CN113350292A - Preparation for improving water dispersibility of lycorine, prolonging half-life period of lycorine and improving antitumor activity of lycorine and application thereof - Google Patents
Preparation for improving water dispersibility of lycorine, prolonging half-life period of lycorine and improving antitumor activity of lycorine and application thereof Download PDFInfo
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- CN113350292A CN113350292A CN202110671410.9A CN202110671410A CN113350292A CN 113350292 A CN113350292 A CN 113350292A CN 202110671410 A CN202110671410 A CN 202110671410A CN 113350292 A CN113350292 A CN 113350292A
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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
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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Abstract
The invention discloses a preparation for improving the water dispersibility of lycorine, prolonging the half-life period of lycorine and improving the anti-tumor activity of lycorine and application thereof. The invention provides application of DSPE-PEG modified lycorine nanoparticles or lycorine nanoparticles in preparation of products for improving lycorine water dispersibility and/or lycorine half-life and/or lycorine anti-tumor activity and/or lycorine anti-tumor cell activity; the DSPE-PEG modified lycorine nanoparticles consist of DSPE-PEG and the lycorine nanoparticles loaded by the DSPE-PEG. The DSPE-PEG modified lycorine nanoparticles have good stability, water dispersibility is obviously better than that of lycorine, half-life period is prolonged by more than 1 time than that of lycorine, toxicity is obviously lower than that of lycorine, and in-vivo and in-vitro anti-tumor activity is obviously better than that of lycorine.
Description
Technical Field
The invention relates to a preparation for improving the water dispersibility of lycorine, prolonging the half-life period of lycorine and improving the anti-tumor activity of lycorine and application thereof.
Background
Cancer is one of the diseases with the highest morbidity and mortality worldwide, and seriously harms the physical health of human beings. With the development of cancer in recent decades, radiotherapy and chemotherapy have become the main treatment methods for advanced cancer, but most anticancer drugs have poor water dispersibility, low bioavailability and no specific selectivity.
Lycorine is a pyrrolophenanthridine ring type alkaloid extracted from Amaryllidaceae plants, and has a structural formula shown in figure 1. Lycorine has a variety of pharmacological effects including anti-tumor, anti-viral, anti-malarial and anti-inflammatory. But lycorine has poor water dispersibility, low bioavailability, short half-life and poor targeting property.
Disclosure of Invention
Experiments of the inventor prove that the lycorine has poor water dispersibility, low bioavailability and short half-life.
The technical problem to be solved by the invention is how to improve the water dispersibility of lycorine and/or prolong the half-life of lycorine and/or improve the targeting property of lycorine and/or improve the anti-tumor activity of lycorine.
In order to solve the technical problems, the invention provides application of lycorine nanoparticles or DSPE-PEG modified lycorine nanoparticles in preparation of products (such as medicines, vaccines, health products and/or foods) for improving water dispersibility of lycorine and/or prolonging half-life period of lycorine and/or improving antitumor activity of lycorine and/or improving antitumor cell activity of lycorine; the DSPE-PEG modified lycorine nanoparticles consist of DSPE-PEG and the lycorine nanoparticles loaded by the DSPE-PEG.
In the above application, the tumor may be a solid tumor, and the tumor cell may be a solid tumor cell. The solid tumor can be liver cancer, colorectal cancer, colon cancer, rectal cancer and/or breast cancer, and the solid tumor cell can be liver cancer cell, colorectal cancer cell, colon cancer cell, rectal cancer cell and/or breast cancer cell.
In the above application, the diameter of the lycorine nanoparticles can be 42-47nm, such as 46.35 nm; the diameter of the DSPE-PEG modified lycorine nanoparticle can be 48-55nm, such as 52.54 nm.
In the above application, the PDI of the lycorine nanoparticles may be 0.229, and the PDI of the DSPE-PEG modified lycorine nanoparticles may be 0.140.
The lycorine nanoparticles can be prepared as described below, and the DSPE-PEG loaded lycorine nanoparticles can be prepared as described below.
A method for preparing lycorine nanoparticles comprises adding an ethanol solution of lycorine to water to obtain lycorine nanoparticles.
In the above method for preparing lycorine nanoparticles, the concentration of lycorine in the lycorine ethanol solution may be 2.9849mM, the solute of the lycorine ethanol solution is lycorine, and the solvent is ethanol. The lycorine ethanol solution is prepared at 60 ℃. The lycorine ethanol solution is added into water, and the ratio of the lycorine ethanol solution to the water meets the requirement that 0.86g of lycorine is added into 9mL of water.
The method for preparing the DSPE-PEG modified lycorine nanoparticles comprises the step of reacting the lycorine nanoparticles with the DSPE-PEG to obtain the DSPE-PEG modified lycorine nanoparticles.
In the method for preparing the DSPE-PEG modified lycorine nanoparticles, the ratio of the lycorine nanoparticles to the DSPE-PEG meets the mass ratio of the lycorine nanoparticles to the DSPE-PEG of 2.1041: 1. The reaction time may be 1 hour. And dispersing the lycorine nano particles into liquid by using water to obtain lycorine nano particle liquid. The DSPE-PEG is dispersed into liquid by water to obtain DSPE-PEG solution. The solute of the DSPE-PEG solution is DSPE-PEG, and the solvent is water (such as ultrapure water).
Herein, the DSPE-PEG may be DSPE-PEG 3000.
The application of the lycorine nano-particles or the DSPE-PEG modified lycorine nano-particles in preparing nano-preparations also belongs to the protection scope of the invention.
The lycorine nano-particles or the DSPE-PEG modified lycorine nano-particles also belong to the protection scope of the invention.
The nanometer preparation containing the lycorine nanometer particles or the DSPE-PEG modified lycorine nanometer particles also belongs to the protection scope of the invention.
Herein, the nano-formulation may be an anti-tumor formulation.
In the above anti-tumor preparation, the tumor may be a solid tumor. The solid tumor can be liver cancer, colorectal cancer, colon cancer, rectal cancer and/or breast cancer.
Herein, the active ingredient of the anti-tumor product, the anti-tumor cell product and the anti-tumor preparation can be the lycorine nanoparticles or the DSPE-PEG modified lycorine nanoparticles; the active ingredients of the anti-tumor products, anti-tumor cell products and anti-tumor preparations may also contain other biological or non-biological components, and the other active ingredients of the products can be determined by those skilled in the art according to the anti-tumor effect.
The anti-tumor or/and anti-tumor cell may be embodied to inhibit growth and/or proliferation of the tumor or/and tumor cell, to inhibit migration and/or invasion of the tumor or/and tumor cell.
The nanometer preparation is prepared by conventional pharmaceutical method and has particle diameter of 1-200 nm.
Herein, water dispersibility refers to the degree to which a dispersoid is dispersed by water (dispersant).
The lycorine nano particles or the DSPE-PEG modified lycorine nano particles are carrier-free nano particles, and have high drug loading (the carrier can increase toxicity and has low drug loading), and the drug loading can be 73.92 +/-7.3%. The lycorine nano particles or the DSPE-PEG modified lycorine nano particles have simple preparation method and no toxic reagent, and lay a foundation for clinical development. The DSPE-PEG modified lycorine nanoparticles have good stability, water dispersibility is obviously better than that of lycorine, half-life period is prolonged by more than 1 time than that of lycorine, and toxicity is obviously lower than that of lycorine. The DSPE-PEG modified lycorine nanoparticles have in-vivo and in-vitro anti-tumor activity obviously better than that of lycorine, and provide a new theoretical basis for the treatment of cancer and clinical application of the lycorine nanoparticles.
Drawings
FIG. 1 shows the structural formula of lycorine.
FIG. 2 is a graph showing the particle size analysis of lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles (DSPE-PEG lycorine nanoparticles for short) in example 1.
FIG. 3 shows the stability of DSPE-PEG modified lycorine nanoparticles, abbreviated as "DSPE-PEG 0.2 mg/mL", "DSPE-PEG 0.4 mg/mL" and "DSPE-PEG 0.6 mg/mL", for 0 and 3 days.
FIG. 4 is a graph of the stability of lycorine nanoparticles modified with 0.4mg/mL DSPE-PEG solution.
A is transmission electron microscope photo of the particle after the lycorine nano particle liquid (left picture) with the lycorine content of 0.086g/L and the lycorine nano particle liquid (right picture) modified by 0.078g/L DSPE-PEG are kept stand for 24 hours at 25 ℃.
B is lycorine nano particle liquid (Ly NPS) with lycorine content of 0.086g/L and lycorine nano particle liquid (DS-Ly NPS) modified by 0.078g/L DSPE-PEG, and the liquid and the diameter are respectively kept still for 0, 1, 2, 3, 4, 5 and 6 days at 25 ℃.
C is 0.078g/L DSPE-PEG modified lycorine nanoparticle liquid which is respectively kept stand at 25 ℃ for 0, 1, 2, 3, 4, 5 and 6 days at Zeta potential.
D is PDI of 0.078g/L DSPE-PEG modified lycorine nanoparticle liquid which is respectively kept stand at 25 ℃ for 0, 1, 2, 3, 4, 5 and 6 days.
FIG. 5 shows the results of plate cloning experiments. The three columns from left to right are respectively a control phase, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 6 shows the Western Blot method for detecting the expression levels of the apoptosis-related marker proteins, namely, Cleaved caspase-3, Bax and Bcl-2 proteins. Three lanes from left to right are respectively a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
Fig. 7 shows the results of the scratch test for inhibiting proliferation and migration of tumor cells. The three columns from left to right are respectively a control phase, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 8 shows the results of experiments on tumor cell migration inhibition. The three columns from left to right are respectively a control phase, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 9 shows the results of experiments for inhibiting tumor cell invasion. The three columns from left to right are respectively a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 10 shows Western Blot analysis for detecting the expression levels of LC-3 protein and p62 protein, which are markers associated with autophagy. Three lanes from left to right are respectively a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 11 shows Western Blot to detect the expression levels of the metastasis marker proteins E-cadherin and Vimentin. Three lanes from left to right are respectively a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group.
FIG. 12 shows the experimental results of in situ hepatoma mice.
FIG. 13 shows the experimental results of orthotopic colorectal cancer mice.
FIG. 14 is a pharmacokinetic profile. The concentration of lycorine is plotted on the ordinate.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the human hepatocellular carcinoma cell line HepG2 (abbreviated as human hepatocellular carcinoma HepG2 cell) was the ATCC product. Human breast cancer MCF-7 cells are ATCC products. The human colorectal adenocarcinoma (colotectal adenocarinoma) cell line SW480 (briefly, human colorectal cancer SW480 cells) was the ATCC product.
Lycorine (Lycorine) (purity > 98%) in the following examples was purchased from shanghai source leaf biotechnology (shanghai, china).
The DSPE-PEG in the following examples is DSPE-PEG3000 (distearoylphosphatidylethanolamine-polyethylene glycol 3000) which is a product of SinOPEG, Xiamen.
Example 1 preparation of lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles
1. The preparation of the lycorine nano particles and the DSPE-PEG modified lycorine nano particles comprises the following steps:
0.025g of lycorine powder is weighed and added into 29.036mL of absolute ethanol, and the mixture is placed in a water bath kettle at 60 ℃ and heated until the lycorine powder is dissolved, so that a lycorine ethanol solution with the concentration of 0.861g/L (hereinafter referred to as 0.861g/L lycorine ethanol solution) is obtained. Adding 9mL of ultrapure water into a round-bottom flask, placing the round-bottom flask on a magnetic stirrer (DF-101S, Waihua instruments, Inc., Guest), adding a rotor of the magnetic stirrer into the round-bottom flask, adjusting the rotating speed of the magnetic stirrer to 20, and adding 1mL of 0.861g/L lycorine ethanol solution into the round-bottom flask at one time to obtain the lycorine nano-particle liquid with the lycorine content of 0.086 g/L. The powder of DSPE-PEG was dissolved in ultrapure water at 25 ℃ to obtain a DSPE-PEG solution having a DSPE-PEG concentration of 0.4 mg/mL. Adding 1mL of the DSPE-PEG solution into a round-bottom flask containing 10mL of 0.086g/L lycorine nanoparticle liquid at 25 ℃, combining the DSPE-PEG with the lycorine nanoparticles through hydrophobic force, placing the round-bottom flask on a magnetic stirrer (DF-101S, Daihua instruments Limited liability company, Inc., in China), adding a rotor of the magnetic stirrer into the round-bottom flask, and adjusting the rotating speed of the magnetic stirrer to 20 to obtain the DSPE-PEG modified lycorine nanoparticle liquid with the lycorine content of 0.078g/L (hereinafter, 0.078g/L of the DSPE-PEG modified lycorine nanoparticle liquid or the lycorine nanoparticle liquid modified by 0.4mg/mL of the DSPE-PEG solution).
2. Particle size of lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles
And (3) measuring the particle diameters and PDIs of the lycorine nano particles and the DSPE-PEG modified lycorine nano particles by using a Malvern particle sizer on 0.086g/L lycorine nano particle liquid prepared in the step (1) and 0.078g/L DSPE-PEG modified lycorine nano particle liquid.
The results show that the lycorine nanoparticles and the DSPE-PEG modified lycorine nanoparticles can generate a Tyndall effect, and particle size detection results (figure 2) show that the particle size (diameter) of the lycorine nanoparticles is 46.35nm, the particle size (diameter) of the DSPE-PEG modified lycorine nanoparticles is 52.54nm, the PDI of the lycorine nanoparticles is 0.229, and the PDI of the DSPE-PEG modified lycorine nanoparticles is 0.140, so that the good monodispersity is shown.
3. Stability of lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles
3.1 modification of lycorine nanoparticles with 0.2mg/mL DSPE-PEG solution
The same procedure as in step 1 was repeated except that the 0.4mg/mL DSPE-PEG solution in step 1 was replaced with the 0.2mg/mL DSPE-PEG solution, to obtain a liquid of DSPE-PEG-modified lycorine nanoparticles having a lycorine content of 0.078g/L in terms of lycorine (hereinafter referred to simply as "DSPE-PEG0.2mg/mL") modified with the 0.2mg/mL DSPE-PEG solution.
3.2 modification of lycorine nanoparticles with 0.4mg/mL DSPE-PEG solution
In the same manner as in step 1, a liquid of DSPE-PEG modified lycorine nanoparticles (hereinafter referred to as "DSPE-PEG 0.4 mg/mL") modified with 0.4mg/mL of DSPE-PEG solution and having a lycorine content of 0.078g/L, based on lycorine, was obtained.
3.3 modification of lycorine nanoparticles with 0.6mg/mL DSPE-PEG solution
The same procedure as in step 1 was repeated except that the 0.4mg/mL DSPE-PEG solution in step 1 was replaced with the 0.6mg/mL DSPE-PEG solution, to obtain a liquid of DSPE-PEG-modified lycorine nanoparticles having a lycorine content of 0.078g/L in terms of lycorine (hereinafter referred to as "DSPE-PEG0.6 mg/mL") modified with the 0.6mg/mL DSPE-PEG solution.
3.4 stability of DSPE-PEG modified lycorine nanoparticles, abbreviated as "DSPE-PEG 0.2 mg/mL", "DSPE-PEG 0.4 mg/mL" and "DSPE-PEG 0.6 mg/mL", after standing for 3 days
The liquid of DSPE-PEG modified lycorine nano particles which are abbreviated as 'DSPE-PEG 0.2 mg/mL', 'DSPE-PEG 0.4 mg/mL' and 'DSPE-PEG 0.6 mg/mL' and have the lycorine content of 0.078g/L is respectively kept stand at 25 ℃ for 0 hour and 72 hours, and the stability degree is measured by a Malvern particle sizer.
The results show that upon standing at 25 ℃ for 3 days, the lycorine nanoparticles modified with 0.2mg/mL DSPE-PEG solution had aggregated (PDI ═ 1.0), while the lycorine nanoparticles modified with 0.4mg/mL DSPE-PEG solution and the lycorine nanoparticles modified with 0.6mg/mL DSPE-PEG solution did not change significantly (fig. 3), and thus 0.4mg/mL DSPE-PEG modified lycorine nanoparticles were selected for subsequent studies.
3.5 modification of stability of lycorine nanoparticles with 0.4mg/mL DSPE-PEG solution
0.025g of lycorine powder is weighed and added into 29.036mL of absolute ethanol, and the mixture is placed in a water bath kettle at 60 ℃ and heated until the lycorine powder is dissolved, so that a lycorine ethanol solution with the concentration of 0.861g/L (hereinafter referred to as 0.861g/L lycorine ethanol solution) is obtained. Adding 9mL of ultrapure water into a round-bottom flask, placing the round-bottom flask on a magnetic stirrer (DF-101S, Waihua instruments, Inc., Guest), adding a rotor of the magnetic stirrer into the round-bottom flask, adjusting the rotating speed of the magnetic stirrer to 20, and adding 1mL of 0.861g/L lycorine ethanol solution into the round-bottom flask at one time to obtain the lycorine nano-particle liquid with the lycorine content of 0.086 g/L.
Standing the lycorine nano particle liquid with the lycorine content of 0.086g/L and the lycorine nano particle liquid modified by 0.078g/L DSPE-PEG in the step 1 at 25 ℃ for 24 hours respectively, placing 5 mu L of nano particles on a copper net of a transmission electron microscope, naturally airing at room temperature (25 ℃), and observing the size and the shape of the lycorine nano particles and the DSPE-PEG modified lycorine nano particles for 24 hours by the Transmission Electron Microscope (TEM).
The lycorine nano particle liquid with the lycorine content of 0.086g/L and the lycorine nano particle liquid modified by 0.078g/L DSPE-PEG in the step 1 are respectively kept stand for 0, 1, 2, 3, 4, 5 and 6 days at 25 ℃, a Malvern particle size analyzer is used for continuously measuring for 7 days, and the particle size (diameter) of the nano particles and the stability of Zeta potential are detected.
The results show that the lycorine nanoparticle liquid with the lycorine content of 0.086g/L has aggregated already at 24h (the left graph of A in FIG. 4), the lycorine nanoparticle liquid modified by 0.078g/L DSPE-PEG has no aggregation at 24h, is round and has uniform particle size and single distribution (the right graph of A in FIG. 4), and the particle size (B in FIG. 4) and the Zeta potential of the lycorine nanoparticle liquid modified by 0.078g/L DSPE-PEG are relatively stable within 7 days (C in FIG. 4), which indicates that the DSPE-PEG modification increases the stability of the lycorine nanoparticles.
The inventors have attempted to modify lycorine nanoparticles with BSA (bovine serum albumin), but the modification was not successful.
Example 2 lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles improve water dispersibility of lycorine
0.078g/L of the DSPE-PEG modified lycorine nanoparticle liquid in the example 1 is put into a refrigerator with the temperature of minus 80 ℃ and is put into a freeze dryer to be freeze-dried into powder state, and the DSPE-PEG modified lycorine nanoparticle is obtained. And dispersing the DSPE-PEG modified lycorine nano particles by using ultrapure water as a dispersing agent. The result shows that the water dispersibility of the DSPE-PEG modified lycorine nano particles is 0.32032 mg/mL. Lycorine is not dispersible in water.
Example 3 lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles increase the half-life of lycorine
To study the concentration of lycorine and DSPE-PEG modified lycorine nanoparticles in plasma samples, LC-MS/MS analysis was used.
SD rats weighing 200 + -10 g were randomly divided into 2 groups of lycorine (Ly) and DSPE-PEG modified lycorine nanoparticle (DS-Ly NPS) 6 animals each after being adaptively fed for one week. In the lycorine group, 0.2mL of lycorine liquid (the dispersing agent is physiological saline and the dispersoid is lycorine) is injected into the tail vein of each mouse at one time, so that the administration dosage of the mouse is 5mg of lycorine/kg of body weight; each mouse of the group of DSPE-PEG modified lycorine nanoparticle was injected with 0.2mL of DSPE-PEG modified lycorine nanoparticle liquid (the dispersant was physiological saline, and the dispersoid was the DSPE-PEG modified lycorine nanoparticles of example 2) in a tail vein at one time so that the administration dose of the mouse was 5mg of lycorine per kg of body weight in terms of lycorine. Respectively taking 0.2mL of blood from the inner canthus of the eye 0.83h, 0.16h, 0.25h, 0.5h, 0.75h, 1h, 2h, 4h, 6h, 12h and 24h after injection, loading the blood into a 1.5mL tube containing heparin, centrifuging at 3000rpm for 15min, sucking 100 mu l of blood plasma, placing the blood plasma into a 1.5mL centrifuge tube, adding acetonitrile (containing 0.1% formic acid), whirling, mixing uniformly, standing on ice for 1h, 12000g, centrifuging at 4 ℃ for 20min, sucking supernatant, placing the supernatant into another 1.5mL centrifuge tube, blowing and drying by blowing nitrogen, adding 50mL of methanol for redissolving, after 1h, 12000g, centrifuging at 4 ℃ for 20min, sucking supernatant, and detecting the content of lycorine in the blood plasma at different times by adopting a liquid chromatography-mass spectrometry method. Wherein, the mobile phase in the liquid chromatogram is 5% acetonitrile water solution, and lycorine methanol solution with each concentration is prepared to make a standard curve for quantitative analysis of lycorine content.
0.2mL of blood was taken from the canthus 24h after injection and placed in a 1.5mL tube. Centrifuge at 3000rpm for 15 minutes and place the top portion of serum into another 1.5mL tube. Glutamate pyruvate transaminase (ALT) and aspartate Aminotransferase (AST) levels are determined.
Data were processed using SPSS11.5 statistical software and results were expressed as mean. + -. standard deviation using One-way ANOVA test, with a significant difference compared to the lycorine group (P < 0.05).
The results show that the half-lives (t) of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group1/2z) is 1.068. + -. 0.205h and 2.13. + -. 1.058 h. Shows that the DSPE-PEG modified lycorine nano particles can obviously prolong half of the length of the lycorine nano particles compared with the lycorineThe time of the aging period is prolonged by more than 1 time, and the toxicity is reduced (figure 14).
Example 4 increasing antitumor Activity of lycorine and DSPE-PEG modified lycorine nanoparticles
1. Cell culture:
inoculating human liver cancer HepG2 cells, human colorectal cancer SW480 cells and human breast cancer MCF-7 cells in DMEM complete medium (DMEM medium + 10% FBS +100U/mL penicillin +10 ug/mL streptomycin, which is a medium obtained by adding FBS, penicillin and streptomycin to DMEM, wherein the DMEM complete medium contains 10% FBS, 100U/mL penicillin and 10 ug/mL streptomycin), placing at 37 ℃ and 5% CO2The cells are subcultured when the cell density is 80% -90%, the culture medium in the bottle is discarded, 1mL of pancreatin is added for about 2min, DMEM is added to complete the culture medium to stop digestion, and the mixture is centrifuged at 1000rpm/min and 4 ℃ for 5 min. And (3) removing the supernatant, adding a DMEM complete culture medium to blow the cells uniformly, transferring the cells to a new culture bottle, transferring the cells 2 to 3 times every week, and culturing the cells until the logarithmic phase of the cells is reached to be used for cell experiments.
2. Preparing DMEM complete medium and lycorine medium and DMEM complete medium and DSPE-PEG modified lycorine nanoparticle medium
Weighing lycorine powder, adding into anhydrous ethanol, placing in a water bath kettle at 60 deg.C, heating to dissolve to obtain lycorine ethanol solution with lycorine concentration of 3mM, and adding into culture medium as mother liquor. The liquid obtained by diluting the mother solution with DMEM complete medium is DMEM complete medium + lycorine medium. The liquid obtained by adding 0.078g/L of the DSPE-PEG modified lycorine nanoparticle liquid obtained in step 1 of example 1 to DMEM complete medium is DMEM complete medium + DSPE-PEG modified lycorine nanoparticle medium.
3. Plate cloning experiment
Taking human liver cancer HepG2 cells, human colorectal cancer SW480 cells and human breast cancer MCF-7 cells in logarithmic growth phase according to 1 × 103Inoculating the inoculum size of each cell/well in 6-well plate, changing the solution once in 2-3 days, culturing with DMEM complete culture medium for 5 days,the method is divided into 3 groups: a control group, a lycorine group (lycorine solution group) and a DSPE-PEG modified lycorine nanoparticle group (DSPE-PEG lycorine nanoparticle group), each group having 3 holes. 2000. mu.l DMEM complete medium was added to each well of the control group; 2000. mu.l DMEM complete medium + lycorine medium (lycorine content is 10. mu.M) is added into each well of lycorine group, and 2000. mu.l DMEM complete medium + DSPE-PEG modified lycorine nanoparticle medium (lycorine content is 10. mu.M) is added into each well of DSPE-PEG modified lycorine nanoparticle group.
After 48h, the medium was aspirated, fresh DMEM complete medium was added, and the culture was terminated when macroscopic colonies were obtained. Discarding the culture medium, washing with PBS for 2-3 times, adding 4% paraformaldehyde, fixing for 30min, discarding the fixing solution, adding 0.1% crystal violet, dyeing for 30min, washing with clear water, air drying under natural condition, taking pictures and counting the number of clones. Data were analyzed using SPSS19.0 statistical software. The results are expressed as (mean ± sd), the comparison between two pairs was examined by one-way anova analysis and LSD, wherein P < 0.01 indicates significant difference from the control group, P < 0.001 indicates significant difference from the control group, # (P < 0.05) indicates significant difference from the lycoris radiata alkali solution group, and # (P < 0.01) indicates significant difference from the lycoris radiata alkali solution group.
The results show that the experiment adopts a plate cloning experiment to explore the influence of lycorine nanoparticles and DSPE-PEG modified lycorine nanoparticles on the proliferation of liver cancer HepG2 cells, human breast cancer MCF-7 cells and colorectal cancer SW480 cells, and the results are shown in figure 5.
Western Blot Western Blot method for detecting expression levels of apoptosis-related marker proteins, namely, cleaned caspase-3, Bax and Bcl-2 proteins
Collecting liver cancer HepG2 cell and colorectal cancer SW480 cell in logarithmic growth phaseAnd breast cancer MCF-7 cells at 2X 105The density of individual cells/well was plated in 6-well plates at 37 ℃ with 5% CO2After being cultured in a constant temperature incubator for 24 hours, the culture medium is divided into 3 groups: a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, wherein each group has 3 holes. A control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, wherein each group has 3 holes. Adding 1000 mul of DMEM complete culture medium into each well of the control group; each well of lycorine group is added with 1000 mul of DMEM complete medium + lycorine medium (lycorine content is 10 mul), and each well of DSPE-PEG modified lycorine nanoparticle group is added with 1000 mul of DMEM complete medium + DSPE-PEG modified lycorine nanoparticle medium (lycorine content is 10 mul).
After further culturing for 48h, the 6-well plate was removed. Liver cancer HepG2 cells, breast cancer MCF-7 cells and colorectal cancer SW480 cells were digested with trypsin and collected in 1.5mL centrifuge tubes, respectively, placed in a pre-precooled centrifuge, centrifuged at 12000g at 4 ℃ for 5min, centrifuged and the supernatant aspirated, added to a pre-prepared lysate (RIPA lysate: PMFS: phosphatase inhibitor: 1000: 10: 5), frozen and thawed 5 times with liquid nitrogen, placed in a precooled centrifuge, centrifuged at 12000g at 4 ℃ for 5min, the supernatant was transferred to a new 1.5mL centrifuge tube, the protein concentration was determined using BCA kit (A solution: B solution: 50: 1), 4 Xloading buffer was added, placed at 100 ℃, 5min, cooled at room temperature and stored at-20 ℃. The total proteins of these three treatment groups were electrophoresed and analyzed by Western blot using the following antibodies for clear caspase-3, Bax, Bcl-2 and Actin (as internal controls): clean Caspase 3(D175) antibody, Bax antibody, Bcl-2 antibody and Actin antibody.
The result shows that after the lycorine nanoparticles modified by the lycorine and the DSPE-PEG are used for treating, the proliferation capacities of the liver cancer HepG2 cells, the colorectal cancer SW480 cells and the breast cancer MCF-7 cells are reduced, and the result is presumed to be closely related to apoptosis. In order to investigate whether the growth effects of lycorine and DSPE-PEG modified lycorine nanoparticles on inhibiting the growth of liver cancer HepG2 cells, breast cancer MCF-7 cells and colorectal cancer SW480 cells are related to promoting apoptosis, Western Blot experiment is further used for detecting, and after lycorine and DSPE-PEG modified lycorine nanoparticles are given, the expression levels of apoptosis-related marker proteins, namely Cleaved caspase-3, Bax and Bcl-2 proteins are observed.
Through the research of an immune protein blotting (Western bolt) experiment result, the apoptosis-related protein Cleaved caspase-3 of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group is found to be obviously improved in Bax expression level, Bcl-2 protein expression level and Bax/Bcl-2 ratio after the lycorine and DSPE-PEG modified lycorine nanoparticle group is administered, compared with a control group (figure 6).
5. Inhibition of tumor cell proliferation and migration (scratch test)
Firstly, placing a ruler and a marker pen on an ultra-clean bench for ultraviolet irradiation for 30min, drawing three transverse lines on the back of a 6-well plate by the marker pen with the ruler at intervals of 0.5-1cm, and taking liver cancer HepG2 cells, colorectal cancer SW480 cells and human breast cancer MCF-7 cells in logarithmic growth phase according to 5 multiplied by 105The density of each cell/well was inoculated in 6-well plates at 37 ℃ with 5% CO2After culturing for 24 hours in the constant temperature incubator, a 200 μ L pipette tip was used to draw a straight line in a vertical well plate, and washed three times with PBS to remove the scratched cells, which were divided into 3 groups: a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, wherein each group has 3 holes. Adding 1000 mul of DMEM complete culture medium into each well of the control group; each well of lycorine group is added with 1000 mul of DMEM complete medium + lycorine medium (lycorine content is 10 mul), and each well of DSPE-PEG modified lycorine nanoparticle group is added with 1000 mul of DMEM complete medium + DSPE-PEG modified lycorine nanoparticle medium (lycorine content is 10 mul).
Continuing at 37 ℃ with 5% CO2The culture was carried out in a constant temperature incubator for 48 hours, and samples were taken and photographed at 0 hour and 48 hours. After the pictures are opened by using Image J software, 6 to 8 horizontal lines are randomly drawn, and the mean value of the distances between cells is calculated to obtain the percentage of wound healing distance between the scratches of the cells. The experiment was performed in triplicate. Data were analyzed using SPSS19.0 statistical software. The results are expressed as (mean. + -. standard deviation) and the comparison between two is carried out using the one-way varianceAnalysis and LSD.
As a result: scratch models are respectively established on a control group of liver cancer HepG2 cells, breast cancer MCF-7 cells and colorectal cancer SW480 cells, lycorine solution and DSPE-PEG modified lycorine nanoparticle groups, and changes of two groups of scratch areas are respectively observed at 0 h and 48h, and the results are shown in figure 7. After 48 hours, the wound healing distances among scratches of the liver cancer HepG2 cells of the control group, the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 46.88%, 26.22% and 8.77% respectively, which indicates that the proliferation and migration inhibition rates of the lycorine group on the liver cancer HepG2 cells are 39.45%; the proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to the liver cancer HepG2 cells is 49.66%. The proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to liver cancer HepG2 cells is 1.26 times that of the lycorine group.
After 48 hours, the wound healing distances among scratches of the breast cancer MCF-7 cells of the control group, the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 57.69%, 23.07% and 12.96% respectively, which indicates that the proliferation and migration inhibition rates of the lycorine group on the breast cancer MCF-7 cells are 17.18%; the proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to the breast cancer MCF-7 cells is 29.06%. The proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to breast cancer MCF-7 cells is 1.69 times that of the lycorine group.
After 48 hours, the wound healing distances among scratches of the colorectal cancer SW480 cells of the control group, the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 56.60%, 31.1% and 8% respectively, which indicates that the proliferation and migration inhibition rate of the lycorine group to the colorectal cancer SW480 cells is 20.20%; the proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group on colorectal cancer SW480 cells is 39.76%. The proliferation and migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group on colorectal cancer SW480 cells is 1.97 times that of the lycorine group.
The DSPE-PEG modified lycorine nanoparticles are proved to obviously inhibit the proliferation and migration of tumor cells compared with lycorine.
6. Experiment for inhibiting migration of tumor cells (transwell method)
Culturing liver cancer HepG2 cell, colorectal cancer SW480 cell and breast cancer MCF-7 cell to logarithmic phase, digesting the cells with trypsin, washing the cells with PBS 2-3 times, centrifuging to remove the supernatant, and re-suspending the cells to 1 × 10 with serum-free DMEM medium5Individual cells/mL. Respectively placing 200 μ L of liver cancer HepG2 cell, colorectal cancer SW480 cell and breast cancer MCF-7 cell suspension in upper chamber of Transwell, adding serum-containing culture medium (DMEM complete culture medium) 600 μ L in lower chamber, placing at 37 deg.C and 5% CO2After culturing for 24h in the constant temperature incubator, the culture medium in the upper chamber is discarded, and the culture medium is divided into 3 groups: a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, each group having 3 small chambers. Control group 200. mu.l DMEM complete medium was added to each upper chamber; 200 mul of DMEM complete medium and lycorine medium (the content of lycorine is 10 mul) are added into each upper chamber of the lycorine group, 200 mul of DMEM complete medium and DSPE-PEG modified lycorine nanoparticle medium (the content of lycorine is 10 mul) are added into each hole of the DSPE-PEG modified lycorine nanoparticle group to continue culturing for 48 h. After the culture is finished, sucking the upper layer liquid of the chamber, taking out the chamber by using a pair of tweezers, wiping off the upper layer cells of the chamber by using a cotton swab, washing the upper layer cells for 2 times by using PBS (phosphate buffer solution), fixing the upper layer cells for 20min by using 4% paraformaldehyde, dyeing the upper layer cells for 15-30min by using 0.5% crystal violet (dissolved by using 2% ethanol or PBS), washing the redundant dyes by using clear water, naturally drying the cells with the bottom surfaces facing upwards, and observing and taking a picture under a microscope. The number of cells passing through the membrane in 5 different visual fields, i.e., upper, lower, left, and right, was selected under a 200-fold optical microscope, and the average value was calculated, and the ability to inhibit the migration of tumor cells was calculated according to the following formula. The migration inhibition rate (1-mean number of migrated cells in experimental group/mean number of migrated cells in control group) × 100%.
The experiment was performed in triplicate. Data were analyzed using SPSS19.0 statistical software. Results are expressed as (mean ± sd) and comparisons between pairs were examined using one-way anova and LSD.
The results showed that the number of cells that pass through the upper ventricle was significantly reduced for the lycorine group and the DSPE-PEG modified lycorine nanoparticle group, and the reduction was more significant for the DSPE-PEG modified lycorine nanoparticle group, compared to the control group (fig. 8). The migration inhibition rates of the lycorine group and the liver cancer HepG2 cells of the DSPE-PEG modified lycorine nanoparticle group are 38.64 +/-3.61 percent and 53.26 +/-4.71 percent respectively; the migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to the liver cancer HepG2 cells is 1.37 times that of the lycorine group. The migration inhibition rates of the colorectal cancer SW480 cells of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are respectively 20.69 +/-3.56 percent and 31.22 +/-1.13 percent; the migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group on colorectal cancer SW480 cells is 1.51 times that of the lycorine group. The migration inhibition rates of the mammary cancer MCF-7 cells of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 22.13 +/-7.9 percent and 49.76 +/-34.50 percent respectively; the migration inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to breast cancer MCF-7 cells is 2.24 times that of the lycorine group. The lycorine nano particles obviously inhibit the migration of tumor cells compared with lycorine.
7. Experiment for inhibiting invasion of tumor cell (transwell method)
Diluting 50mg/L Matrigel with serum-free DMEM medium at a ratio of 1:8 for coating bottom membrane of Transwell chamber, culturing at 37 deg.C for 30min to polymerize Matrigel, sucking out excessive liquid, culturing HepG2 cell, SW480 cell and MCF-7 cell to logarithmic growth phase, digesting the HepG2 cell, SW480 cell and MCF-7 cell with trypsin, washing the cells with PBS 2-3 times, centrifuging to suck out supernatant, and re-suspending the cells with serum-free DMEM medium to 1 × 105Individual cells/mL. 200. mu.L of the cell suspension was placed in the upper chamber of a Transwell, 600. mu.L of serum-containing medium (DMEM complete medium) was added to the lower chamber, and the mixture was incubated at 37 ℃ with 5% CO2After culturing for 24h in the constant temperature incubator, the culture medium in the upper chamber is discarded, and the culture medium is divided into 3 groups: control group, lycorine group and DSPE-PEG modified lycorine nano-particlesGroups, 3 cells each. Control group 200. mu.l DMEM complete medium was added to each upper chamber; 200 mul of DMEM complete medium and lycorine medium (the content of lycorine is 10 mul) are added into each upper chamber of the lycorine group, 200 mul of DMEM complete medium and DSPE-PEG modified lycorine nanoparticle medium (the content of lycorine is 10 mul) are added into each hole of the DSPE-PEG modified lycorine nanoparticle group to continue culturing for 48 h. After the culture is finished, sucking the upper layer liquid of the chamber, taking out the chamber by using a pair of tweezers, wiping off the upper layer cells of the chamber by using a cotton swab, washing the upper layer cells for 2 times by using PBS (phosphate buffer solution), fixing the upper layer cells for 20min by using 4% paraformaldehyde, dyeing the upper layer cells for 15-30min by using 0.5% crystal violet (dissolved by using 2% ethanol or PBS), washing away redundant dyes by using clear water, naturally drying the cells in the air with the bottom surface facing upwards, observing the cells under a microscope, randomly selecting a visual field for taking a picture and counting the number of the cells. At random 5 fields were counted under high power (x 200) and averaged. The invasion inhibitory ability of MANF-shRNA to tumor cells was calculated as follows. The invasion inhibition rate was (1-mean number of transmembrane cells in experimental group/mean number of transmembrane cells in control group) × 100%. The experiment was performed in triplicate. Data were analyzed using GraphPad Prism statistical software. Results are expressed as (mean ± sd) and comparisons between pairs were examined using one-way anova and LSD.
The results showed that the number of cells that pass through the upper ventricle was significantly reduced for the lycorine group and the DSPE-PEG modified lycorine nanoparticle group, and the reduction was more significant for the DSPE-PEG modified lycorine nanoparticle group, compared to the control group (fig. 9). The invasion inhibition rates of the lycorine group and the liver cancer HepG2 cells of the DSPE-PEG modified lycorine nanoparticle group are 27.92 +/-8.69 percent and 50.40 +/-5.27 percent respectively; the invasion inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to liver cancer HepG2 cells is 1.80 times that of the lycorine group. The invasion inhibition rates of the colorectal cancer SW480 cells of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 29.14 +/-0.20 percent and 44.18 +/-3.22 percent respectively; the invasion inhibition rate of the DSPE-PEG modified lycorine nanoparticle group on colorectal cancer SW480 cells is 1.51 times that of the lycorine group. The invasion inhibition rates of the mammary cancer MCF-7 cells of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are 39.52 +/-3.90 percent and 57.61 +/-5.43 percent respectively; the invasion inhibition rate of the DSPE-PEG modified lycorine nanoparticle group to breast cancer MCF-7 cells is 1.46 times that of the lycorine group. The lycorine nano particles are compared with lycorine, so that the invasion of tumor cells is obviously inhibited.
8. Promoting effect on tumor cell autophagy
Dividing liver cancer HepG2 cells, colorectal cancer SW480 cells and breast cancer MCF-7 cells into a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, respectively treating the liver cancer HepG2 cells, the colorectal cancer SW480 cells and the breast cancer MCF-7 cells for 48h by using lycorine and DSPE-PEG modified lycorine nanoparticles, and respectively detecting the expression conditions of autophagy marker proteins LC-3 and p62 of the three groups of cells by using a Westerrn Blot in the same step 5.1 as a specific experimental method. The antibodies used were SQSTM1/p62(D5E2) antibody, LC-3B (D11) antibody and anti- β -Actin (A5441) antibody (Cell Signaling technologies (Danvers, MA, USA) product) and were subjected to Western blot analysis for the content of p62, LC-3B and Actin (as internal reference). The Westerrn Blot result shows (fig. 10) that compared with a control group, the expressions of LC-3 proteins of lycorine and DSPE-PEG modified lycorine nanoparticles are increased, the expression of p62 protein is reduced, and the effect of the DSPE-PEG modified lycorine nanoparticles is more obvious, which indicates that the DSPE-PEG modified lycorine nanoparticles have a stronger effect on promoting autophagy of liver cancer HepG2 cells, colorectal cancer SW480 cells and breast cancer MCF-7 cells than the lycorine, thereby inhibiting the occurrence and development of tumors.
9. Western Blot Western Blot method for detecting expression levels of transfer marker proteins E-cadherin and Vimentin
Dividing the liver cancer HepG2 cells, the colorectal cancer SW480 cells and the breast cancer MCF-7 cells into a control group, a lycorine group and a DSPE-PEG modified lycorine nanoparticle group, respectively treating the liver cancer HepG2 cells, the colorectal cancer SW480 cells and the breast cancer MCF-7 cells for 48h by using the lycorine group and the DSPE-PEG modified lycorine nanoparticles, and respectively detecting the expression conditions of three groups of cell transfer marker proteins E-cadherin and Vimentin by using a Westerrn Blot in the same step 4 in a specific experimental method. The results of Westerrn Blot show that compared with a control group, the expressions of E-cadherin proteins of the lycorine group and the DSPE-PEG modified lycorine nanoparticle group are increased, the expression of Vimentin protein is reduced, and the effect of the DSPE-PEG modified lycorine solution group is more obvious, which indicates that the DSPE-PEG modified lycorine nanoparticles have stronger inhibiting effect on the transfer of liver cancer HepG2 cells, colorectal cancer SW480 cells and breast cancer MCF-7 cells than the lycorine solution group, thereby inhibiting the occurrence and development of tumors (figure 11).
10. In vivo experiments in mice
10.1 preparation of the medicament
Weighing lycorine powder, adding into anhydrous ethanol, placing in a water bath at 60 deg.C, heating to dissolve to obtain lycorine ethanol solution with lycorine concentration of 3mM, storing in equal parts at 4 deg.C, and diluting with PBS to obtain lycorine liquid.
The DSPE-PEG modified lycorine nanoparticles of example 2 were dispersed in PBS and the resulting liquid was called DSPE-PEG modified lycorine nanoparticle liquid.
10.2 in situ liver cancer mouse experiment
One week after adapted feeding of Kunming mice, 5 × 10 mice prepared for suspension in PBS6H22 mouse liver cancer cells of each/mL are injected into a mouse body in a subcutaneous injection mode to construct a tumor-bearing mouse model. Observing the state of mouse and subcutaneous tumor size every day, killing the mouse when the tumor grows to a certain size, taking off subcutaneous tumor, and cutting into 1mm tumor size3The size of the product is placed in normal saline for later use.
Ordering Kunming mice of 4 weeks old in advance, adaptively feeding for one week, anesthetizing the mice, opening the abdominal cavity by operation, placing 2-3 tumor small blocks on the liver of the mice by using straight forceps, suturing the abdominal cavity of the mice by using an operation line, smearing iodine solution for disinfection, and observing the awakening condition of the mice the next day. The mice were normally bred for two weeks, and the surviving mice weighing 41.24 ± 5.25g were randomly divided into three groups, 5 mice each, control group, lycorine group (lycorine group), and DSPE-PEG modified lycorine nanoparticle group (DSPE-PEG lycorine nanoparticle group). Each mouse of the lycorine group was injected with 0.2mL of lycorine liquid every two days of tail vein injection, so that the administration dose of the mouse was 5mg of lycorine/kg of body weight; each mouse of the DSPE-PEG modified lycorine nanoparticle group is injected with 0.2mL of DSPE-PEG modified lycorine nanoparticle liquid every two days of tail vein injection, so that the administration dose of the mouse is 5mg of lycorine/kg of body weight calculated by the lycorine; each mouse in the control group was administered every two days with 0.2mL PBS injected into the tail vein every two days, and after 22 days, the mice were sacrificed (all animal experiments were performed under approval of the animal care and use committee of the tianjin medical university), and tissues and organs such as lung and liver were collected. The data were processed using SPSS11.5 statistical software, and the results were expressed as mean. + -. standard deviation, and as One-way ANOVA test, where a (P < 0.05) indicates significant difference from the control group, a (P < 0.01) indicates significant difference from the control group, and a # (P < 0.05) indicates significant difference from the lycoris radiata alkali solution group.
As a result: establishing a mouse in-situ liver cancer model, carrying out treatment on the mouse by using lycorine and DSPE-PEG modified lycorine nanoparticles for 22 days, anesthetizing the mouse, dissecting and photographing, and observing the growth condition of liver tumors in each group of mice. The results showed that the size and number of tumors grown by the lycorine group and the DSPE-PEG modified lycorine nanoparticle group were all inhibited compared to the control group (fig. 12 (a) and (b)), and lung metastasis of liver cancer in mice could be inhibited to some extent (fig. 12 (c) and (d)), and the change in body weight of mice during treatment was as in fig. 12 (e)), with no significant difference between the groups. Compared with the control group, the volume of the tumor grown by the lycorine group and the DSPE-PEG modified lycorine nanoparticle group is 160.1 +/-119.388 mm3、28.7±22.25mm3And 4.2. + -. 2.86mm3. The lycorine nano particles are compared with lycorine, so that the growth and the metastasis of tumor liver cancer are obviously inhibited.
Mice are sacrificed at the fourth week of administration, livers are taken out, and the change conditions of liver tissue autophagy, apoptosis and EMT marker protein expression of different groups of mice are analyzed through immunohistochemical staining and Western Blot Western Blot experiments, so that the apoptosis, autophagy and EMT processes regulated by the DSPE-PEG modified lycorine nanoparticles are further analyzed. Western Blot results show that the expression of the DSPE-PEG modified lycorine nanoparticle group is higher than that of lycorine autophagy marker protein LC-3, the expression of p62 is reduced, the protein level of EMT marker protein E-Cadherin is obviously increased, and the expression of apoptosis marker proteins, namely, Cleaved caspase-3 and Bax protein is reduced (figure 12 (f)). Immunohistochemistry experimental results showed that the levels of DSPE-PEG modified lycorine nanoparticles group E-Cadherin, LC-3 protein were increased and Ki67 protein was decreased compared to the lycorine group (fig. 12 (g)).
10.3 in situ colorectal cancer experiments
C57BL/6J mice 5 weeks old were acclimatized and fed for one week, 10 mice were randomly selected as a control group and intraperitoneally injected with physiological saline, and the remaining mice were intraperitoneally injected with a single dose of a mutagen azomethane (AOM, Sigma-Aldrich)10mg/kg, and 5 days after normal drinking water (drinking ultrapure water), 2.5% DSS water (liquid obtained by adding dextran sulfate sodium salt (DSS) to DSS in a mass content of 2.5% to the ultrapure water) was administered for 5 days, and after that, normal drinking water (drinking ultrapure water) was administered for 14 days, which was the first cycle and three cycles during which diet was normal. After drinking normal water (drinking ultrapure water) in the second circulation, dead mice were removed, the remaining mice with a weight of 24.96 ± 1.78g were divided into three groups (model group, lycorine group (lycorine solution group), DSPE-PEG modified lycorine nanoparticle group (DSPE-PEG lycorine nanoparticle group), 5 mice per group, 0.2mL of lycorine liquid was intravenously injected every two days into the tail of each mouse of the lycorine group, so that the administration dose of the mice was 5mg of lycorine/kg of body weight, 0.2mL of DSPE-PEG modified lycorine nanoparticle liquid was intravenously injected every two days into the tail of each mouse of the DSPE-PEG modified lycorine nanoparticle group, so that the administration dose of the mice was 5mg of lycorine/kg of body weight, 0.2mL of PBS was intravenously injected every two days into each mouse of the model group, every two days, administration was performed every once, after 22 days (fig. 13 (a)), the mice were decapped and sacrificed (all animal experiments were performed with approval of the animal care and use committee of the tianjin university of medicine) to collect tissue organs such as the colorectal. Data were analyzed using SPSS19.0 statistical software. The results are expressed as (mean ± sd), the comparison between two pairs was examined by one-way anova analysis and LSD, where x (P < 0.05) indicates significant difference from the control group, x (P < 0.001) indicates very significant difference from the control group, $ (P < 0.05) indicates significant difference from the model group, and # (P < 0.05) indicates significant difference from the lycoris radiata alkali solution group.
As a result: studies showed that the model group had a significantly shorter colorectal length compared to the control group, and that the colorectal length was significantly restored after administration of lycorine and DSPE-PEG modified lycorine nanoparticles, while the length restored by DSPE-PEG modified lycorine nanoparticles was most significant (fig. 13 (b), (d)). In FIG. 13 (d), Ly represents a lycorine group, and DS-Ly NPs represent a DSPE-PEG modified lycorine nanoparticle group.
The number of tumors in the model group was significantly increased compared to the control group, and the number of tumors was gradually decreased after administration of lycorine and DSPE-PEG modified lycorine nanoparticles, which were the least numerous (fig. 13 (c)). The overall body weight of the mice decreased during DSS administration. Treatment with DSPE-PEG modified lycorine nanoparticles gradually restored the lost body weight over time (fig. 13 (f), Mod model group, Con control group). The Western Blot result shows that compared with the control group, the expressions of mouse autophagy protein p62 and EMT-related protein Vimentin of the model group are increased, the expressions of apoptosis-related proteins clear caspase-3, autophagy-related protein LC-3 and EMT-related protein E-Cadherin are all reduced, the autophagy, apoptosis and transfer protein amounts are recovered after the treatment of the lycorine and DSPE-PEG modified lycorine nanoparticles, and the treatment effect of the DSPE-PEG modified lycorine nanoparticles is more obvious (figure 13 (h)). Observed through an HE staining experiment, the control group has regular gland arrangement, contains abundant goblet cells, the model group has disordered gland arrangement, the number of the goblet cells is reduced compared with the normal group, after the lycorine and the DSPE-PEG modified lycorine nano particles are used for treatment, the pathological state is improved, the glands tend to be arranged regularly, the number of the goblet cells is increased, and the effect is more obvious after the DSPE-PEG modified lycorine nano particles are used for treatment (fig. 13 (e)).
The immunohistochemical result shows that the expression of Ki67 protein in intestinal mucosa interstitial substance of a mouse in a control group is lower, the expression of the protein in a model group is obviously increased compared with the control group, the protein expression of Ki67 is reduced after lycorine and DSPE-PEG modified lycorine nano particles are administered compared with the model group, and the recovery of the DSPE-PEG modified lycorine nano particles is obvious; the intestinal mucosa interstitial LC-3 and E-Cadherin proteins of the mice in the normal group are highly expressed, compared with the model group, the LC-3 and E-Cadherin proteins are obviously reduced, the LC-3 and E-Cadherin proteins are recovered after the lycorine solution and the DSPE-PEG modified lycorine nano particles are administered, and the DSPE-PEG modified lycorine nano particles are obviously recovered (fig. 13 (g)).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
- The application of the DSPE-PEG modified lycorine nanoparticles or lycorine nanoparticles in preparing products for improving the water dispersibility of lycorine and/or prolonging the half-life of lycorine and/or improving the antitumor activity of lycorine and/or improving the antitumor cell activity of lycorine; the DSPE-PEG modified lycorine nanoparticles consist of DSPE-PEG and the lycorine nanoparticles loaded by the DSPE-PEG.
- 2. Use according to claim 1, characterized in that: the tumor is a solid tumor, and the tumor cell is a solid tumor cell.
- 3. Use according to claim 1 or 2, characterized in that: the solid tumor is liver cancer, colorectal cancer, colon cancer, rectal cancer and/or breast cancer, and the solid tumor cell is liver cancer cell, colorectal cancer cell, colon cancer cell, rectal cancer cell and/or breast cancer cell.
- 4. Use according to any one of claims 1 to 3, characterized in that: the diameter of the lycorine nano particles is 42-47nm, and the particle size of the DSPE-PEG modified lycorine nano particles is 48-55 nm.
- 5. Use according to any one of claims 1 to 4, characterized in that: the lycorine nanoparticles prepared according to the method of claim 6, the DSPE-PEG loaded lycorine nanoparticles prepared according to the method of claim 7.
- 6. A process for the preparation of the lycorine nanoparticles for use according to any one of claims 1 to 4, comprising adding an ethanolic solution of lycorine to water to obtain lycorine nanoparticles.
- 7. A method of making the DSPE-PEG modified lycorine nanoparticles of claims 1-4 comprising reacting lycorine nanoparticles with DSPE-PEG to obtain DSPE-PEG modified lycorine nanoparticles.
- 8. Use of the lycorine nanoparticles or the DSPE-PEG modified lycorine nanoparticles of any one of claims 1-5 for the preparation of a nano-formulation.
- 9. Use according to claim 8, characterized in that: the nanometer preparation is an anti-tumor preparation.
- 10. Any one of the following products:1) lycorine nanoparticles according to any one of claims 1-5,2) the DSPE-PEG modified lycorine nanoparticles of any one of claims 1-5,3) a nano-formulation comprising the lycorine nanoparticles of any one of claims 1-5,4) a nano-formulation comprising DSPE-PEG modified lycorine nanoparticles as claimed in any one of claims 1 to 5.
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