WO2023082243A1 - 一种菜籽肽及其在制备药物纳米载体方面的应用 - Google Patents

一种菜籽肽及其在制备药物纳米载体方面的应用 Download PDF

Info

Publication number
WO2023082243A1
WO2023082243A1 PCT/CN2021/130617 CN2021130617W WO2023082243A1 WO 2023082243 A1 WO2023082243 A1 WO 2023082243A1 CN 2021130617 W CN2021130617 W CN 2021130617W WO 2023082243 A1 WO2023082243 A1 WO 2023082243A1
Authority
WO
WIPO (PCT)
Prior art keywords
hcpt
drug
mixed solution
rapeseed peptide
tumor
Prior art date
Application number
PCT/CN2021/130617
Other languages
English (en)
French (fr)
Inventor
何荣
王志高
鞠兴荣
袁建
邹智鹏
黄家强
王明洁
Original Assignee
南京财经大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京财经大学 filed Critical 南京财经大学
Publication of WO2023082243A1 publication Critical patent/WO2023082243A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • 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
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of biomedical materials, in particular to a rapeseed peptide and its application in the preparation of drug nanocarriers.
  • nanomaterial drug delivery system has gradually become a new field of nanomedicine.
  • Some nanomaterials such as micelles, liposomes, hydrogels, magnetic particles and other nanomaterials have been applied in succession.
  • hydrophilic traditional hydrogels lack ligands that interact with hydrophobic drugs, and their ability to load and control drug release is limited. Poor sex and other shortcomings.
  • amphiphilic polypeptide As a new type of carrier material, amphiphilic polypeptide has a small molecular weight, can be degraded and absorbed by the human body, has good biocompatibility and excellent self-assembly performance, and at the same time avoids the disadvantages of high molecular materials that are difficult to degrade and have large toxic and side effects , has become one of the hotspots in the research of self-assembled materials in the world.
  • amphiphilic polypeptides that have good drug loading effect and can significantly reduce the IC 50 of the drug.
  • the purpose of the present invention is to provide a kind of rapeseed peptide, which is an excellent nano-carrier of anti-tumor drugs, has a high drug embedding rate, significantly reduces the IC 50 of the drug, and reduces the dosage of the drug.
  • Another object of the present invention is to provide the application of the above-mentioned rapeseed peptide in the preparation of drug nanocarriers.
  • the present invention also provides a method for preparing an antitumor drug using the polypeptide as a carrier.
  • the method is simple, and the prepared antitumor drug has good pH and dual responsiveness of CathB, and the drug loading effect is good.
  • the release rate of nanocarriers in the environment is higher than that in the physiological environment, and the release rate is moderate and the stability is good.
  • the present invention provides the following solutions:
  • a rapeseed peptide the sequence of which is shown in SEQ ID NO:1.
  • the invention also provides the application of the rapeseed peptide in the preparation of drug nanocarriers.
  • the drug is hydroxycamptothecin.
  • the mass ratio of rapeseed peptide to hydroxycamptothecin is 5-15:1.
  • the present invention also provides a method for preparing an antitumor drug using the polypeptide as a carrier, comprising the following steps:
  • the mass ratio of rapeseed peptide to hydroxycamptothecin is 5-15:1.
  • the mass concentration of rapeseed peptide in the mixed solution A is 5-15 mg/mL.
  • the volume ratio of the phosphate buffer solution to the mixed solution A is 1:8-15, and the mass percentage of Tween 80 in the mixed solution B is 0.5-1.5%.
  • the present invention after removing the chloroform in the mixed solution B in step (3), it is filtered to obtain the anti-tumor nano-medicine.
  • the filter membrane used in the filtration has a pore size of 0.4-0.8 ⁇ m.
  • the rapeseed peptide provided by the invention is an excellent nano-carrier of anti-tumor drugs, has a high drug embedding rate, significantly reduces the IC 50 of the drug, and reduces the dosage of the drug.
  • the antitumor drug prepared by adopting the rapeseed peptide SVIRPPL of the present invention has good dual responsiveness of pH and CathB, high drug carrier specificity, good drug loading effect, and the release rate of nanocarriers in lysosomes and tumor microenvironments is higher than that in Under physiological environmental conditions, the drug release rate is moderate and the stability is good.
  • the invention creatively uses the amphiphilic polypeptide rapeseed peptide SVIRPPL as a carrier material, avoids the aggregation of protein nanocarriers in the liver, improves the tumor penetration of nanocarriers, reduces biological recognition in vivo, and improves the efficiency of targeted transport.
  • Figure 1 shows the dynamic light scattering particle size of anti-tumor nano-drugs prepared by rapeseed peptide C stored at 4°C for different periods of time.
  • the ordinate is the average particle size, and the abscissa is time, in days.
  • Figure 2 shows the PDI changes of antitumor drugs prepared using rapeseed peptide C stored at 4°C for different periods of time, the abscissa is time, and the unit is day.
  • Fig. 3 is a transmission electron microscope (TEM) image of rapeseed peptide C blank nanocarrier (A) and antitumor drug (B) without HCPT.
  • TEM transmission electron microscope
  • Figure 4 Effects of different HCPT embedding concentrations of anti-tumor drugs and different concentrations of HCPT aqueous solution on the survival rate of HepG2 tumor cells, where HCPT represents HCPT aqueous solution, SVIRPPL-HCPT/NP represents anti-tumor drugs, and the abscissa is the concentration of HCPT aqueous solution or anti-tumor drug. Embedding concentration of HCPT in tumor drugs. There are significant differences between different letters.
  • Figure 5 Effects of different HCPT embedding concentrations of anti-tumor drugs and different concentrations of HCPT aqueous solution on the survival rate of MKN-28 tumor cells, where HCPT represents HCPT aqueous solution, SVIRPPL-HCPT/NP represents anti-tumor drugs, and the abscissa is the concentration of HCPT aqueous solution Or the embedding concentration of HCPT in antitumor drugs, and the ordinate is the survival rate of MKN-28 tumor cells. There are significant differences between different letters.
  • Figure 6 Effects of anti-tumor drugs with different HCPT embedding concentrations and different concentrations of HCPT aqueous solution on the survival rate of A549 tumor cells, where HCPT represents HCPT aqueous solution, SVIRPPL-HCPT/NP represents anti-tumor drugs, and the abscissa is the concentration of HCPT aqueous solution or anti-tumor drug.
  • concentration of HCPT embedded in the tumor drug, the vertical axis is the survival rate of A549 tumor cells. There are significant differences between different letters.
  • Figure 7 Effects of antitumor drugs with different HCPT embedding concentrations and different concentrations of HCPT aqueous solutions on the survival rate of MCF-7 tumor cells, where HCPT represents HCPT aqueous solutions, SVIRPPL-HCPT/NP represents antitumor drugs, and the abscissa is the concentration of HCPT aqueous solutions Or the embedding concentration of HCPT in antitumor drugs, and the vertical axis is the survival rate of MCF-7 tumor cells. There are significant differences between different letters.
  • SVIRPPLNP is the SVIRPPLNP group
  • HCPT is the HCPT aqueous solution group
  • SVIRPPL-HCPT is the SVIRPPL-HCPT/NP group.
  • Fig. 10 Bioluminescence imaging of mice in each group before the first injection of D-luciferin (denoted as day 0), and on the 7th and 15th day after the first injection of D-luciferin.
  • Saline is the normal saline group
  • SVIRPPLNP is the SVIRPPLNP group
  • HCPT is the HCPT aqueous solution group
  • SVIRPPL-HCPT NP is the SVIRPPL-HCPT/NP group.
  • Fig. 11 Fluorescent quantitative analysis of liver tumors in each group of mice before the first injection of D-luciferin (denoted as day 0), on the 7th day, the 15th day and the 19th day after the first D-luciferin injection.
  • Saline is the normal saline group
  • SVIRPPLNP is the SVIRPPLNP group
  • HCPT is the HCPT aqueous solution group
  • SVIRPPL-HCPT NP is the SVIRPPL-HCPT/NP group.
  • Figure 12 shows the distribution of antitumor drugs in the whole body and isolated organs at different times after Cy5.5-labeled SVIRPPL-HCPT/NP injection.
  • Rapeseed peptide C was prepared by Jiepeptide Company according to the conventional solid-state synthesis method, and used for the experiments in the present invention.
  • the antitumor drug is prepared by adopting rapeseed peptide C, comprising the following steps:
  • step (2) The mixed solution A obtained in step (1) was added dropwise at a speed of 8mL/h to 10mL of PBS phosphate buffer solution (purchased from Suleibao Company) with a concentration of 0.01M and pH of 7.4, and then Add Tween 80 and stir for 8 hours at 4°C and 600r/min to obtain a uniform mixed solution B.
  • the volume percent concentration of Tween 80 in the mixed solution B is 1%.
  • the PBS phosphate buffer solution was magnetically stirred at 600 r/min.
  • step (3) After ultrasonically dispersing the mixed liquid B obtained in step (2) at 4°C for 1 min, stir at 25°C with a rotation speed of 100 r/min to remove chloroform by volatilization. After the chloroform was removed, the antitumor drug (abbreviated as SVIRPPL-HCPT/NP) was prepared by filtering through a membrane with a pore size of 0.45 ⁇ m. The power of ultrasound is 300KW.
  • Rapeseed peptide C blank nanocarriers without HCPT (abbreviated as SVIRPPLNP) were prepared according to the preparation method of antitumor drugs, except that 0.1 mL of water containing 1 mg of HCPT was replaced by 0.1 mL of water.
  • This example is used to illustrate the characterization of the antitumor drug prepared in Example 1.
  • Malvern Zetasizer Nano ZS instrument He-He as laser: 633nm; scattering angle: 173° was used to detect the dynamic light scattering particle size and polydispersity index (PDI) of the antitumor drug prepared in Example 1 stored at 4°C.
  • PDI polydispersity index
  • the assay method of embedding rate is as follows: take 1mL of the medicine after step (3) volatilizes and removes the chloroform in Example 1, and take the precipitate after centrifugation to obtain unembedded HCPT, add 1mL of chloroform solution to the precipitate , prepared into HCPT solution, using a spectrophotometer to measure the concentration of HCPT at 367nm, and calculating the content of unembedded HCPT in the antitumor drug to calculate the embedding rate of HCPT.
  • the formula for calculating the embedding rate is as follows:
  • Example 1 It is found through experimental testing that the antitumor drug prepared in Example 1 is a transparent solution. When it is just prepared, the average particle diameter is 178nm, and the average PDI value is 0.26; After being stored for 30 days, the average particle size was 195nm, and the PDI was less than 0.3, indicating that the antitumor drug can maintain good colloidal stability. Through detection, it was found that the embedding rate of the antitumor drug to HCPT was 78.5%.
  • This example is used to illustrate the antitumor activity of the antitumor drug prepared in Example 1 on four kinds of tumor cells.
  • an antineoplastic drug with an HCPT embedding concentration of 7.8 ⁇ M was prepared, and then diluted with 0.01M, pH7.4 PBS phosphate buffer to an HCPT embedding concentration
  • Antitumor drugs with six different concentrations of 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, and 1 ⁇ M were used as samples.
  • rapeseed peptide C blank nanocarrier without HCPT Prepare the rapeseed peptide C blank nanocarrier without HCPT according to the method of Example 1, the only difference is that 0.1 mL of water is used to replace 0.1 mL of an aqueous solution containing 1 mg of HCPT (hydroxycamptothecin), that is, no HCPT is added; then, Dilute with 0.01M, pH7.4 PBS buffer solution to form rapeseed peptide C blank nanocarriers corresponding to anti-tumor drugs with HCPT embedding concentrations of 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M and 1 ⁇ M, as samples .
  • HCPT was dissolved in PBS phosphate buffer solution with a concentration of 0.01M and pH7.4, and HCPT aqueous solutions with different concentrations of 0.01 ⁇ M, 0.05 ⁇ M, 0.1 ⁇ M, 0.25 ⁇ M, 0.5 ⁇ M, and 1 ⁇ M were prepared as samples.
  • Liver cancer cell HepG2 gastric cancer cell MKN-28, lung cancer cell A549 and breast cancer cell MCF- 7 (purchased from Jiangsu Shenji Biological Co., Ltd.) were inoculated into a 96 In the well plate, some wells that were not inoculated were left as blank wells, so a total of four 96-well plates were inoculated and incubated overnight at 37°C.
  • the 96-well plates inoculated with various tumor cells were cultured, and sample wells (including antitumor drugs of various embedding concentrations, blank nanocarriers of rapeseed peptide C, and sample wells of HCPT aqueous solutions of various concentrations), control wells and blank wells were set up. .
  • IC 50 of antineoplastic drugs on liver cancer cell HepG2 gastric cancer cell MKN-28, lung cancer cell A549 and breast cancer cell MCF-7 were 0.17, 0.18, 0.25 and 0.27 ⁇ M respectively.
  • IC 50 values of rapeseed peptide C blank nanocarriers against liver cancer cell HepG2, gastric cancer cell MKN-28, lung cancer cell A549 and breast cancer cell MCF-7 were 0.57, 0.81, 0.97 and 1.10 mM, respectively.
  • rapeseed peptide C as a nanocarrier can effectively reduce the IC 50 value of HCPT on tumor cells, effectively improve the inhibitory effect of antitumor drugs on tumor cells, and reduce the biological toxicity caused by high HCPT concentration.
  • This example is used to illustrate the release curve of HCPT in the antitumor drug prepared in Example 1 at different pHs and with or without Cath B.
  • Example 1 The in vitro release characteristics of the antitumor drug prepared in Example 1 were studied by standard dialysis method. Put 5 mL of the antineoplastic drug prepared in Example 1 into a dialysis bag (MWCO: 500 Da), then place the dialysis bag in solutions 1, 2, 3, and 4 (Table 1), and dialyze with stirring at 100 rpm/min at 37°C When the solution outside the bag was investigated at different pHs and with or without Cath B (cathepsin B), the release curve of HCPT in the antitumor drug prepared in Example 1.
  • the original solution refers to the solution in the initial state outside the dialysis bag, that is, solutions in Table 1).
  • Example 1 According to the preparation method of Example 1, it can be seen that the concentration of HCPT in the antitumor drug prepared in Example 1 is 100 ⁇ g/mL, and according to the embedding rate (78.5%) calculated in Example 2, it can be calculated that there is 0.4 mg of HCPT.
  • the antitumor drug prepared in Example 1 showed a pH-dependent release characteristic, and the release rate of the antitumor drug under lysosome (pH 5.0 and containing CathB) and tumor microenvironment (pH 6.5) was higher than It is higher in the physiological environment (pH 7.4), and the antitumor drug is released slowly in pH 7.4, showing good in vitro stability.
  • the release of the HCPT aqueous solution in the dialysis bag in each solution has no pH dependence, and the release rate reaches 70% within 5 hours through the dialysis bag rapidly.
  • antineoplastic drugs When antineoplastic drugs are added to CathB at pH 5.0, the release of HCPT from antineoplastic drugs will be further accelerated, and the release efficiency of antineoplastic drugs in CathB for 48h is twice that of its release efficiency at pH 7.4, indicating that acidic conditions It will destroy the combination of rapeseed peptide in anti-tumor drugs and HCPT and other drugs, resulting in the rapid release of drugs.
  • antitumor drugs Through the acid-dependent properties of protease CathB, especially in very acidic lysosomes and weakly acidic tumor microenvironment, antitumor drugs will achieve accelerated drug release through the response of pH and CathB.
  • mice Male NOD SCID mice (6 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised under pathogen-free conditions. Open the abdomen below the left costal margin of the mouse to expose the left lobe of the liver, and inject 30 ⁇ L of a suspension containing 1 ⁇ 10 6 HepG2 cells to construct orthotopic liver cancer. Three weeks later, when the tumor volume reached 100 mm 3 , the treatment experiment was performed.
  • mice In order to evaluate the in vivo therapeutic effect of SVIRPPL-HCPT/NP, tumor-bearing mice were randomly divided into four groups: normal saline group, HCPT aqueous solution group, and SVIRPPLNP (rapeseed peptide C blank nanocarrier without HCPT, prepared in Example 1) group and SVIRPPL-HCPT/NP (prepared in Example 1) group. Mice in each group were administered via tail vein injection, administered once every three days, and administered 4 times during the treatment period.
  • Each administration method is as follows: the HCPT aqueous solution group is administered according to the HCPT dose of 5.0 mg/kg with the HCPT aqueous solution; kg for administration; the normal saline group was given the same volume of normal saline; the SVIRPPLNP group was given the same volume of rapeseed peptide C blank nanocarriers without HCPT (prepared in Example 1).
  • tumor tissue was taken for slice analysis.
  • the removed tumor tissue was fixed in 10% neutral formalin, dehydrated with graded ethanol solution, and embedded in paraffin. Subsequently, the sections were sectioned with a tissue microtome, stained with hematoxylin-eosin (H&E), and mounted with resin. Finally, the pathological changes of tumor tissue were observed by optical microscope.
  • the normal saline group was moderately differentiated hepatocellular carcinoma, the cancer cells were flaky, diffusely arranged, and deeply stained with nuclei and cytoplasm; the SVIRPPL NP group and the HCPT aqueous solution group were mildly differentiated hepatocellular carcinoma, and there were clusters of cancer cells and normal adjacent tumors.
  • the nucleus of the cancer cells was deeply stained; in the SVIRPPL-HCPT/NP group, only a small amount of cancer cells were distributed, and there was a certain degree of inflammatory infiltration.
  • mice in each group were injected with D-luciferin at a dose of 150 mg/kg every seven days for a total of 3 injections. Meanwhile, the growth of liver cancer tumors in mice was observed by bioluminescent imaging. Bioluminescence imaging before the first injection of D-luciferin (denoted as 0 days), 7 days and 15 days after the first injection of D-luciferin (Figure 10) showed that SVIRPPL-HCPT/NP can effectively inhibit the growth of liver cancer tumors , the bioluminescent intensity was significantly lower than that of the saline-treated group.
  • mice Male NOD SCID mice (6 weeks old) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised under pathogen-free conditions. Open the abdomen below the left costal margin of the mouse to expose the left lobe of the liver, and inject 30 ⁇ L of a suspension containing 1 ⁇ 10 6 HepG2 cells to construct orthotopic liver cancer. When the tumor volume reached 200mm 3 , in vivo fluorescence imaging experiments and biodistribution experiments were started.
  • SVIRPPL-HCPT/NP the antitumor drug prepared in Example 1
  • IVIS LuminaXRIII the near-infrared imaging system
  • the Cy5.5-labeled SVIRPPL-HCPT/NP was injected into the tumor-bearing mice at the tail vein at a dose of 5 mg/kg body weight of HCPT.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Optics & Photonics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Manufacturing & Machinery (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Inorganic Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供了一种菜籽肽及其在制备药物纳米载体方面的应用，涉及生物医药材料技术领域。该菜籽肽序列如SEQ ID NO:1所示，可以应用于制备药物纳米载体。还提供了以所述多肽为载体制备抗肿瘤药物的方法，包括以下步骤：将所述菜籽肽和抗肿瘤药物依次溶解在三氯甲烷中，得到混合液A。将所述混合液A逐滴加入到磷酸缓冲液中，然后再加入吐温80，搅拌均匀，得到混合液B。除去混合液B中三氯甲烷，制得抗肿瘤药物。所述菜籽肽对药物包埋率高，显著降低了药物的IC 50，减少药物使用剂量。

Description

一种菜籽肽及其在制备药物纳米载体方面的应用 技术领域
本发明涉及生物医药材料技术领域，尤其是涉及一种菜籽肽及其在制备药物纳米载体方面的应用。
背景技术
近年来，药物治疗仍是很多疾病治疗的主要手段。但由于许多药物存在选择性差、毒副作用大、药物释放不可控等问题，因此导致治疗效果不理想。为提高药物疗效，往往要增加药物浓度和给药次数，但过多的药物会损害正常的组织和器官，导致长期治疗效果欠佳。
随着纳米医学技术和高分子材料的发展，纳米材料传输药物体系逐渐成为一个新的纳米医学领域，一些纳米材料如胶束、脂质体、水凝胶、磁性颗粒等纳米材料相继被应用于药物传输领域，但亲水性的传统水凝胶缺乏与疏水性药物相互作用的配体，它对药物的装载与控释能力均有限，同时高分子材料还存在着毒副作用大、生物相容性差等缺点。两亲性多肽作为一种新型载体材料，分子量小，可被人体降解吸收，具有良好的生物相容性和优良的自组装性能，同时避开了高分子材料的难降解、毒副作用大的缺点，成为国际上的自组装材料方面研究的热点之一。但是，现有技术中缺乏载药效果好、能够显著降低药物IC 50的两亲性多肽。
发明内容
本发明的目的是提供一种菜籽肽，是优良的抗肿瘤药物的纳米载体，对药物包埋率高，显著降低了药物的IC 50，减少药物使用剂量。
本发明的另一目的是提供上述菜籽肽在制备药物纳米载体方面的应用。
本发明还提供以所述多肽为载体制备抗肿瘤药物的方法，该方法简单，制备得到的抗肿瘤药物具有良好的pH和CathB的双重响应性，载药效果好、在溶酶体及肿瘤微环境下纳米载体的释放速率比在生理环境条件下更高，释药速度适中且稳定性好。
为实现这一目的，本发明提供如下方案：
一种菜籽肽，序列如SEQ ID NO:1所示。
本发明还提供所述菜籽肽在制备药物纳米载体方面的应用。
在本发明中，所述药物为羟基喜树碱。
在本发明中，菜籽肽与羟基喜树碱的质量比为5-15:1。
本发明还提供以所述多肽为载体制备抗肿瘤药物的方法，包括以下步骤：
(1)将所述菜籽肽和抗肿瘤药物依次溶解在三氯甲烷中，得到混合液A；
(2)将所述混合液A逐滴加入到磷酸缓冲液中，然后再加入吐温80，搅拌均匀，得到混合液B。
(3)除去混合液B中三氯甲烷，制得抗肿瘤药物。
在本发明中，所述菜籽肽和羟基喜树碱的质量比为5-15:1。
在本发明中，所述混合液A中菜籽肽的质量浓度为5-15mg/mL。
在本发明中，所述磷酸缓冲液与混合液A的体积比为1:8-15，混合液B中吐温80的质量百分含量为0.5-1.5％。
在本发明中，步骤(3)中除去混合液B中的三氯甲烷后，过滤，得到抗肿瘤纳米药物。
在本发明中，过滤中使用的过滤膜的孔径为0.4-0.8μm。
本发明提供的菜籽肽，是优良的抗肿瘤药物的纳米载体，对药物包埋率高，显著降低了药物的IC 50，减少了药物的使用剂量。采用本发明菜籽肽SVIRPPL制备的抗肿瘤药物具有良好的pH和CathB的双重响应性，药物载体特异性高、载药效果好、在溶酶体及肿瘤微环境下纳米载体的释放速率比在生理环境条件下更高，释药速度适中且稳定性好。本发明创造性的将两亲多肽菜籽肽SVIRPPL作为载体材料，避免了蛋白质纳米载体在肝脏的聚集，提高纳米载体的肿瘤渗透力，减少体内生物识别，进而提高靶向运输效率。
附图说明
图1显示了采用菜籽肽C制备的抗肿瘤纳米药物在4℃储藏不同时间的动态光散射粒径，纵坐标是平均粒径，横坐标是时间，单位是天。
图2显示了采用菜籽肽C制备的抗肿瘤药物在4℃储藏不同时间的PDI变化，横坐标是时间，单位是天。
图3是无HCPT的菜籽肽C空白纳米载体(A)与抗肿瘤药物(B)的透射电镜(TEM)图。
图4不同HCPT包埋浓度的抗肿瘤药物和不同浓度HCPT水溶液对HepG2肿瘤细胞存活率的影响，其中HCPT表示HCPT水溶液，SVIRPPL-HCPT/NP表示抗肿瘤药物，横坐标为HCPT水溶液的浓度或者抗肿瘤药物中HCPT的包埋浓度。不同字母之间表示具有显著性差异。
图5不同HCPT包埋浓度的抗肿瘤药物和不同浓度HCPT水溶液对MKN-28肿瘤细胞存活率的影响，其中HCPT表示HCPT水溶液，SVIRPPL-HCPT/NP表示抗肿瘤药物，横坐标为HCPT水溶液的浓度或者抗肿瘤药物中HCPT的包埋浓度，纵坐标为MKN-28肿瘤细胞存活率。不同字母之间表示具有显著性差异。
图6不同HCPT包埋浓度的抗肿瘤药物和不同浓度HCPT水溶液对A549肿瘤细胞存活率的影响，其中HCPT表示HCPT水溶液，SVIRPPL-HCPT/NP表示抗肿瘤药物，横坐标为HCPT水溶液的浓度或者抗肿瘤药物中HCPT的包埋浓度，纵坐标为A549肿瘤细胞存活率。不同字母之间表示具有显著性差异。
图7不同HCPT包埋浓度的抗肿瘤药物和不同浓度HCPT水溶液对MCF-7肿瘤细胞存活率的影响，其中HCPT表示HCPT水溶液，SVIRPPL-HCPT/NP表示抗肿瘤药物，横坐标为HCPT水溶液的浓度或者抗肿瘤药物中HCPT的包埋浓度，纵坐标为MCF-7肿瘤细胞存活率。不同字母之间表示具有显著性差异。
图8 SVIRPPL-HCPT/NP在不同pH与CathB的HCPT药物释放曲线。
图9是各组治疗后的肝肿瘤组织的病理切片，其中Saline是生理盐水组，SVIRPPLNP是SVIRPPLNP组，HCPT是HCPT水溶液组，SVIRPPL-HCPT是SVIRPPL-HCPT/NP组。
图10各组小鼠在首次注射D-荧光素前(记为0天)、首次注射D-荧光素后第7天和第15天时的生物发光成像。其中Saline是生理盐水组，SVIRPPLNP是SVIRPPLNP组，HCPT是HCPT水溶液组，SVIRPPL-HCPT NP是SVIRPPL-HCPT/NP组。
图11各组小鼠在首次注射D-荧光素前(记为0天)、首次注射D-荧光素后第7天、第15天和第19天时的肝肿瘤的荧光定量分析。其中Saline是生理盐水组，SVIRPPLNP是SVIRPPLNP组，HCPT是HCPT水溶液组，SVIRPPL-HCPT NP是SVIRPPL-HCPT/NP组。不同字母之间表示具有显著性差异。
图12显示了Cy5.5标记的SVIRPPL-HCPT/NP注射后不同时间抗肿瘤药物在全身和离体器官中的分布情况。
具体实施方式
下面结合具体实例及附图，进一步阐述本发明，但本发明的实施方式不仅限于此。
以下实施例中如无特殊说明，所使用原料均来源于市售，所采用方法均为本领域技术人员公知的常规操作方法。
实施例1
将菜籽蛋白采用碱性蛋白酶水解并分离后，发现序列为SVIRPPL的小肽(SEQ ID NO:1)，该小肽是一种两亲性多肽，命名为菜籽肽C。由杰肽公司按照常规的固态合成方法，制备菜籽肽C，用于本发明中的实验。
采用菜籽肽C制备抗肿瘤药物，包括如下步骤：
(1)将10mg菜籽肽C粉末加入1mL三氯甲烷中，搅拌至溶解，得到菜籽肽C的三氯甲烷溶液；再将0.1mL含有1mg的HCPT(羟基喜树碱)的水溶液按8mL/h速度缓慢滴加到菜籽肽C的三氯甲烷溶液中，在15℃、避光条件下超声至HCPT完全溶解，得到混合液A。超声条件：功率为70kW。
(2)将步骤(1)获得的混合液A按8mL/h的速度，全部滴加到10mL浓度为0.01M、pH为7.4的PBS磷酸盐缓冲液(购买于索莱宝公司)中，然后加入吐温80，在4℃、转速为600r/min条件下搅拌8h，得到体系均一的混合液B。混合液B中吐温80的体积百分浓度为1％。滴加过程中，PBS磷酸盐缓冲液中以600r/min进行磁力搅拌。
(3)将步骤(2)获得的混合液B在4℃超声分散1min后，在25℃、转速为100r/min条件下搅拌，以挥发去除三氯甲烷。去除三氯甲烷后，采用孔径为0.45μm的膜过滤，即制备获得抗肿瘤药物(缩写为SVIRPPL-HCPT/NP)。超声的功率为300KW。
按照抗肿瘤药物的制备方法制备无HCPT的菜籽肽C空白纳米载体(缩写为SVIRPPLNP)，不同之处仅在于以0.1mL水替代0.1mL含有1mg的HCPT的水溶液。
实施例2
本实施例用于说明实施例1制备的抗肿瘤药物的表征。
使用Malvern Zetasizer Nano ZS仪器(He-He作为激光器：633nm；散射角：173°)检测实施例1制备的抗肿瘤药物在4℃储藏的动态光散射粒径和多分散指数(PDI)。包埋率的测定方法如下：取1mL实施例1中步骤(3)挥发除去三氯甲烷后的药物，离心后取沉淀，即得未包埋的HCPT，在该沉淀中加入1mL三氯甲烷溶液，制备成HCPT溶液，在367nm下使用分光光度计测定HCPT的浓度，通过计算得到抗肿瘤药物中未包埋HCPT的含量，用以计算HCPT的包埋率。包埋率计算公式如下：
Figure PCTCN2021130617-appb-000001
经实验测试发现，实施例1制备的抗肿瘤药物是透明的溶液，刚制备好时，平均粒径为178nm，平均PDI值为0.26；贮藏实验结果如图1-图2所示，在4℃下储藏30天后，平均粒径为195nm，PDI小于0.3，说明该抗肿瘤药物能保持较好的胶体稳定性。通过检测发现，该抗肿瘤药物对HCPT的包埋率为78.5％。
该抗肿瘤药物和无HCPT的菜籽肽C空白纳米载体制备结束后6h，通过透射电镜分析，结果如图3所示，可以看到空载体和抗肿瘤药物的形状近似为球形，颗粒分布均匀，为直径160～180nm的球体，与动态光散射测定结果相一致，进一步说明了两亲性菜籽肽C可通过自组装形成具有较好稳定性的纳米载药载体。
实施例3
本实施例用于说明实施例1制备的抗肿瘤药物对四种肿瘤细胞的抗肿瘤活性。
根据实施例2计算的包埋率，参照实施例1的方法制得HCPT包埋浓度为7.8μM的抗肿瘤药物，然后用0.01M、pH7.4的PBS磷酸盐缓冲液稀释成HCPT包埋浓度为0.01μM、0.05μM、0.1μM、0.25μM、0.5μM、1μM六种不同浓度的抗肿瘤药物，作为样品。按照实施例1的方法制备无HCPT的菜籽肽C空白纳米载体，不同之处仅在于采用0.1mL水替代0.1mL含有1mg的HCPT(羟基喜树碱)的水溶液，即不添加HCPT；然后，用0.01M、pH7.4的PBS缓冲液稀释成HCPT包埋浓度为0.01μM、0.05μM、0.1μM、0.25μM、0.5μM、1μM的抗肿瘤药物对应的菜籽肽C空白纳米载体，作为样品。另外，将HCPT溶于浓度为 0.01M、pH7.4的PBS磷酸盐缓冲液中，制备0.01μM、0.05μM、0.1μM、0.25μM、0.5μM、1μM不同浓度的HCPT水溶液，作为样品。
将肝癌细胞HepG2、胃癌细胞MKN-28、肺癌细A549和乳腺癌细胞MCF-7四种肿瘤细胞(购买于江苏申基生物有限公司)以5×10 3个/孔的密度分别接种到一个96孔板中，留若干不接种的孔作为空白孔，因此共接种了4个96孔板，在37℃孵育过夜。培养接种有各肿瘤细胞的96孔板，均设置样品孔(包括各包埋浓度的抗肿瘤药物，各菜籽肽C空白纳米载体，各浓度的HCPT水溶液的样品孔)、对照孔和空白孔。每个样品孔(铺有细胞)中分别加入100μL样品。每个对照孔(铺有细胞)中分别加入100μL浓度为0.01M、pH7.4的PBS磷酸盐缓冲液替代样品。每个空白孔中无细胞，仅加入0.01M、pH7.4的PBS磷酸盐缓冲液100μL。将四个96孔板置于37℃恒温CO2培养箱中，孵育24h后，弃去上清，用0.01M、pH7.4的PBS磷酸盐缓冲液洗去残留的液体，每个孔内加入1mg/mL的MTT(四甲基偶氮唑蓝)溶液120μL；置于37℃恒温CO2培养箱中继续培养4h后，弃上清，在每孔中加入100μL的二甲基亚砜，于37℃恒温CO 2培养箱中震荡20min，然后在波长490nm的条件下测各孔的吸光度(OD值)，计算各浓度抗肿瘤药物、各菜籽肽C空白纳米载体、各浓度的HCPT水溶液干预后的肿瘤细胞存活率。
Figure PCTCN2021130617-appb-000002
结果如图4-7所示，HCPT水溶液和抗肿瘤药物都表现出了明显的浓度依赖性的肿瘤细胞增殖抑制特性。与HCPT水溶液相比，抗肿瘤纳米药物显著增强了HCPT对上述四种肿瘤细胞的抑制作用，说明菜籽肽C是优良的载体，可以有效地提高HCPT的抗肿瘤效果，提高其生物利用度。
通过计算，发现抗肿瘤药物对肝癌细胞HepG2、胃癌细胞MKN-28、肺癌细A549和乳腺癌细胞MCF-7四种细胞的IC 50分别为0.17、0.18、0.25和0.27μM，HCPT水溶液对将肝癌细胞HepG2、胃癌细胞MKN-28、肺癌细A549和乳腺癌细胞MCF-7四种细胞的IC 50分别为0.45μM、0.37μM、0.46μM和0.48μM。
菜籽肽C空白纳米载体对肝癌细胞HepG2、胃癌细胞MKN-28、肺癌细A549和乳腺癌细胞MCF-7四种细胞的IC 50分别为0.57、0.81、0.97和1.10mM。
上述结果说明了菜籽肽C作为纳米载体能够有效降低HCPT对肿瘤细胞的IC 50值，可有效提高抗肿瘤药物对肿瘤细胞的抑制作用，降低HCPT浓度大引起的生物毒性。
实施例4
本实施例用于说明在不同pH和有无Cath B时，实施例1制备的抗肿瘤药物中HCPT的释放曲线。
采用标准透析法对实施例1制备的抗肿瘤药物的体外释放特性进行研究。将实施例1制备的抗肿瘤药物5mL装在透析袋中(MWCO：500Da)，然后将透析袋分别置于溶液1、2、3、4(表1)中，37℃下100rpm/min搅拌透析袋外的溶液，考察不同pH和有无Cath B(组织蛋白酶B)时，实施例1制备的抗肿瘤药物中HCPT的释放曲线。在1、2、4、8、12、16、24、36及48h时，分别取透析袋外溶液2mL，然后加入2mL的原溶液(此处原溶液是指透析袋外初始状态的溶液，即表1中溶液)。将每次取出的溶液通过紫外分光光度计在367nm下测量HCPT的含量，并绘制出HCPT的释放动力学曲线。
根据实施例1制备方法可知，实施例1制备的抗肿瘤药物中HCPT浓度为100μg/mL，根据实施例2测算的包埋率(78.5％)，可计算出每5mL抗肿瘤药物中包埋有0.4mg的HCPT。为了证明在透析袋外检测到的HCPT不是由于透析袋本身造成的，同时设置对照：取0.4mgHCPT溶于5mL、0.01M的PBS磷酸缓冲盐溶液中，装在透析袋中(MWCO：500Da)，分别置于溶液1、2、3、4(见表1)中，然后采用上述相同方法测量HCPT水溶液在透析袋中的释放特性，绘制出HCPT的释放动力学曲线。
表1透析袋外的溶液的组成
Figure PCTCN2021130617-appb-000003
如图8所示，实施例1制备的抗肿瘤药物表现出了pH依赖性释放特性，在溶酶体(pH 5.0且含有CathB)及肿瘤微环境(pH 6.5)下抗肿瘤药物的释放速率比在生理环境(pH 7.4)条件下更高，在pH 7.4中抗肿瘤药物释放缓慢，表现 出良好的体外稳定性。透析袋中的HCPT水溶液在各溶液中的释放不具有pH依赖性，其快速透过透析袋在5h内释放率高达到70％。抗肿瘤药物在pH 5.0的条件下加入CathB时会进一步加速HCPT从抗肿瘤药物中的释放，并且抗肿瘤药物在CathB中48h的释放效率是其在pH 7.4中释放效率的两倍，说明酸性条件会破坏抗肿瘤药物中菜籽肽与HCPT等药物之间的结合，导致了药物的快速释放。通过蛋白酶CathB的酸依赖特性，特别是在非常酸性的溶酶体和弱酸性肿瘤微环境中，抗肿瘤药物将通过pH和CathB的响应实现药物的加速释放。
实施例5小鼠实验
1.SVIRPPL-HCPT/NP的治疗实验
雄性NOD SCID小鼠(6周龄)购自北京维通利华实验动物技术有限公司，并在无病原体的条件下饲养。沿小鼠的左肋缘下方开腹，暴露出肝脏左叶，注射30μL含有1×10 6个HepG2细胞的悬液，进行原位肝癌的构建。三周后当肿瘤体积达到100mm 3时，进行治疗实验。
为评估SVIRPPL-HCPT/NP的体内治疗效果，将荷瘤小鼠随机分为四组：生理盐水组，HCPT水溶液组，SVIRPPLNP(无HCPT的菜籽肽C空白纳米载体，实施例1制备)组和SVIRPPL-HCPT/NP(实施例1制备)组。各组小鼠通过尾静脉注射给药，每三天给药一次，治疗期间共给药4次。每次给药方法如下：HCPT水溶液组以HCPT水溶液按照HCPT剂量为5.0mg/kg进行给药；SVIRPPL-HCPT/NP组以SVIRPPL-HCPT/NP(实施例1制备)按照HCPT剂量为5.0mg/kg进行给药；生理盐水组给予相同体积的生理盐水；SVIRPPLNP组给予相同体积的无HCPT的菜籽肽C空白纳米载体(实施例1制备)。
在最后一次给药后，每组随机取一只小鼠处死，取肿瘤组织进行切片分析。将取出的肿瘤组织在10％中性福尔马林中固定，用梯度乙醇溶液脱水，石蜡包埋。随后用组织切片机切片，制片，用苏木精-伊红(H&E)染色，树脂封片。最后使用光学显微镜观察肿瘤组织病理变化。
结果如图9，肝肿瘤组织的病理切片分析发现，生理盐水组处理对肿瘤组织无明显影响，在经过SVIRPPL NP、HCPT水溶液和SVIRPPL-HCPT/NP组治疗后，肿瘤组织出现不同程度的坏死。由于HCPT在体内易被清除，导致HCPT在肿瘤处的积累较少，HCPT水溶液组的肿瘤组织染色后仅出现部分组织的坏 死，范围较小。SVIRPPL-HCPT/NP组肿瘤组织染色后出现大范围的肿瘤组织坏死，SVIRPPL NP组肿瘤组织也出现了少部分的坏死组织。生理盐水组为中度分化肝细胞癌，癌细胞呈片状，弥漫性排列，核质深染；SVIRPPL NP组与HCPT水溶液组为轻度分化肝细胞癌，可见团状癌细胞与癌旁正常组织，癌细胞胞核深染；SVIRPPL-HCPT/NP组仅有少量癌细胞分布，存在一定炎症浸润。上述结果表明了SVIRPPL-HCPT/NP能够有效地转运至肿瘤处并抑制肿瘤细胞的生长。
为了充分了解SVIRPPL-HCPT/NP在荷HepG2肝肿瘤小鼠体内的治疗效果，在最后一次给药后，各组小鼠每七天注射一次150mg/kg剂量的D-荧光素，共注射3次。其间，通过生物发光成像观察小鼠肝癌肿瘤的生长。首次注射D-荧光素前(记为0天)、首次注射D-荧光素后第7天和第15天时的生物发光成像(图10)显示，SVIRPPL-HCPT/NP能够有效抑制肝癌肿瘤的增长，生物荧光强度明显低于生理盐水治疗组。肝肿瘤的荧光定量分析(图11)后发现，在第7天SVIRPPL-HCPT/NP治疗后小鼠肿瘤部位的荧光与治疗之前相比并没有显著上升，而生理盐水组，HCPT水溶液组和SVIRPPL NP组均有明显上升，且SVIRPPL-HCPT/NP组的平均荧光强度显著低于其他各组，活体成像结果再次证明了SVIRPPL-HCPT/NP具有良好的抗肿瘤能力。
2.SVIRPPL-HCPT/NP的生物分布研究
雄性NOD SCID小鼠(6周龄)购自北京维通利华实验动物技术有限公司，并在无病原体的条件下饲养。沿小鼠的左肋缘下方开腹，暴露出肝脏左叶，注射30μL含有1×10 6个HepG2细胞的悬液，进行原位肝癌的构建。当肿瘤体积达到200mm 3，开始活体荧光成像实验和生物分布实验。
活体荧光成像实验和生物分布实验的方法如下：用近红外荧光菁染料Cy5.5活性酯对SVIRPPL-HCPT/NP(实施例1制备的抗肿瘤药物)进行标记，使用近红外成像系统IVIS LuminaXRⅢ(ex/em＝680nm/700nm)分析抗肿瘤药物在全身和离体器官中的分布。将Cy5.5标记的SVIRPPL-HCPT/NP以HCPT给药剂量为5mg/kg体重，在尾静脉处注射荷瘤小鼠，在注射后不同时间，使用2％异氟烷麻醉小鼠，并利用IVIS LuminaXRⅢ成像仪在ex/em＝680nm/700nm进行全身成像。
结果如图12，在注射2h后，SVIRPPL-HCPT/NP在肿瘤部位便可监测到，8 h后SVIRPPL-HCPT/NP在肿瘤部位的积累量达到最高，24h后SVIRPPL-HCPT/NP在肿瘤部位仍然可以监测到并持续到了48h。这些结果说明SVIRPPL-HCPT/NP可以有效靶向到肿瘤部位并在肿瘤处积聚。

Claims (10)

  1. 一种菜籽肽，序列如SEQ ID NO:1所示。
  2. 权利要求1所述菜籽肽在制备药物纳米载体方面的应用。
  3. 根据权利要求2所述应用，其特征在于所述药物为羟基喜树碱。
  4. 根据权利要求3所述应用，其特征在于菜籽肽与羟基喜树碱的质量比为5-15:1。
  5. 一种以权利要求1所述多肽为载体制备抗肿瘤药物的方法，其特征在于包括以下步骤：
    (1)将权利要求1所述菜籽肽和抗肿瘤药物依次溶解在三氯甲烷中，得到混合液A；
    (2)将所述混合液A逐滴加入到磷酸缓冲液中，然后再加入吐温80，搅拌均匀，得到混合液B。
    (3)除去混合液B中三氯甲烷，制得抗肿瘤药物。
  6. 根据权利要求5所述方法，其特征在于所述菜籽肽和羟基喜树碱的质量比为5-15:1。
  7. 根据权利要求6所述方法，其特征在于所述混合液A中菜籽肽的质量浓度为5-15mg/mL。
  8. 根据权利要求7所述方法，其特征在于所述磷酸缓冲液与混合液A的体积比为1:8-15，混合液B中吐温80的质量百分含量为0.5-1.5％。
  9. 根据权利要求8所述方法，其特征在于步骤(3)中除去混合液B中的三氯甲烷后，过滤，得到抗肿瘤纳米药物。
  10. 根据权利要求9所述方法，其特征在于过滤中使用的过滤膜的孔径为0.4-0.8μm。
PCT/CN2021/130617 2021-11-11 2021-11-15 一种菜籽肽及其在制备药物纳米载体方面的应用 WO2023082243A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111331274.5 2021-11-11
CN202111331274.5A CN114409729B (zh) 2021-11-11 2021-11-11 一种菜籽肽及其在制备药物纳米载体方面的应用

Publications (1)

Publication Number Publication Date
WO2023082243A1 true WO2023082243A1 (zh) 2023-05-19

Family

ID=81265042

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/130617 WO2023082243A1 (zh) 2021-11-11 2021-11-15 一种菜籽肽及其在制备药物纳米载体方面的应用

Country Status (3)

Country Link
CN (1) CN114409729B (zh)
LU (1) LU504660B1 (zh)
WO (1) WO2023082243A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165080A (zh) * 2024-05-15 2024-06-11 山东第二医科大学 响应性小分子肽、纳米载药载体及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080254505A1 (en) * 2007-04-16 2008-10-16 Novozymes A/S Whey protein hydrolysate
CN101917866A (zh) * 2007-04-16 2010-12-15 索莱有限责任公司 具有改善的感官特性和物理特性的蛋白质水解产物组合物
CN104489237A (zh) * 2014-12-26 2015-04-08 南京财经大学 一种改性菜籽蛋白、微胶囊及制备方法
CN107619844A (zh) * 2017-10-13 2018-01-23 南京财经大学 一种用β‑环糊精包埋菜籽多肽的方法
CN110483812A (zh) * 2019-09-05 2019-11-22 南京财经大学 一种菜籽蛋白基纳米凝胶及其应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2227511T3 (es) * 1992-04-15 2005-04-01 Sembiosys Genetics Inc. Proteinas con cuepo aceitoso como vectores de peptidos de gran valor en vegetales.
US20060064773A1 (en) * 2004-06-28 2006-03-23 Pioneer Hi-Bred International, Inc. Cell cycle polynucleotides and polypeptides and methods of use
ES2384060B1 (es) * 2010-03-24 2013-09-23 Lipotec S.A. Cápsulas de nanopartículas lipídicas.
EP2399885A1 (en) * 2010-06-22 2011-12-28 Ulrich Dietz Device and method for solubilizing, separating, removing and reacting carboxylic acids in aqueous or organic solutions by means of micro- or nanoemulsification
ES2625847T3 (es) * 2011-11-04 2017-07-20 Lipotec, S.A. Péptidos que inhiben la actividad de receptores activados y su uso en composiciones cosméticas o farmacéuticas
CN102676621B (zh) * 2012-04-28 2014-03-19 南京财经大学 一种降血压菜籽肽及其制备方法和应用
US20130337710A1 (en) * 2012-06-14 2013-12-19 Basf Corporation Treated non-woven fabric comprising functional additive and a method of preparing a treated non-woven fabric
CA2898513A1 (en) * 2015-07-27 2017-01-27 Stephan HEATH Methods, products, and systems relating to making, providing, and using nanocrystalline (nc) products comprising nanocrystalline cellulose (ncc), nanocrystalline (nc) polymers and/or nanocrystalline (nc) plastics or other nanocrystals of cellulose composites or structures, in combination with other materials
EP3898987A1 (en) * 2018-12-20 2021-10-27 Basf Plant Science Company GmbH Native delivery of biomolecules into plant cells using ionic complexes with cell-penetrating peptides

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080254505A1 (en) * 2007-04-16 2008-10-16 Novozymes A/S Whey protein hydrolysate
CN101917866A (zh) * 2007-04-16 2010-12-15 索莱有限责任公司 具有改善的感官特性和物理特性的蛋白质水解产物组合物
CN104489237A (zh) * 2014-12-26 2015-04-08 南京财经大学 一种改性菜籽蛋白、微胶囊及制备方法
CN107619844A (zh) * 2017-10-13 2018-01-23 南京财经大学 一种用β‑环糊精包埋菜籽多肽的方法
CN110483812A (zh) * 2019-09-05 2019-11-22 南京财经大学 一种菜籽蛋白基纳米凝胶及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG LIFENG, ZHANG JING, YUAN QIANG, XIE HUIHUI, SHI JIAYI, JU XINGRONG: "Separation and purification of an anti-tumor peptide from rapeseed (Brassica campestris L.) and the effect on cell apoptosis", FOOD & FUNCTION, R S C PUBLICATIONS, GB, vol. 7, no. 5, 1 January 2016 (2016-01-01), GB , pages 2239 - 2248, XP093066424, ISSN: 2042-6496, DOI: 10.1039/C6FO00042H *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118165080A (zh) * 2024-05-15 2024-06-11 山东第二医科大学 响应性小分子肽、纳米载药载体及应用

Also Published As

Publication number Publication date
LU504660B1 (en) 2023-11-07
CN114409729B (zh) 2023-06-20
LU504660A1 (en) 2023-07-05
CN114409729A (zh) 2022-04-29

Similar Documents

Publication Publication Date Title
Liu et al. A peptide‐network weaved nanoplatform with tumor microenvironment responsiveness and deep tissue penetration capability for cancer therapy
Jia et al. Plasma membrane activatable polymeric nanotheranostics with self-enhanced light-triggered photosensitizer cellular influx for photodynamic cancer therapy
Mao et al. A biomimetic nanocomposite made of a ginger-derived exosome and an inorganic framework for high-performance delivery of oral antibodies
Li et al. Reduction breakable cholesteryl pullulan nanoparticles for targeted hepatocellular carcinoma chemotherapy
US11938186B2 (en) Thermosensitive hydrogel for cancer therapeutics and methods of preparation thereof
Wang et al. Laser-triggered polymeric lipoproteins for precision tumor penetrating theranostics
Khan et al. Chondroitin sulfate-based redox-responsive nanoparticles for melanoma-targeted drug delivery
Das et al. Preparation of a size selective nanocomposite through temperature assisted co-assembly of gelatin and pluronic F127 for passive targeting of doxorubicin
Wang et al. Mitochondria-targeting folic acid-modified nanoplatform based on mesoporous carbon and a bioactive peptide for improved colorectal cancer treatment
Lee et al. Gold-stabilized carboxymethyl dextran nanoparticles for image-guided photodynamic therapy of cancer
WO2023082243A1 (zh) 一种菜籽肽及其在制备药物纳米载体方面的应用
Yan et al. A ROS-responsive biomimetic nano-platform for enhanced chemo-photodynamic-immunotherapy efficacy
Xu et al. A tumor acidity-driven transformable polymeric nanoassembly with deep tumor penetration and membrane-anchoring capability for targeted photodynamic therapy
Zhou et al. Ros-responsive galactosylated-nanoparticles with doxorubicin entrapment for triple negative breast cancer therapy
Wu et al. Tumor homing-penetrating and nanoenzyme-augmented 2D phototheranostics against hypoxic solid tumors
Yuan et al. Regulating tumor-associated macrophage polarization by cyclodextrin-modified PLGA nanoparticles loaded with R848 for treating colon cancer
CN113648415A (zh) 一种用于靶向治疗肿瘤的有机金属纳米载药颗粒及其制备方法
WO2021114605A1 (zh) 一种基于糖基金属框架材料的肝靶向治疗药物及制备方法
Geng et al. Reshaping the tumor microenvironment for increasing the distribution of glucose oxidase in tumor and inhibiting metastasis
CN106606783B (zh) 一种靶向共递释光敏剂与化疗药物的药物递释系统
CN110755379A (zh) 一种能抗耐药肿瘤的靶向载药系统及其制备方法
CN110279661A (zh) 一种纳米颗粒组合物递送系统、制备方法及其用途
CN102716137B (zh) 荧光纳米钻石-阿霉素复合物的制备方法和应用
Wang et al. Functionalized Tumor Cell Membrane-Camouflaged Photo-Activatable Nanoparticle for Spatiotemporal Antitumor Therapy
Zeng et al. A cascade dual-targeted nanocarrier for enhanced alectinib delivery to ALK-positive lung cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21963690

Country of ref document: EP

Kind code of ref document: A1