大小和表面形貌，TEM图如图3所示，其平均直径为2.66±0.45nm，并且分散性良好。使用zeta电位仪测得金纳米簇的表面带正电荷(如图4所示)，zeta电位为19.9±0.8mV。
(2)向所述金纳米簇溶液加入NGF siRNA，在25℃下涡旋搅拌30min，得到所述GNC-siRNA。其中，NGF siRNA与金纳米簇(金纳米簇的质量由ICP-MS测量)的质量比为3:1。如图1下图所示，通过静电吸附作用，将带有负电荷的NGF siRNA吸附在带正电荷的金纳米簇的表面。该GNC-siRNA的zeta电位见图5。
图6为XPS能谱仪测定的GNC(来自步骤(1))和GNC-siRNA(来自步骤(2))的表面XPS图谱。由该图谱可知，GNC没有P的峰，说明不含磷元素，GNC-siRNA中出现P的峰，说明GNC-siRNA含磷元素(P元素来自于核酸)，即GNC表面静电吸附有NGF siRNA。
第二至第十实施例
本实施例用于说明本发明的GNC-siRNA及其制备方法。
按照与第一实施例相同的方法制备第二至第十实施例的GNC-siRNA，区别仅在于，步骤(2)中的NGF siRNA与金纳米簇的质量比分别为0.5:1、1:1、1.5:1、2:1、2.5:1、3.5:1、4:1、5:1、100:1。
使用zeta电位仪测量上述GNC-siRNA的zeta电位，结果见图5。其中，将步骤(2)的金纳米簇溶液作为质量比0:1。从图5中可看出，当NGF siRNA/GNC质量比大于3时，溶液的zeta电位不变，表明siRNA达到饱和状态。因此，GNC-siRNA的siRNA负载量最高可达3000mg siRNA/g金纳米簇，相当于226μmol siRNA/g金纳米簇。
由此可知，在第一至第十实施例所得的GNC-siRNA中，NGF siRNA与金纳米簇的质量比分别为3:1、0.5:1、1:1、1.5:1、2:1、2.5:1、3:1、3:1、3:1、3:1。
第十一实施例
本实施例用于说明本发明的GNC-siRNA及其制备方法。
通过以下步骤制备本发明的GNC-siRNA(技术路线如图1所示)：
(1)在24℃下向HAuCl4溶液加入还原剂GSH和CR9，其中HAuCl4为100mM，GSH为50mM，CR9为50mM，相当于HAuCl4与GSH的摩尔 比为1:0.5，HAuCl4与CR9的摩尔比为1:0.5。然后加热至70℃并恒温搅拌12h，得到金纳米簇溶液。金纳米簇溶液的颜色呈浅黄绿色。使用TEM观察金纳米簇的大小和表面形貌，其平均直径为2.41nm，并且分散性良好。使用zeta电位仪测得金纳米簇的表面带正电荷，zeta电位为20.3mV。
(3)向所述金纳米簇溶液加入NGF siRNA，在24℃下涡旋搅拌15min，得到所述GNC-siRNA。其中，NGF siRNA与金纳米簇(金纳米簇的质量由ICP-MS测量)的质量比为3:1。
本实施例的GNC-siRNA的XPS图谱存在P峰，说明GNC表面静电吸附有NGF siRNA。本实施例所得的GNC-siRNA中，NGF siRNA与金纳米簇的质量比为3:1。
第十二实施例
本实施例用于说明本发明的GNC-siRNA及其制备方法。
通过以下步骤制备本发明的GNC-siRNA(技术路线如图1所示)：
(1)在26℃下向HAuCl4溶液加入还原剂GSH和CR9，其中HAuCl4为100mM，GSH为300mM，CR9为150mM，相当于HAuCl4与GSH的摩尔比为1:3，HAuCl4与CR9的摩尔比为1:1.5。然后加热至70℃并恒温搅拌36h，得到金纳米簇溶液。金纳米簇溶液的颜色呈浅黄绿色。使用TEM观察金纳米簇的大小和表面形貌，其平均直径为2.89nm，并且分散性良好。使用zeta电位仪测得金纳米簇的表面带正电荷，zeta电位为20.1mV。
(3)向所述金纳米簇溶液加入NGF siRNA，在26℃下涡旋搅拌45min，得到所述GNC-siRNA。其中，NGF siRNA与金纳米簇(金纳米簇的质量由ICP-MS测量)的质量比为3:1。
本实施例的GNC-siRNA的XPS图谱存在P峰，说明GNC表面静电吸附有NGF siRNA。本实施例所得的GNC-siRNA中，NGF siRNA与金纳米簇的质量比为3:1。
第十三实施例
本实施例用于说明本发明的GNC-siRNA及其制备方法。
通过以下步骤制备本发明的GNC-siRNA(技术路线如图1所示)：
(1)在25℃下向HAuCl4溶液加入还原剂GSH和CR9，其中HAuCl4为100mM，GSH为250mM，CR9为100mM，相当于HAuCl4与GSH的摩尔比为1:2.5，HAuCl4与CR9的摩尔比为1:1。然后加热至70℃并恒温搅拌 24h，得到金纳米簇溶液。金纳米簇溶液的颜色呈浅黄绿色。使用TEM观察金纳米簇的大小和表面形貌，其平均直径为2.75nm，并且分散性良好。使用zeta电位仪测得金纳米簇的表面带正电荷，zeta电位为19.6mV。
(3)向所述金纳米簇溶液加入NGF siRNA，在25℃下涡旋搅拌60min，得到所述GNC-siRNA。其中，NGF siRNA与金纳米簇(金纳米簇的质量由ICP-MS测量)的质量比为3:1。
本实施例的GNC-siRNA的XPS图谱存在P峰，说明GNC表面静电吸附有NGF siRNA。本实施例所得的GNC-siRNA中，NGF siRNA与金纳米簇的质量比为3:1。
第一试验例
本试验例用于验证不同siRNA在血清中的稳定性。
在含有10％血清(v/v)的培养基中培养游离NGF siRNA和第一实施例的GNC-siRNA，使siRNA的初始浓度均为100nM，孵育0min、15min、30min、45min和60min后，提取溶液各5μl，加入到聚丙烯酰胺凝胶电泳槽中，在160V电压下跑胶45min，并用GelRed对核酸进行染色和紫外显影，评价siRNA核酸的位置和分布。结果如图7所示，表明本发明的GNC-siRNA在血清中的稳定性比游离siRNA的稳定性更强。
第二试验例
本试验例用于验证不同siRNA在胰腺癌细胞中的摄取量。
用Cy5荧光分子(激发/发射＝649/670nm)标记siRNA，形成Cy5-siRNA。在培养皿中培养胰腺癌细胞Panc-1细胞至80％细胞融合度，在培养基中分别加入游离siRNA和第一实施例的GNC-siRNA，使siRNA的初始浓度均为100nM。培养1h后，清洗细胞，用4％多聚甲醛固定细胞，清洗细胞，然后利用共聚焦激光显微镜观察Panc-1癌细胞对Cy5-siRNA的摄取情况。结果如图8所示，表明本发明的GNC-siRNA在胰腺癌细胞中的摄取量明显高于游离siRNA的细胞摄取量。
第三试验例
本试验例用于验证不同siRNA在胰腺癌细胞中的NGF基因沉默效应。
在培养皿中培养胰腺癌细胞Panc-1细胞至70％～80％融合度，在培养基中分别加入游离siRNA和第一实施例的GNC-siRNA，使siRNA的初始浓度 均为100nM，并且以未处理组、GNC-nsRNA(结合nonsense siRNA的金纳米簇。其中siRNA的初始浓度相同均为100nM)组作为对照。培养48h后，清洗细胞。利用PCR表征细胞中NGF mRNA的表达水平，利用蛋白印迹表征细胞中NGF蛋白的表达水平。结果分别如图9和图10所示，表明相对于游离siRNA，本发明的GNC-siRNA显著降低了胰腺癌细胞中的NGF mRNA表达和NGF蛋白表达。
第四试验例
本试验例用于验证不同siRNA在小鼠体内的循环时间。
用Cy5荧光分子(激发/发射＝649/670nm)标记siRNA，得到Cy5-siRNA。小鼠尾静脉分别注射游离siRNA和第一实施例的GNC-siRNA，使siRNA的注射量相同，每只小鼠注射30μg Cy5-siRNA。尾静脉注射0h、1h、2h、3h、6h后，分别收集小鼠血液约150μl。之后将100μl血液转移到PCR小管中，采用CRI Maestro 2多光谱小动物活体成像系统分析游离siRNA和第一实施例的GNC-siRNA在血液中的荧光强度，采用激发和发射光谱为649nm和670nm。结果如图11所示，表明本发明的GNC-siRNA在血清中的稳定性增强，比游离siRNA的体内循环时间明显增加。
第五试验例
本试验例用于验证不同siRNA在小鼠体内的肿瘤靶向性。
用Cy5荧光分子(激发/发射＝649/670nm)标记siRNA。在荷载皮下肿瘤的Balb/c裸鼠中，采用尾静脉分别注射游离siRNA和第一实施例的GNC-siRNA，使siRNA的注射量相同，均为每只小鼠注射30μg Cy5-siRNA，以生理盐水注射组作为对照。尾静脉注射0h、3h、6h、24h后，采用CRI Maestro 2多光谱小动物活体成像系统分析游离siRNA和第一实施例的GNC-siRNA在小鼠体内分布，采用CRI Maestro 2成像系统的黄色滤光片，采集从650nm到800nm的荧光发射信号。结果如图12所示。
注射siRNA 24h之后，解剖小鼠并取出肿瘤组织，用上述成像系统进行体外成像，观察游离siRNA和GNC-siRNA在肿瘤部位的荧光分布。结果如图13所示。
以上结果均表明，本发明的GNC-siRNA在胰腺肿瘤中的摄取量明显高于游离siRNA的摄取量。
第六试验例
本试验例用于验证不同siRNA在小鼠中的抗肿瘤效果。
在Balb/c裸鼠中，在胰腺的胰头部位种植1×106个标记有荧光素酶的胰腺癌细胞(Panc-1-luc)，构建原位胰腺癌肿瘤模型。大约2周后，形成原位的胰腺肿瘤。对小鼠进行尾静脉注射游离siRNA和第一实施例的GNC-siRNA，保持注射的siRNA量均为每只小鼠30μg siRNA，并且以生理盐水注射组、GNC注射组(GNC注射量相同)、GNC-nsRNA注射组(GNC和siRNA的注射量相同)作为对照，每两天注射一次，一共注射7次。采用小动物体内生物发光成像法(原理：荧光素酶与荧光素底物的特异性反应并发出光子)，观察小鼠中原位胰腺癌的生长，观测到的光子强度与肿瘤的生长大小成正比。28天后，牺牲并解剖小鼠，取出胰腺及周围的肠系膜，用生物发光成像法观察肿瘤在肠系膜的转移。之后取出胰腺中的原位肿瘤，并对肿瘤组织进行称重。
原位胰腺肿瘤模型的构建、给药时间和小鼠牺牲时间如图14所示。
图15为实验过程中小鼠的体重变化，结果显示给药过程中小鼠的体重变化不明显，表明不同的给药方式对小鼠没有造成明显的毒性。
图16为生物发光法标记的原位胰腺肿瘤的生长结果。图17为原位胰腺肿瘤的发光强度的统计结果。图18为实验结束后收集的原位胰腺肿瘤的体外照片。图19为原位胰腺肿瘤的称重统计结果。上述结果均表明本发明的GNC-siRNA能显著抑制小鼠原位胰腺肿瘤的生长。
图20为用生物发光法标记的原位胰腺肿瘤在肠系膜的转移结果。图21为肠系膜上转移的胰腺肿瘤的发光强度的统计结果。上述结果表明本发明的GNC-siRNA能显著抑制胰腺肿瘤的转移。
第七试验例
本试验例用于验证不同siRNA在小鼠体内的胰腺肿瘤中的NGF基因沉默效应。
取第六试验例中的肿瘤组织，利用PCR表征细胞中NGF mRNA的表达水平，利用蛋白印迹表征细胞中NGF蛋白的表达水平。结果分别如图22和图23所示，表明相对于游离siRNA，本发明的GNC-siRNA显著降低了胰腺肿瘤组织中的NGF mRNA表达和NGF蛋白表达。
试剂盒
本发明还提供了一种试剂盒，所述试剂盒包括本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇。
该试剂盒中还可以包括用于PCR扩增、载体构建等所需的各种试剂，包括但不限于扩增缓冲液、引物、模板DNA、酶等。
此外，所述试剂盒中还可包括使用说明书和/或使用/分析软件。
药物组合物
本发明还提供了一种药物组合物，所述药物组合物包含药学上可接受的载体，以及本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇。所述NGF siRNA联合金纳米簇在该药物组合物中可以为有效量的或治疗有效量的。
如本文所用，“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，“药学上可接受的”的成分是适用于人和/或动物(如哺乳动物和禽类)而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。“药学上可接受的载体”是指用于给药的载体，可以包括各种赋形剂和稀释剂等。
本发明的药物组合物可以含有安全有效量的本发明的NGF siRNA联合金纳米簇作为活性成分以及药学上可接受的载体。这类载体可以包括但不限于：生理盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物的剂型可以依需要制备为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、稀释剂等。例如用生理盐水或含有葡萄糖和其他辅料的水溶液通常以常规方法进行制备。该药物组合物更适宜在无菌条件下制造。根据上述实验结果，由于本发明的NGF siRNA联合金纳米簇在血清中的稳定性较好，因而适合制备为注射剂，尤其是静脉注射剂。
本发明所述的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数，例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，在绝大部分情况下，当本发明的活性成分每天可以以约0.01mg-50mg/kg动物体重(较 佳的1mg-5mg/kg动物体重)的剂量给予，能得到令人满意的效果。更为具体的，在一种实施方案中，剂量为：30μg/小鼠(～20g)，即～1.5mg/kg；一般，尾静脉注射siRNA的范围在1μg-400μg/小鼠之间，即给药量是0.05-20mg/kg，如此类推。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明所述的药学上可接受的载体包括但不限于：水、生理盐水、脂质体、脂质、蛋白、蛋白-抗体缀合物、肽类物质、纤维素、纳米凝胶、或其组合。载体的选择通常应与给药方式相匹配，这是本领域的普通技术人员所熟知的。
本发明还提供了所述药物组合物的应用，其用于制备预防和/或治疗癌症的药物。优选地，所述癌症为NGF过度表达而引发的癌症，例如可以为胰腺癌。
预防和/或治疗方法
本发明还提供了一种预防和/或治疗癌症的方法，该方法包括向需要的对象给予治疗有效量的本发明第一方面的NGF siRNA联合金纳米簇或按照本发明第二方面的方法而制备的NGF siRNA联合金纳米簇或本发明第三方面的药物组合物。
本发明的所述NGF siRNA联合金纳米簇或药物组合物可以通过胃肠道、鼻腔、气管、肺、非病灶部位的静脉或表皮、皮内、皮下、心内、肌肉、骨髓、腹腔、硬膜外、口腔、舌下、眼部、直肠、阴道、尿道、耳道等途径给药。优选施用方式或给药方式包括：口服、呼吸道、注射、透皮、粘膜、或腔道给药。
其中，所述口服给药方式包括吞服、含化等。所述呼吸道给药方式包括吸入方式，例如超声雾化吸入、氧气雾化吸入、手压式雾化吸入等。所述注射给药方式包括动脉注射、静脉注射、肌肉注射、心内注射、皮内注射等。所述透皮给药或经皮给药方式，包括离子导入法、电致孔透皮法等。所述粘膜给药方式包括鼻粘膜给药、口腔粘膜给药、眼粘膜给药、直肠粘膜给药、子宫给药以及阴道粘膜给药等。所述腔道给药方式包括直肠给药、阴道给药、尿道给药、鼻腔给药、耳道给药等。根据本发明的上述试验结果可知，NGF siRNA联合金纳米簇在血清中的稳定性较好，因而尤其适合通过动脉注射或静脉注射给药。
在本发明提及的所有文献(包括专利文献或非专利文献)都在本发明中引用作为参考，就如同每一篇文献被单独引用作为参考那样。尽管本发明已进行了一定程度的描述，明显地，在不脱离本发明的精神和范围的条件下，可进行各个条件的适当变化。可以理解，本发明不限于所述实施方案，而归于权利要求的范围，其包括所述每个因素的等同替换。
Figure PCTCN2016087364-appb-000001
Figure PCTCN2016087364-appb-000002

Claims (10)

  1. 一种NGF siRNA联合金纳米簇，其特征在于，所述NGF siRNA联合金纳米簇包含金纳米簇和NGF siRNA，并且所述金纳米簇与所述NGF siRNA之间以静电吸附作用相结合；
    优选地，NGF siRNA与金纳米簇的质量比为1.5～3.5:1，优选为2～3:1，最优选为3:1。
  2. 根据权利要求1所述的NGF siRNA联合金纳米簇，其特征在于：
    所述金纳米簇的平均直径为1.63～3.57nm，优选为2.2～3.1nm；
    所述金纳米簇的表面带正电荷；
    所述金纳米簇的电位为19～21mV，优选为19.1～20.7mV；和/或
    所述NGF siRNA的序列为：
    siRNA sense 5’-CCACAGACAUCAAGGGCAA dTdT-3’，
    siRNA antisense 3’-dTdT GGUGUCUGUAGUUCCCGUU-5’。
  3. 权利要求1或2所述的NGF siRNA联合金纳米簇的制备方法，其特征在于，该方法包括以下步骤：
    (1)向HAuCl4溶液加入还原剂，加热并恒温搅拌，得到金纳米簇溶液；
    (2)向所述金纳米簇溶液加入NGF siRNA并常温搅拌，得到所述NGFsiRNA联合金纳米簇；
    所述还原剂优选为谷胱甘肽和多肽分子，所述多肽分子优选为CRRRRRRRRR。
  4. 根据权利要求3所述的制备方法，其特征在于，在步骤(1)中：
    HAuCl4与谷胱甘肽的摩尔比为1:0.5～3，优选为1:1.5，HAuCl4与CRRRRRRRRR的摩尔比为1:0.5～1.5，优选为1:0.75；
    优选地，在室温下，优选在24～26℃下，更优选在25℃下，向HAuCl4溶液加入还原剂；和/或
    优选地，加热恒温的温度为70℃；恒温搅拌的时间优选为12～36h，更优选为24h。
  5. 根据权利要求3或4所述的制备方法，其特征在于，在步骤(2)中：
    所述NGF siRNA与金纳米簇的质量比为0.5～100:1，优选为1.5～5:1，更优选为2～3.5:1，最优选为3:1；
    所述常温为22～28℃，优选为24～26℃；
    所述搅拌为涡旋搅拌；和/或
    所述搅拌的时间为15～90分钟，优选为15～45分钟，最优选为30分钟。
  6. 一种药物组合物，其特征在于，所述药物组合物包含药学上可接受的载体，以及权利要求1或2所述的NGF siRNA联合金纳米簇或按照权利要求3至5中任一项所述的方法制备的NGF siRNA联合金纳米簇；
    所述药物组合物的剂型优选为注射剂，更优选为静脉注射剂。
  7. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1或2所述的NGF siRNA联合金纳米簇或按照权利要求3至5中任一项所述的方法制备的NGF siRNA联合金纳米簇。
  8. 权利要求1或2所述的NGF siRNA联合金纳米簇、按照权利要求3至5中任一项所述的方法制备的NGF siRNA联合金纳米簇或权利要求6所述的药物组合物在制备用于预防和/或治疗癌症的药物中的应用；
    所述癌症优选为胰腺癌；和/或
    所述药物优选为注射剂，更优选为静脉注射剂。
  9. 一种预防和/或治疗癌症的方法，所述方法包括向需要的对象给予治疗有效量的如权利要求1或2所述的NGF siRNA联合金纳米簇、按照权利要求3至5中任一项所述的方法制备的NGF siRNA联合金纳米簇或权利要求6所述的药物组合物；
    所述癌症优选为胰腺癌；和/或
    所述给予方式优选为注射，更优选为静脉注射。
  10. 一种用于预防和/或治疗癌症的NGF siRNA联合金纳米簇，所述NGFsiRNA联合金纳米簇为权利要求1或2所述的NGF siRNA联合金纳米簇或按照权利要求3至5中任一项所述的方法制备的NGF siRNA联合金纳米簇。
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