CN116370436A - Application of emodin polymer lipid hybrid nanoparticles - Google Patents
Application of emodin polymer lipid hybrid nanoparticles Download PDFInfo
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- CN116370436A CN116370436A CN202310252352.5A CN202310252352A CN116370436A CN 116370436 A CN116370436 A CN 116370436A CN 202310252352 A CN202310252352 A CN 202310252352A CN 116370436 A CN116370436 A CN 116370436A
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- emodin
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- polymer lipid
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- A—HUMAN NECESSITIES
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- 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
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
- A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
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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
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- 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
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Abstract
The invention discloses an application of emodin polymer lipid hybrid nano-particles, which are used for IL-6/JAK2/STAT3 signal path inhibitors; the emodin polymer lipid hybrid nanoparticle consists of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3-4:20-25:40-50:8-9. The emodin polymer lipid hybrid nanoparticle provided by the invention has good hydrophilicity, can stably exist in blood circulation and accumulate at a tumor site, enters breast cancer drug-resistant cells through endocytosis, degrades lipid shells and polymer coating layers through endosomes or lysosomes, releases free emodin, and influences the expression of IL-6/JAK2/STAT3 signal pathway proteins through being combined with a hydrophobic pocket of a target receptor, so that the emodin polymer lipid hybrid nanoparticle can be used for IL-6/JAK2/STAT3 signal pathway inhibitors, can be further used for signal pathway regulators for improving breast cancer drug resistance, and improves the breast cancer treatment effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, relates to a new application of emodin polymer lipid hybridization nanoparticles, and in particular relates to an application of emodin polymer lipid hybridization nanoparticles serving as IL-6/JAK2/STAT3 signal pathway inhibitors.
Background
Breast cancer is one of the common malignant tumors of women, more than 130 thousands of new cases of breast cancer occur annually in the world, and more than 50 tens of thousands of people die due to the breast cancer. The main treatment method of the breast cancer is chemotherapy, but due to the fact that the breast cancer is classified into various types, the problem of multi-drug resistance (MDR) is often induced, so that the sensitivity of tumors to chemotherapeutic drugs is reduced, and the chemotherapy is failed.
At present, research for overcoming tumor MDR is mainly focused on tumor cells per se, such as inhibiting up-regulation of efflux pump proteins by changing multi-drug resistance genes, reducing drug efflux, inhibiting metabolic activity, enhancing drug activity, or regulating expression of apoptosis genes, inducing apoptosis of tumor cells, etc., but the effect is not ideal because the generation of tumor cell drug resistance is not only caused by the change of endogenous cell, but also involves the change of microenvironment in which the tumor cell is located.
The Tumor Microenvironment (TME) refers to a local steady-state environment composed of tumor cells, fibroblasts, mesenchymal cells, vascular endothelial cells, smooth muscle cells, inflammatory/immune cells, extracellular matrix and the like, and is significantly different from the normal tissue microenvironment, and is mainly characterized by hypoxia, subacidity, reducibility, low vascular density and the like. Tumor microenvironment plays an important role in the tumorigenic development process of immune escape, distant metastasis and the like, and researches show that TME participates in the formation process of tumor MDR: TME blocks the penetration of chemotherapeutic drugs into tumor tissues by forming physiological barriers, influences the drug efficacy and promotes tumor resistance, and a large number of immune cells, fibroblasts and active mediators in TME have correlation with tumor MDR occurrence. For example, tumor cells activate JAK/STAT3 signaling pathway and anti-apoptosis gene bcl-2 by secreting IL-6, secrete CXCL12, activate Wnt/beta-catenin pathway, induce epithelial transformation of breast cancer cells to interstitium, thereby inducing breast cancer cell resistance, but few reports exist at present on using IL-6/JAK2/STAT3 signaling pathway inhibitors for reversing breast cancer resistance, mainly because monoclonal antibody drugs are expensive and have limited curative effects.
The inhibition of IL-6/JAK2/STAT3 signaling pathway by natural components has become a new idea for solving the above-mentioned problems, and among many natural components, emodin (EMO, C1) 5 H 10 O 5 ) Is a natural anthraquinone derivative, has the effects of resisting inflammation, inhibiting bacteria, regulating immunity, resisting tumor and the like, and the anti-tumor action mechanism comprises the steps of inducing DNA damage of tumor cells, inducing ROS to generate, inhibiting MMP expression, inhibiting the activity of Akt and NF- κB signal paths, reducing Bcl-2/Bax ratio, increasing Caspase-3/9 activation and the like. Emodin has the potential of treating breast cancer, but has the problems of poor water solubility and low bioavailability.
In order to solve the technical problems, the water solubility and the bioavailability of the emodin can be improved by combining the emodin with polymer lipid hybrid nanoparticles (PLNs), and the PLNs have the advantages of both liposome and nanoparticles, and have the characteristics of controllable particle size, stable structure, high drug loading capacity, sustainable release of drugs, easy surface modification, spontaneous formation and the like.
However, there is no report on the use of emodin polymer lipid hybrid nanoparticles for inhibitors of the IL-6/JAK2/STAT3 signaling pathway.
In view of this, it is necessary to provide a new use of emodin polymer lipid hybrid nanoparticles.
Disclosure of Invention
Therefore, the invention aims to solve the technical problems in the background technology, and provides application of the emodin polymer lipid hybrid nanoparticle.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the invention provides an application of an emodin polymer lipid hybridization nanoparticle, wherein the emodin polymer lipid hybridization nanoparticle is used for an IL-6/JAK2/STAT3 signal pathway inhibitor; wherein the emodin polymer lipid hybrid nanoparticle consists of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3-4:20-25:40-50:8-9.
Preferably, the emodin polymer lipid hybrid nanoparticle is used for preparing a drug for reversing the drug resistance of MCF-7/ADR breast cancer cells.
Preferably, the emodin polymer lipid hybrid nanoparticles are used as IL-6/JAK2/STAT3 signaling pathway inhibitors of MCF-7/ADR breast cancer cells.
Preferably, the emodin and the polylactic acid-glycolic acid copolymer form an organic phase, the lipid material is encapsulated outside the organic phase, and the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is modified on the surface of the lipid material.
Preferably, the emodin polymer lipid hybrid nanoparticle consists of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3:20:40:9.
Preferably, the lipid material is at least one of soybean phospholipid, soybean lecithin, dilauroyl lecithin, dimyristoyl phosphatidylcholine or (2, 3-dioleoyl-propyl) -trimethylamine.
Preferably, the emodin polymer lipid hybrid nanoparticle is prepared by the following steps:
s1, mixing emodin with a polylactic acid-glycolic acid copolymer according to a proportion, and then dissolving the mixture by using an organic solvent to form an organic phase;
s2, respectively weighing lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to a proportion, dissolving the lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in an emulsifier, heating and stirring the mixture until a water phase is formed;
and S3, adding the organic phase into the water phase, heating, stirring, centrifuging, and filtering the supernatant with a water system filter membrane to obtain the emodin polymer lipid hybrid nanoparticle.
Preferably, in the step S2, the heating temperature is 65-75 ℃, the stirring speed is 750-850rpm, and the stirring time is 2-4min.
Preferably, in the step S3, the heating temperature is 65-75 ℃, the stirring speed is 750-850rpm, the stirring time is 40-60min, the rotational speed of the centrifugation is 900-1100rpm, and the centrifugation time is 4-6min.
Preferably, the medicament is an injection.
Compared with the prior art, the technical scheme of the invention has the following advantages:
the invention provides an application of an emodin polymer lipid hybridization nanoparticle, which is used for an IL-6/JAK2/STAT3 signal path inhibitor; wherein the emodin polymer lipid hybrid nanoparticle consists of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3-4:20-25:40-50:8-9. The emodin polymer lipid hybrid nanoparticle provided by the invention has good hydrophilicity, can stably exist in blood circulation and accumulate at a tumor site, enters breast cancer drug-resistant cells through endocytosis, degrades lipid shells and polymer coating layers through endosomes or lysosomes, releases free emodin, and influences the expression of IL-6/JAK2/STAT3 signal pathway proteins through being combined with a hydrophobic pocket of a target receptor, so that the emodin polymer lipid hybrid nanoparticle can be used for IL-6/JAK2/STAT3 signal pathway inhibitors, can be further used for signal pathway regulators for improving breast cancer drug resistance, and improves the breast cancer treatment effect.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which
FIGS. 1 (a) - (b) are appearance morphology diagrams of dispersions and lyophilized powders of emodin polymer lipid-hybrid nanoparticles provided in example 4 of the present invention;
FIG. 2 is a linear regression plot of EMO standard solution;
FIG. 3 is a particle size distribution plot of E-PLNs;
FIG. 4 is a Zeta potential diagram of an E-PLNs;
FIG. 5 is a graph of UV absorption spectra of EMO, PLNs, EMO-PLNs physical mixtures, E-PLNs;
FIG. 6 is an infrared scan spectrum of EMO, PLNs, EMO-PLNs physical mixtures, E-PLNs;
FIG. 7 is an X-ray diffraction pattern of a EMO, PLNs, EMO-PLNs physical mixture, E-PLNs;
FIG. 8 is a differential scanning calorimetric profile of a EMO, PLNs, EMO-PLNs physical mixture, E-PLNs;
FIG. 9 is a confocal imaging of MCF-7 and MCF-7/ADR cells;
FIGS. 10 (a) - (b) are graphs of flow cytometry testing of MCF-7 and MCF-7/ADR cells;
FIG. 11 is a graph of fluorescence intensity assays for MCF-7 and MCF-7/ADR cells;
FIG. 12 is a chart of toxicity test of DOX on MCF-7 and MCF-7/ADR cells;
FIGS. 13 (a) - (c) are graphs of cytotoxicity assays with EMO, E-PLNs, alone and in combination with DOX, respectively;
FIG. 14 is an MCF-7/ADR apoptosis laser confocal image;
FIGS. 15 (a) - (b) are graphs of MCF-7/ADR apoptosis flow assays;
FIGS. 16 (a) - (b) are Western blot charts;
FIGS. 17 (a) - (b) are graphs showing the detection of DOX accumulation in MCF-7/ADR cells treated with different dosing groups;
FIGS. 18 (a) - (b) are graphs showing the measurement of MCF-7/ADR cell protein expression by treatment of different dosing groups;
FIGS. 19 (a) - (b) are graphs showing the effect of E-PLNs in combination with DOX on the expression of IL-6 and IFN-gamma in MCF-7/ADR cells;
FIG. 20 is a graph of the effect of IL-6 on MCF-7/ADR cell viability;
FIGS. 21 (a) - (b) are graphs showing the reversal of IL-6 induced apoptosis protein in MCF-7/ADR by E-PLNs;
FIGS. 22 (a) - (b) are graphs showing apoptosis induced by DOX in MCF-7/ADR cells following IL-6 induction;
FIGS. 23 (a) - (b) are graphs showing the number of apoptosis when DOX+EMO and DOX+E-PLNs were added;
FIGS. 24 (a) - (b) are graphs showing drug resistance protein expression following IL-6 induction of MCF-7/ADR cells;
FIGS. 25 (a) - (b) are graphs showing drug resistance protein expression following IL-6+DOX induction of MCF-7/ADR cells;
FIGS. 26 (a) - (b) are graphs showing the levels of MCF-7/ADR cell JAK2/STAT3 pathway protein expression following administration of E-PLNs in combination with DOX;
FIGS. 27 (a) - (b) are graphs showing the levels of JAK2/STAT3 pathway protein expression following the combined administration of DOX with EMO, E-PLNs under IL-6 intervention.
Detailed Description
The invention is further illustrated by the following examples. It should be understood that the methods described in the examples of the present invention are only for illustrating the present invention, and not for limiting the present invention, and that simple modifications to the preparation methods of the present invention under the concept of the present invention are within the scope of the present invention as claimed. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1
The present embodiment provides an application of an emodin polymer lipid hybrid nanoparticle, specifically, the application of an emodin polymer lipid hybrid nanoparticle (E-PLNs) as an IL-6/JAK2/STAT3 signal path inhibitor, wherein the emodin polymer lipid hybrid nanoparticle is composed of emodin, polylactic acid-glycolic acid copolymer (PLGA), a lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) according to a mass ratio of 4:25:50:9, and the lipid material is preferably soybean phospholipid.
The emodin and PLGA form an organic phase, the soybean phospholipid is encapsulated outside the organic phase, and DSPE-PEG2000 is modified on the surface of the soybean phospholipid.
The rheum emodin polymer lipid hybrid nano-particles are prepared through the following steps:
S1, mixing emodin with a polylactic acid-glycolic acid copolymer according to a proportion, and dissolving the mixture with acetone to form an organic phase;
s2, respectively weighing lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to a proportion, dissolving the lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in a Pluronic F68 emulsifier, heating the mixture in a water bath at 65 ℃ and stirring the mixture for 4min at a rotating speed of 750rmp to form a water phase;
and S3, uniformly dripping the organic phase obtained in the step S1 into the water phase obtained in the step S2, heating in a water bath at 65 ℃ and stirring for 60min at a rotating speed of 750rmp, centrifuging for 6min at a rotating speed of 900rpm, and freeze-drying the supernatant after passing through a water-based filter membrane to obtain the emodin polymer lipid hybrid nano-particles.
The application of the emodin polymer lipid hybrid nanoparticle provided by the embodiment is that the emodin polymer lipid hybrid nanoparticle (E-PLNs) is a nano preparation obtained by loading emodin on a polymer core of the polymer lipid hybrid nanoparticle, compared with free Emodin (EMO), the emodin polymer lipid hybrid nanoparticle has better bioavailability and tumor targeting, has good hydrophilicity, can be uniformly dispersed in an aqueous solution in a small particle form, can stably exist in blood circulation, and can be accumulated in a tumor site due to large interstitial pressure and increased gaps among vascular endothelial cells, so that the emodin polymer lipid hybrid nanoparticle with the particle size smaller than 200nm is allowed to pass through blood vessels and accumulate in the tumor site; at the tumor site, the molecular structure of the emodin is not easily damaged due to the protection effect of the polymer coating the emodin and the lipid shell, the lipid shell is the same as the cell membrane component, so as to be beneficial to penetrating the cell membrane, the emodin polymer lipid hybrid nano-particles enter the tumor cells through endocytosis to play a role, and in the tumor cells, the emodin polymer lipid hybrid nano-particles are firstly degraded by endosomes or lysosomes to release free emodin, and then the emodin is combined with a hydrophobic pocket of a target receptor of the emodin, so that a series of signal transmission related to the target receptor is influenced.
The emodin polymer lipid hybrid nanoparticle enters into breast cancer drug-resistant cells through endocytosis, lipid shells and polymer coating layers are degraded through endosomes or lysosomes, free emodin is released, the free emodin influences the expression of IL-6/JAK2/STAT3 signal pathway proteins through the combination with a hydrophobic pocket of a target receptor, and therefore the emodin polymer lipid hybrid nanoparticle can be used as an IL-6/JAK2/STAT3 signal pathway inhibitor.
The emodin polymer lipid hybrid nanoparticle inhibits the expression of IL-6/JAK2/STAT3 signal pathway proteins, thereby influencing the expression of downstream survivin proteins P53, fas, bax, bcl-2 and drug-resistant proteins P-gp and MRP1, and specifically comprises the following steps: the preparation method has the advantages that P-gp, MRP1 and P53 expression is down-regulated, fas expression is up-regulated, the Bax/Bcl-2 ratio is improved, the effect of reversing the drug resistance of breast cancer cells is realized, and the preparation method can be further used for preparing drugs for reversing the drug resistance of MCF-7/ADR breast cancer cells, wherein the drugs are preferably injections and are administered in an intravenous injection mode.
The emodin polymer lipid hybrid nanoparticle can play a role in inhibiting the activity of IL-6/JAK2/STAT3 signal pathway of MCF-7/ADR breast cancer cells by reducing the expression of JAK2, STAT3, p-JAK2 and p-STAT3 proteins, and can be further applied to IL-6/JAK2/STAT3 signal pathway inhibitors of the MCF-7/ADR breast cancer cells.
In conclusion, the application of the emodin polymer lipid hybrid nano-particles as signal path and molecular network regulator provided by the embodiment is a novel way of breaking through innovation in the technical field of biological medicine disease prevention and treatment, and the novel application of the emodin polymer lipid hybrid nano-particles as signal path regulator in breast cancer resistance is developed for the first time, so that the emodin polymer lipid hybrid nano-particles have the effect of reversing breast cancer resistance, and the treatment effect of breast cancer is improved.
Example 2
The embodiment provides application of an emodin polymer lipid hybridization nanoparticle, in particular application of the emodin polymer lipid hybridization nanoparticle (E-PLNs) serving as an IL-6/JAK2/STAT3 signal pathway inhibitor, and the emodin polymer lipid hybridization nanoparticle is further used for preparing a drug for reversing drug resistance of MCF-7/ADR breast cancer cells and an IL-6/JAK2/STAT3 signal pathway inhibitor applied to the MCF-7/ADR breast cancer cells.
The application of the emodin polymer lipid hybrid nanoparticle provided by the embodiment, wherein the emodin polymer lipid hybrid nanoparticle is composed of emodin, PLGA, a lipid material and DSPE-PEG2000 according to the mass ratio of 3.5:22:45:8.5, and the lipid material is soybean lecithin. The emodin and PLGA form an organic phase, the soybean lecithin is encapsulated outside the organic phase, and DSPE-PEG2000 is modified on the surface of the soybean lecithin.
The rheum emodin polymer lipid hybrid nano-particles are prepared through the following steps:
s1, mixing emodin with PLGA according to a proportion, and dissolving with acetone to form an organic phase;
s2, respectively weighing the lipid material and DSPE-PEG2000 according to a proportion, dissolving in Pluronic F68 emulsifier, heating in a water bath at 70 ℃ and stirring for 3min at 800rmp until a water phase is formed;
and S3, uniformly dripping the organic phase obtained in the step S1 into the water phase obtained in the step S2, heating in a water bath at 70 ℃, stirring for 50min at the rotation speed of 800rmp, centrifuging for 5min at the rotation speed of 1000rpm, and freeze-drying the supernatant after passing through a water-based filter membrane to obtain the emodin polymer lipid hybrid nano-particles.
Example 3
The embodiment provides application of an emodin polymer lipid hybridization nanoparticle, in particular application of the emodin polymer lipid hybridization nanoparticle (E-PLNs) serving as an IL-6/JAK2/STAT3 signal pathway inhibitor, and the emodin polymer lipid hybridization nanoparticle is further used for preparing a drug for reversing drug resistance of MCF-7/ADR breast cancer cells and an IL-6/JAK2/STAT3 signal pathway inhibitor applied to the MCF-7/ADR breast cancer cells.
The application of the emodin polymer lipid hybrid nanoparticle provided by the embodiment, wherein the emodin polymer lipid hybrid nanoparticle is composed of emodin, PLGA, a lipid material and DSPE-PEG2000 according to the mass ratio of 3:20:40:9, and the lipid material is a mixture of dilauroyl lecithin and dimyristoyl phosphatidylcholine. The emodin and PLGA form an organic phase, the lipid material is encapsulated outside the organic phase, and DSPE-PEG2000 is modified on the surface of the lipid material.
The rheum emodin polymer lipid hybrid nano-particles are prepared through the following steps:
s1, mixing emodin with PLGA according to a proportion, and dissolving with acetone to form an organic phase;
s2, respectively weighing the lipid material and DSPE-PEG2000 according to a proportion, dissolving in Pluronic F68 emulsifier, heating in a water bath at 75 ℃ and stirring for 2min at a rotation speed of 850rmp to form a water phase;
and S3, uniformly dripping the organic phase obtained in the step S1 into the water phase obtained in the step S2, heating in a water bath at 75 ℃, stirring for 40min at a rotation speed of 850rmp, centrifuging for 4min at a rotation speed of 1100rpm, and freeze-drying the supernatant after passing through a water-based filter membrane to obtain the emodin polymer lipid hybrid nano-particles.
Example 4
The embodiment provides application of an emodin polymer lipid hybridization nanoparticle, in particular application of the emodin polymer lipid hybridization nanoparticle (E-PLNs) serving as an IL-6/JAK2/STAT3 signal pathway inhibitor, and the emodin polymer lipid hybridization nanoparticle is further used for preparing a drug for reversing drug resistance of MCF-7/ADR breast cancer cells and an IL-6/JAK2/STAT3 signal pathway inhibitor applied to the MCF-7/ADR breast cancer cells.
The application of the emodin polymer lipid hybrid nanoparticle provided by the embodiment, wherein the emodin polymer lipid hybrid nanoparticle consists of emodin, PLGA, a lipid material and DSPE-PEG2000 according to the mass ratio of 3:20:40:9. The emodin and PLGA form an organic phase, the lipid material is encapsulated outside the organic phase, and DSPE-PEG2000 is modified on the surface of the lipid material.
The rheum emodin polymer lipid hybrid nano-particles are prepared through the following steps:
s1, precisely weighing 3mg of emodin and 20mg of PLGA, and dissolving with acetone to form an organic phase;
s2, precisely weighing 40mg of soybean phospholipid and 9mg of DSPE-PEG2000, adding 32mL of 2.5% Pluronic F68 emulsifier, heating in a water bath at 75 ℃ and stirring for 3min at a rotation speed of 800rmp to form a water phase;
and S3, uniformly dripping the organic phase obtained in the step S1 into the water phase obtained in the step S2, heating in a water bath at 75 ℃, stirring for 50min at the rotation speed of 800rmp, centrifuging for 5min at the rotation speed of 1000rpm, and freeze-drying the supernatant after passing through a water-based filter membrane to obtain the emodin polymer lipid hybrid nano-particles.
Experimental example
The emodin polymer lipid hybrid nanoparticles (E-PLNs) used in the following test examples are all E-PLNs provided in example 4.
1. Appearance characterization of emodin Polymer lipid hybrid nanoparticles (E-PLNs)
The dispersion of the emodin polymer lipid hybrid nanoparticles prepared in example 4 was a yellow clear liquid with opalescence as shown in FIG. 1 (a); the lyophilized emodin polymer lipid hybrid nanoparticle powder is shown in fig. 1 (b), the surface of the lyophilized powder is smooth and full, and the result shows that the appearance and the shape of the emodin polymer lipid hybrid nanoparticle prepared in the embodiment 4 are good.
2. Determination of Emodin (EMO) content in emodin Polymer lipid hybrid nanoparticles (E-PLNs)
The content of emodin in the emodin polymer lipid hybrid nanoparticle prepared in example 4 was determined by High Performance Liquid Chromatography (HPLC), and the chromatographic conditions were as follows:
chromatographic column: agilent ZORBAX SB C18 (4.6 mm. Times.250 mm,5 μm); column temperature: 30 ℃; detection wavelength: 290nm; mobile phase: methanol-0.1% phosphoric acid (75:25); flow rate: 1.0mL/min; sample injection amount: 10 mu L.
EMO standard curve: 2.5mg of EMO standard substance is precisely weighed, methanol is taken as a solvent, the solution is fixed in a 50mL brown volumetric flask to obtain 50.0 mug/mL EMO mother liquor, the solution is sequentially diluted into linear solutions of 25.0 mug/mL, 12.5 mug/mL, 6.25 mug/mL, 3.125 mug/mL, 1.5625 mug/mL and 0.781 mug/mL, the EMO content is detected by high performance liquid chromatography, and a standard curve is drawn.
Peak areas of EMO standard solutions with different concentrations were determined by HPLC method, and linear regression was performed with EMO concentration and corresponding peak areaAnalysis gave the EMO linear regression equation as y=35.917x+8.3615 (R 2 =0.9999), the standard solution linear regression curve is shown in fig. 2, and the test result shows that the linear relationship of the EMO solution is good within the concentration range of 0.781 μg/mL to 50.0 μg/mL.
EMO content determination in E-PLNs: 1mL of E-PLNs dispersion is taken, 1mL of methanol is added, and after ultrasonic demulsification and filtration treatment, the EMO content is detected by sample injection high performance liquid chromatography.
3. Determination of EMO encapsulation and drug loading in E-PLNs
1mL of E-PLNs dispersion (E-PLNs content is M) is taken, 1mL of methanol is added, and after ultrasonic demulsification and filtration treatment, the EMO content (Mt) is detected by high performance liquid chromatography. Another 1mL E-PLNs dispersion was placed in an ultrafiltration centrifuge tube (30 kDa) and centrifuged at 12000rpm for 30min, after centrifugation was completed, the solution at the lower part of the ultrafiltration tube was fed to 1mL, and after filtration, EMO content (Mf) was detected by high performance liquid chromatography. And (3) respectively calculating the EMO encapsulation efficiency and the drug loading rate in the E-PLNs by using the formula (1) and the formula (2).
Formula (1): encapsulation Efficiency (EE)% = (Mt-Mf)/mt×100%;
formula (2): drug Loading (DL)% = (Mt-Mf)/mx100%.
3 batches of E-PLNs were prepared by the preparation method in example 4, and the encapsulation efficiency and drug loading of 3 batches of E-PLNs dispersion were measured according to the EMO linear regression equation obtained in experimental example 2, and the results are shown in Table 1:
TABLE 1
The test results show that: the prepared E-PLNs have higher encapsulation efficiency.
4. Determination of particle size and Zeta potential of E-PLNs
1mL of E-PLNs dispersion (diluted to an appropriate concentration with ultrapure water, 1mL was added to a square-well sample tube, and the Polydispersity (PDI), particle size, and Zeta potential were measured by a Zetasizer Nano ZS nm potentiometric particle size analyzer.
As shown in the test results of FIG. 3-4, the E-PLNs have a particle size of (123.1+ -0.32) nm, a Zeta potential of (-28.6+ -0.23) mV, and a polydispersity PDI of 0.135 (< 0.2), indicating that the nanoparticles have a smaller particle size and a uniform particle size distribution.
5. Ultraviolet absorbance detection of E-PLNs
Precisely weighing 2.0mg of EMO standard substance, placing in a 50mL volumetric flask, and diluting with methanol to obtain standard substance solution with concentration of 40 μg/mL; in addition, the empty carrier polymer lipid hybrid nanoparticle (PLNs), E-PLNs and PLNs-EMO physical mixture are precisely weighed, diluted to proper concentration by PBS buffer solution (the mixture is dissolved by proper amount of methanol and then PBS buffer solution is added), methanol and PBS are used as blank control respectively, and the absorbance of each sample is detected by an ultraviolet-visible spectrophotometer (200-800 nm).
The test results are shown in fig. 5, and the test results indicate that: EMO has larger absorption peaks at 225, 290 and 450nm, while empty carrier PLNs have no absorption peaks at the three wavelengths, and EMO-PLNs physical mixtures and E-PLNs have absorption peaks at the three wavelengths, and ultraviolet absorption test patterns prove the existence of EMO and ultraviolet absorption groups thereof in the E-PLNs.
6. Fourier Transform Infrared (FTIR) scan test of E-PLNs
Weighing a proper amount of EMO, PLNs, E-PLNs and PLNs-EMO physical mixture according to the weight ratio of 1:100, respectively mixing with dried potassium bromide powder in mortar, and measuring at 4000-400cm with Fourier transform infrared spectrophotometer -1 Each sample was scanned to analyze the phase state of EMO in E-PLNs.
The FTIR spectra of the EMO, PLNs, EMO-PLNs physical mixture, E-PLNs, are shown in FIG. 6, with the test results showing: the-OH group attached to the aromatic pentacyclic ring in the EMO structure is at 3389cm -1 With a characteristic peak at 1695cm for the c=o functional group -1 And 1596cm -1 With a characteristic peak at 759cm for aromatic C-H groups -1 There is a characteristic peak. PLNs have PLGA-OH extension bands at 3281cm -1 With characteristic peaks, the c=o group of the ester is 1458cm -1 Characteristic peaks are present at the C-H and C-O stretches of DLPC at 2935cm -1 And 1085cm -1 There is a characteristic peak. The spectra of the physical mixture of EMO-PLNs clearly distinguish between PLNs and EMO, whereas in E-PLNsNo characteristic peak of EMO was found in the spectra, indicating that EMO was encapsulated in E-PLNs.
7. X-ray diffraction (XRD) analysis of E-PLNs
Accurately weighing a proper amount of EMO, PLNs, E-PLNs and PLNs-EMO physical mixture, and detecting the X-ray diffraction pattern of the E-PLNs under the condition that the scanning range is 5-90 degrees and the scanning speed is 2 degrees/min.
The XRD patterns of the EMO, PLNs, EMO-PLNs physical mixture, E-PLNs, are shown in FIG. 7. EMO has stronger diffraction peaks at 9.94 degrees, 13.27 degrees, 14.53 degrees, 22.49 degrees, 26.40 degrees and 28.56 degrees, which indicates that EMO exists in a crystal form; the PLNs exist in a crystal form by virtue of the auxiliary materials, and have a plurality of stronger diffraction peaks in an XRD pattern; diffraction peaks of EMO can be clearly seen in the EMO-PLNs physical mixture map, so that the EMO and PLNs states are proved to be simply and physically mixed, while diffraction peaks of EMO in E-PLNs are not obvious, so that the EMO is wrapped in the E-PLNs in an amorphous state.
8. Differential Scanning Calorimeter (DSC) analysis of E-PLNs
Accurately weighing a proper amount of EMO, PLNs, E-PLNs and PLNs-EMO physical mixture, and detecting the relation between the heat absorption rate and the temperature of each sample at the temperature range of 0-600 ℃, namely a DSC curve.
The DSC pattern of EMO, PLNs, EMO-PLNs physical mixture, E-PLNs is shown in FIG. 8. EMO has a distinct characteristic peak at 263.2 ℃, indicating that EMO exists in crystalline form; PLNs have distinct characteristic peaks, but do not affect EMO; E-PLNs have no absorption peak at 263.2 ℃. The results indicated that the EMO dispersed in an amorphous state, consistent with XRD analysis results.
9. Test of emodin polymer lipid hybrid nanoparticles for inhibiting drug resistance of MCF-7/ADR breast cancer cells
9.1 cell culture
MCF-7 cells in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37℃in 5% CO 2 Culturing in an incubator. Doxorubicin hydrochloride (DOX) was added to the medium of MCF-7/ADR cells at a drug concentration of 200ng/mL.
9.2 detection of uptake by cells
Each sample was prepared using coumarin-6 (C-6) instead of EMO, and cells were cultured for 2h according to experimental groups and washed twice with PBS. The cells are divided into two parts, one part of the cells is fixed by 500 mu L of 4% paraformaldehyde for 10min, 500 mu L of host staining solution is added into the removed fixing solution, the cells are fully stained by incubation for 5-10 min in a dark place, PBS is washed twice, and then the cell uptake condition is observed under a laser confocal microscope; another portion of the cells was resuspended in 500. Mu.L of PBS and then centrifuged (2000 rpm,5 min) and the fluorescence intensity of the nanoparticles was measured using a flow cytometer.
The uptake of E-PLNs by MCF-7 and MCF-7/ADR cells is shown in FIG. 9, and FIG. 9 is a laser confocal image, and the test results show that: the uptake of C-6-PLNs in both cells was higher than in free C-6, C-6-PLNs, thereby promoting the uptake of the drug by the cells.
Further quantitative analysis of the average fluorescence intensity of both cells using flow cytometry, as shown in FIGS. 10 (a) - (b) and 11, showed significant differences in the average fluorescence intensity between MCF-7/ADR cells and MCF-7 cells, which may be related to the unique environment of the MCF-7/ADR cells and the efflux protein expression level.
9.3 drug resistance index and cell viability assays
The densities were all 4X 10 4 MCF-7 cells and MCF-7/ADR cells were seeded in 96-well plates for 24h, old culture broth was removed and fresh culture broth containing DOX (200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56. Mu.M), EMO (250, 125, 62.5, 31.25, 15.63, 7.813, 3.906, 1.953. Mu.M) and E-PLNs (500, 250, 125, 62.5, 31.25, 15.63, 7.813, 3.906. Mu.M) at different concentrations was replaced, and after further culturing for 48h, thiazole blue (MTT) solution (20. Mu.L/well) was added to each well for 4h, protected from light. After removal of the medium, dimethyl sulfoxide (DMSO) (150. Mu.L/well) was added, and the mixture was shaken in a constant temperature shaking table in the absence of light for 15min, and the absorbance of each well, i.e., the absorbance (OD) value at a wavelength of 570nm was read by a multifunctional microplate reader. And calculating the cell survival rate of the MCF-7 cells and the MCF-7/ADR cells under different doxorubicin hydrochloride (DOX) concentrations according to a formula (3), and determining the drug resistance index of the MCF-7/DOX cells according to a formula (4).
Equation (3): survival (%) = (OD experimental group-OD blank)/(OD control group-OD blank) ×100%;
equation (4): drug Resistance Index (RI) =dox to drug resistant cells IC50/DOX to sensitive cells IC50.
Cell viability was then examined using the screened EMO and E-PLNs in combination with varying concentrations of DOX, respectively, on MCF-7/ADR cells.
As shown in FIG. 12, the half inhibitory concentration (IC 50) of DOX at various concentrations (1.56-200. Mu.M) was 5.35. Mu.M for MCF-7 cells and 124.1. Mu.M for MCF-7/ADR cells. The results show that: DOX has less toxic effect on MCF-7/ADR cells than on MCF-7 cells, and MCF-7/ADR cells have drug resistance with a drug resistance index RI of 23.2.
As shown in FIGS. 13 (a) - (c), MCF-7/ADR cell viability decreased with increasing concentrations of EMO and E-PLNs (FIGS. 13 (a) - (b)), indicating that EMO and E-PLNs have an inhibitory effect on MCF-7/ADR cell proliferation, and the calculated IC50 value for EMO is 150.5. Mu.M and the IC50 value for E-PLNs is 138.7. Mu.M, indicating that E-PLNs have a stronger inhibitory effect on MCF-7/ADR cell proliferation than EMO. At 30. Mu.M for both EMO and E-PLNs, the cell viability was higher than 90% and the tumor suppressing effect itself interfered with little DOX effect, so this concentration was selected for the subsequent co-administration experiments. EMO and E-PLNs at 30. Mu.M concentration were each combined with DOX at different concentrations to MCF-7/ADR cells (FIG. 13 (c)), with an IC50 value of 32.65. Mu.M for EMO+DOX group and 5.38. Mu.M for E-PLNs+DOX group, indicating that EMO, E-PLNs, and DOX combined administration enhanced DOX toxic effects and that E-PLNs were more pronounced than DOX alone.
9.4 apoptosis detection
After culturing cells in a drug-containing medium for 48h according to the experimental group, the supernatant (apoptotic suspension cells) was collected for use, the cells were digested with an appropriate amount of pancreatin free of ethylenediamine tetraacetic acid (EDTA), the supernatant previously collected was mixed with the digested cell suspension for centrifugation (1000 rpm,5 min) and the cell pellet was collected, and the cells were washed with cold PBS (centrifugation 5min at 2000 rpm) twice. After washing, removing the supernatant, transferring the supernatant to a flow tube, adding 500 mu L of cell staining buffer solution to resuspend cells, and respectively adding 5 mu L of Annexin-V/fluorescein (Annexin-V/FITC) and 5 mu L of Propidium Iodide (PI) dye solution to mix uniformly to obtain a sample group. The control groups are a negative control group, an Annexin-V/FITC single positive group and a PI single positive group, and the groups are incubated for 15min at room temperature in a dark place, and the apoptosis percentage of the groups is detected by a flow cytometer.
Meanwhile, the DAPI staining method is adopted to detect and detect the apoptosis morphology, and the specific method comprises the following steps: MCF-7/ADR cells (3X 10) 5 After overnight incubation in 96-well plates, fresh medium with the corresponding drug concentration was changed, incubation was continued for 48h, after washing twice with PBS, fixation with 1mL of 4% paraformaldehyde solution was performed for 15min, the fixation was removed and washed twice with PBS, 1mL of DAPI dye (0.1. Mu.g/mL) was added to incubate for 20min in the absence of light, after washing the cells with PBS, the cell morphology was observed by laser confocal microscopy.
During apoptosis, cells undergo morphological changes, cell shrinkage, chromatin aggregation, apoptotic body formation, and cytoskeletal disintegration. Nuclei were stained with DAPI and each group of apoptosis morphology features are shown in fig. 14. The Control group does not undergo apoptosis, and the cell nucleus is complete in morphology; the nuclei of some cells of the DOX (10. Mu.M) group were enlarged and cavitation occurred. The nuclei of the DOX+EMO (30. Mu.M) group and DOX+E-PLNs (30. Mu.M) group showed high concentration of chromatin, and the latter also showed a significant decrease in cell number and fragmentation of the nuclei. Obvious apoptosis occurs in DOX+E-PLNs group, which indicates that E-PLNs can enhance the induction of MCF-7/ADR apoptosis by DOX. Flow cytometry analysis results (fig. 15 (a) - (b)) showed that apoptosis rates were 18.49% and 24.48% respectively for the dox+emo (30 μm) and dox+e-PLNs (30 μm) groups, with a significant increase in apoptosis rate compared to the DOX (10 μm) group apoptosis rate (15.52%).
Western blot (Western blot) results (FIGS. 16 (a) - (b)) also showed that the Bax/Bcl-2 ratio was increased for the DOX+EMO (30. Mu.M) and DOX+E-PLNs (30. Mu.M) groups, fas expression was up-regulated, p53 expression was down-regulated, and DOX+E-PLNs groups exhibited more significant effects in affecting apoptotic protein expression. The results show that EMO and E-PLNs can enhance the apoptosis induction effect of DOX on MCF-7/ADR cells, and the E-PLNs have better effect.
9.5 Western blot detection (Western blot)
Extracting total cell protein: MCF-7 cells and MCF-7/ADR cells were treated according to the experimental group dosing for 48h and the collected cell pellet was washed 2 times with PBS. After 1-2s of addition of the pre-formulated cell lysate, the cells were lysed and the procedure was completed on ice. Transferring the cell sediment after lysis into a centrifuge tube, centrifuging (10000 rpm,5 min) at 4 ℃ and collecting supernatant to obtain the total cell protein. mu.L of total protein was pipetted for subsequent BCA assay to determine total protein concentration. The remaining total cellular protein was expressed according to 4:1 (V/V) adding protein buffer solution, denaturing in a metal bath at 100deg.C for 3-5min, packaging, and storing in a refrigerator at-80deg.C for a long time.
Protein quantification (BCA) method to determine total protein concentration: preparing a protein standard solution and a sample solution. 10 mu L to 96-well plates were taken, BCA working solution (200 mu L/well) was added and incubated for 20min after thoroughly mixing with the samples. And (3) measuring an OD value (detection wavelength is 562 nm) by using an enzyme-labeled instrument, calculating the total protein concentration of each sample, and determining the loading amount of the protein of the sample.
Western Blot detection protein expression: each histone was separated by SDS-PAGE (Bio-RAD) electrophoresis at a loading of 30 μg, the bands on the gel after electrophoresis were transferred to PVDF membrane, blocked with 5% nonfat milk powder for 2h, PVDF membrane was placed in primary antibody solution, incubated overnight at 4 ℃, PBST solution was washed 3 times or more, total time was not less than 1h, and then treated with horseradish peroxidase-conjugated secondary antibody for 2h, and likewise PBST solution was washed 3 times or more. And uniformly coating the prepared ECL developing solution on each strip, collecting images by a chemiluminescent imaging system, and analyzing and calculating gray values by Image J software.
The accumulation of DOX in MCF-7/ADR cells treated with different dosing groups (Control, DOX, DOX +EMO, DOX+E-PLNs) was quantified by flow cytometry. As shown in FIGS. 17 (a) - (b), the DOX+EMO (30. Mu.M) group and the DOX+E-PLNs (30. Mu.M) group had higher average fluorescence intensities than the DOX group, and the DOX+E-PLNs had higher average fluorescence intensities than the DOX group, indicating that the E-PLNs could promote uptake of DOX by MCF-7/ADR cells and increase intracellular concentrations thereof. In addition, as shown in fig. 18 (a) - (b), the expression levels of the P-gp and MRP1 proteins in the DOX group were not greatly different from those in the control group, while the expression levels of the dox+emo (30 μm) group and the dox+e-PLNs (30 μm) group were significantly down-regulated, and the down-regulation of the dox+e-PLNs group was more significant, indicating that the co-administration of the DOX and E-PLNs can effectively inhibit the P-gp and MRP1 proteins, reduce the extracellular discharge capacity, and facilitate the aggregation of the DOX in the cell, thereby enhancing the inhibitory effect of the DOX on MCF-7/ADR cells and reversing the DOX resistance.
The experimental results fully show that: EMO and E-PLNs can improve the uptake of DOX in MCF-7/ADR cells, improve the cytotoxicity and apoptosis induction effect of DOX on MCF-7/ADR cells, reduce the expression of drug resistance related proteins, and have more remarkable effect compared with free drugs EMO and E-PLNs.
10. Test of emodin Polymer lipid hybrid nanoparticles for inhibiting activity of IL-6/JAK2/STAT3 signaling pathway of MCF-7/ADR breast cancer cells
10.1 cell culture
MCF-7 cells in RPMI-1640 medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37℃in 5% CO 2 Culturing in an incubator. Doxorubicin hydrochloride (DOX) was added to the medium of MCF-7/ADR cells at a drug concentration of 200ng/mL.
Test the effect of E-PLNs in combination with DOX on the expression of IL-6 and IFN-gamma in MCF-7/ADR cells:
cytokines, one of the important members of the tumor microenvironment, function in paracrine or autocrine ways to initiate downstream signaling pathways associated with tumor progression, thereby exerting a promoting or inhibiting effect in tumor progression. In addition to IL-6, cytokines such as IFN-gamma are also closely related to tumor cell resistance and activate the same downstream signals, and in order to evaluate which cytokines are primarily used in combination with DOX to affect downstream pathways, experiments were performed to analyze and screen the expression of both cytokines and to detect the secretion levels of IL-6 and IFN-gamma by ELISA kits. As shown in FIG. 19 (a), both MCF-7 and MCF-7/ADR cells were autocrine for cytokines IL-6 and IFN-gamma, whereas the secretion level of IL-6 was significantly higher in MCF-7/ADR cells than in IFN-gamma, tending to be highly expressed, indicating that IL-6 was involved in the acquisition of resistance in MCF-7/ADR cells. As shown in FIG. 19 (b), by the administration treatment, it was found that the DOX+E-PLNs significantly inhibited the IL-6 secretion level, whereas the IFN-gamma secretion level was not much different in the 3 groups of cells, indicating that E-PLNs effectively inhibited the IL-6 secretion, with less influence on the IFN-gamma secretion level, compared with the DOX group.
10.2 apoptosis detection
After culturing cells in the drug-containing medium for 48h according to the experimental group, the supernatant (apoptotic suspension cells) was collected for use, the cells were digested with an appropriate amount of pancreatin free of EDTA, the supernatant previously collected was mixed with the digested cell suspension for centrifugation (1000 rpm,5 min) and the cell pellet was collected, and the cells were washed with cold PBS (centrifugation 5min at 2000 rpm) twice. After washing, removing the supernatant, transferring the supernatant to a flow tube, adding 500 mu L of cell staining buffer solution to resuspend cells, and respectively adding 5 mu LAnnexin-V/FITC and 5 mu L of PI staining solution to mix uniformly to obtain a sample group. The control groups are a negative control group, an Annexin-V/FITC single positive group and a PI single positive group, and the groups are incubated for 15min at room temperature in a dark place, and the apoptosis percentage of the groups is detected by a flow cytometer.
To verify the relationship between IL-6 action and E-PLNs action and IL-6, IL-6 induction concentration was screened, exogenous IL-6 pretreatment was given, and apoptosis and drug-resistance related protein expression were detected using each group of drug treatments. As shown in FIG. 20, the MCF-7/ADR cells were cultured in culture medium containing different concentrations of IL-6, and the cell viability increased in a concentration-dependent manner, and the proliferation of cells was significantly decreased when the IL-6 neutralizing antibody was added, further demonstrating that IL-6 has a promoting effect on the proliferation of MCF-7/ADR cells. The cells were treated at different concentrations for 0, 24, 48, 72h, respectively, with the fastest proliferation of cells at 24h, so that IL-6 concentrations of 100ng/mL were selected to induce cells for 24h for subsequent experiments.
10.3 enzyme-Linked immunosorbent (ELISA) detection
After incubation of MCF-7 cells and MCF-7/ADR cells (3X 105 cells/well, 6-well plate) for 24h, the concentration of IL-6 and IFN-gamma in the supernatants was measured using an enzyme-labeled instrument according to ELISA kit instructions.
10.4 Western Blot (Western Blot) detection
Extracting total cell protein: MCF-7 cells and MCF-7/ADR cells were treated according to the experimental group dosing for 48h and the collected cell pellet was washed 2 times with PBS. After 1-2s of addition of the pre-formulated cell lysate, the cells were lysed and the procedure was completed on ice. Transferring the cell sediment after lysis into a centrifuge tube, centrifuging (10000 rpm,5 min) at 4 ℃ and collecting supernatant to obtain the total cell protein. mu.L of total protein was pipetted for subsequent BCA assay to determine total protein concentration. The remaining total cellular protein was expressed according to 4:1 (V/V) adding protein buffer solution, denaturing in a metal bath at 100deg.C for 3-5min, packaging, and storing in a refrigerator at-80deg.C for a long time.
BCA assay to determine total protein concentration: preparing a protein standard solution and a sample solution. 10 mu L to 96-well plates were taken, BCA working solution (200 mu L/well) was added and incubated for 20min after thoroughly mixing with the samples. And (3) measuring an OD value (detection wavelength is 562 nm) by using an enzyme-labeled instrument, calculating the total protein concentration of each sample, and determining the loading amount of the protein of the sample.
Western Blot detection protein expression: each histone was separated by SDS-PAGE (Bio-RAD) electrophoresis at a loading of 30 μg, the bands on the gel after electrophoresis were transferred to PVDF membrane, blocked with 5% nonfat milk powder for 2h, PVDF membrane was placed in primary antibody solution, incubated overnight at 4 ℃, PBST solution was washed 3 times or more, total time was not less than 1h, and then treated with horseradish peroxidase-conjugated secondary antibody for 2h, and likewise PBST solution was washed 3 times or more. And uniformly coating the prepared ECL developing solution on each strip, collecting images by a chemiluminescent imaging system, and analyzing and calculating gray values by Image J software.
IL-6 group apoptotic protein expression was increased compared to the control group, particularly Bcl-2 and P53 proteins (FIGS. 21 (a) - (b)); the IL-6 induced MCF-7/ADR cells produced a resistance to DOX-induced apoptosis with a percentage of apoptosis of 6.19% (FIGS. 22 (a) - (b)); however, when DOX+EMO or DOX+E-PLNs were added, the number of apoptosis was significantly increased, and the percentage of apoptosis after DOX+E-PLNs addition was 20.04% (FIGS. 23 (a) - (b)); bax and Fas protein expression were most up-regulated in the DOX+E-PLNs group compared to the IL-6 group, and on the contrary Bcl-2 and p53 expression levels were down-regulated (FIGS. 21 (a) - (b). It is apparent that DOX-induced apoptosis was inhibited by IL-6 in MCF-7/ADR cells, while E-PLNs addition inhibited the effect of IL-6, reversing the apoptosis resistance phenomenon of cells.
As shown in FIGS. 24 (a) - (b), after IL-6 induction of MCF-7/ADR cells, P-gp and MRP1 expression levels were up-regulated, suggesting that IL-6 is involved in resistance of MCF-7/ADR cells by regulating transporter expression. As shown in FIGS. 25 (a) - (b), the expression of P-gp was not greatly changed in the IL-6+DOX group compared to the IL-6 group, and the expression amount of MRP1 protein was increased, indicating that IL-6 increased MCF-7/ADR DOX resistance by inducing MRP1 protein expression. After the combined administration of DOX and E-PLNs under IL-6 intervention, the expression level of both P-gp and MRP1 proteins was reduced, indicating that E-PLNs could reverse the IL-6 induced increase in MRP1 protein expression and thus the MCF-7/ADR cell resistance. Thus, reversal of drug resistance by E-PLNs is associated with inhibition of IL-6 signaling.
Furthermore, the JAK2/STAT3 pathway is a downstream signal activated by IL-6 and plays an important role in regulating biological activities such as cell growth, protein expression and the like. As shown in FIGS. 26 (a) - (b), the levels of protein expression in the JAK2/STAT3 pathway in MCF-7/ADR cells were down-regulated following administration of E-PLNs in combination with DOX, whereas DOX alone did not, suggesting that the synergistic effect of E-PLNs on DOX might be associated with the JAK2/STAT3 pathway. As shown in FIGS. 27 (a) - (b), after co-administration of DOX with EMO or E-PLNs under IL-6 intervention, the levels of JAK2, STAT3, p-JAK2, p-STAT3 protein expression were significantly down-regulated, and the effect of co-administration of E-PLNs with DOX was more pronounced. The results indicate that E-PLNs can inhibit the activity of IL-6/JAK2/STAT3 signaling pathway, thereby reversing DOX resistance of MCF-7/ADR cells.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. Use of an emodin polymer lipid hybrid nanoparticle, wherein the emodin polymer lipid hybrid nanoparticle is used for an IL-6/JAK2/STAT3 signaling pathway inhibitor; wherein the emodin polymer lipid hybrid nanoparticle consists of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3-4:20-25:40-50:8-9.
2. The use of the emodin polymer lipid hybrid nanoparticle according to claim 1, wherein the emodin polymer lipid hybrid nanoparticle is used for preparing a medicament for reversing MCF-7/ADR breast cancer cell resistance.
3. Use of the emodin polymer lipid hybrid nanoparticle according to claim 1 or 2, wherein the emodin polymer lipid hybrid nanoparticle is for use as an IL-6/JAK2/STAT3 signaling pathway inhibitor in MCF-7/ADR breast cancer cells.
4. Use of the emodin polymer lipid hybrid nanoparticle according to claim 3, wherein the emodin forms an organic phase with the polylactic acid-glycolic acid copolymer, the lipid material is encapsulated outside the organic phase, and the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is modified on the surface of the lipid material.
5. The use of the emodin polymer lipid hybrid nanoparticle according to claim 4, wherein the emodin polymer lipid hybrid nanoparticle is composed of emodin, polylactic acid-glycolic acid copolymer, lipid material and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to the mass ratio of 3:20:40:9.
6. The use of the emodin polymer lipid hybrid nanoparticle according to claim 5, wherein the lipid material is at least one of soybean phospholipid, soybean lecithin, dilauroyl lecithin, dimyristoyl phosphatidylcholine or (2, 3-dioleoyl-propyl) -trimethylamine.
7. Use of the emodin polymer lipid hybrid nanoparticle according to any one of claims 4 to 6, wherein the emodin polymer lipid hybrid nanoparticle is prepared by the steps of:
S1, mixing emodin with a polylactic acid-glycolic acid copolymer according to a proportion, and then dissolving the mixture by using an organic solvent to form an organic phase;
s2, respectively weighing lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 according to a proportion, dissolving the lipid materials and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in an emulsifier, heating and stirring the mixture until a water phase is formed;
and S3, adding the organic phase into the water phase, heating, stirring, centrifuging, and filtering the supernatant with a water system filter membrane to obtain the emodin polymer lipid hybrid nanoparticle.
8. The use of the emodin polymer lipid hybrid nanoparticle according to claim 7, wherein in the step S2, the heating temperature is 65-75 ℃, the stirring speed is 750-850rpm, and the stirring time is 2-4min.
9. The use of the emodin polymer lipid hybrid nanoparticle according to claim 8, wherein in the step S3, the heating temperature is 65-75 ℃, the stirring speed is 750-850rpm, the stirring time is 40-60min, the rotational speed of the centrifugation is 900-1100rpm, and the centrifugation time is 4-6min.
10. The use of emodin polymer lipid hybrid nanoparticles according to claim 2, wherein the medicament is an injection.
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