CN114949256B - Silk fibroin nano medicine carrying system and preparation method and application thereof - Google Patents
Silk fibroin nano medicine carrying system and preparation method and application thereof Download PDFInfo
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- CN114949256B CN114949256B CN202210647114.XA CN202210647114A CN114949256B CN 114949256 B CN114949256 B CN 114949256B CN 202210647114 A CN202210647114 A CN 202210647114A CN 114949256 B CN114949256 B CN 114949256B
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Classifications
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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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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Abstract
The invention discloses a silk fibroin nano medicine carrying system and a preparation method and application thereof, wherein the preparation method of the nano medicine carrying system is as follows: (1) degumming cocoons to obtain silk fibroin fibers; (2) Dissolving silk fibroin fibers, dialyzing, and freeze-drying to obtain freeze-dried silk fibroin; (3) Dissolving freeze-dried silk fibroin in water and adding DOX to obtain DOX silk fibroin mixed solution; (4) Dripping the DOX silk fibroin mixed solution into an acetone solution containing the hypoxia inducible factor inhibitor to obtain silk fibroin nano particles loaded with the drug and the hypoxia inducible factor inhibitor; (5) And carrying out folic acid targeted modification on the silk fibroin nano-particles loaded with the drugs and the hypoxia inducible factor inhibitor to obtain silk fibroin nano-drug-loaded particles. The co-drug-loaded FA targeted silk fibroin nano-particles prepared by the invention can obviously increase the retention of a chemotherapeutic drug DOX in multi-drug resistant breast cancer cells, effectively inhibit proliferation of the multi-drug resistant breast cancer cells, and overcome the problem of drug resistance generated when doxorubicin is singly administered.
Description
Technical Field
The invention belongs to the technical field of nano-drugs, and in particular relates to a silk fibroin nano-drug loading system and a preparation method and application thereof.
Background
The incidence of breast cancer in women is first in the world, severely threatening the health of women. The current breast cancer treatment modes mainly comprise operation treatment, radiotherapy and chemotherapy. Among them, chemotherapy has been an important role in breast cancer treatment, and is to inhibit or kill tumor cells by acting on different links of tumor cell growth and proliferation through chemotherapeutic drugs.
The anthracycline chemotherapeutic drug is a basic stone for breast cancer chemotherapy, and the doxorubicin hydrochloride (Doxorubicin hydrochloride, DOX) is a classical and effective Encycline chemotherapeutic drug, has extremely high cost performance and is currently the first-line drug for breast cancer chemotherapy. DOX exerts its effects by intercalating DNA and inhibiting topoisomerase I and II, resulting in DNA damage and formation of reactive oxygen species, activating caspases and causing apoptosis. However, with the wide range of doxorubicin use, tumor cells develop multi-drug resistance.
The multi-drug resistance refers to that cancer cells generate drug resistance to an anti-tumor drug and drug resistance to other non-similar drugs, which is one of the main reasons for failure of tumor chemotherapy, and the problem also seriously affects the chemotherapeutic effect of DOX, so that the multi-drug resistance of tumors is a great problem to be solved in the current tumor treatment.
Disclosure of Invention
The invention aims at: provides a silk fibroin nano drug-loading system to solve the problem of multi-drug resistance of doxorubicin in the breast cancer treatment process.
The technical scheme adopted by the invention is as follows:
a silk fibroin nano drug-loading system comprises a carrier, a drug and a targeting molecule, wherein the carrier is silk fibroin nano particles, the drug is DOX and hypoxia inducible factor inhibitor which are co-carried on silk fibroin, the targeting molecule is FA (Folic acid), and the FA is grafted on the silk fibroin nano particles loaded with the DOX and the hypoxia inducible factor inhibitor to form the silk fibroin nano drug-loading particles.
Further, the hydrated particle size of the silk fibroin nano-drug-loaded particles is 100-300nm.
Still further, the hypoxia inducible factor inhibitor is one or more of PX478, oltipraz, LW6, acriflavine.
The preparation method of the silk fibroin nano drug-loading system comprises the following steps:
cutting cocoon shells into pieces, degumming to obtain silk fibroin fibers, dissolving the silk fibroin fibers, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
dissolving freeze-dried silk fibroin in water to form a silk fibroin solution, adding DOX to obtain a mixed solution of DOX and silk fibroin, and then dripping the mixed solution into an acetone solution containing an hypoxia inducible factor inhibitor to obtain silk fibroin nano particles loaded with a drug and the hypoxia inducible factor inhibitor;
and carrying out FA targeting modification on the silk fibroin nano particles to obtain silk fibroin nano drug-loaded particles.
Further, the hypoxia inducible factor inhibitor is PX478, the drug-loaded silk fibroin nanoparticles are PX478-DOX-SFNPs, and the silk fibroin nanoparticle drug-loaded particles are FA-PX478-DOX-SFNPs.
Further, after the silk fibroin fibers are dissolved in a ternary solution, dialysis and freeze-drying treatment are carried out, wherein the ternary solution comprises calcium chloride, ethanol and water in a molar ratio of 1:2:8, and 1g of silk fibroin fibers are correspondingly 15-25mL of ternary solution.
Further, the concentration of the freeze-dried silk fibroin after water dissolution is 5-20mg/mL, the concentration of DOX after adding silk fibroin solution is 0.1-0.5mg/mL, and the concentration of PX478 after mixing with acetone solution is 6.25-50 mug/mL.
Further, the FA grafting to the surface of the silk fibroin nanoparticle is as follows:
dissolving EDC and NHS in 0.1mol/mL MES solution to make the concentrations of EDC and NHS be 2-15mg/mL, then adding FA to make the concentration of FA be 50-100 mug/mL, and reacting for 1-3h to obtain primary solution;
adding NaOH to regulate the pH value of the primary solution to 7-8 to obtain a secondary solution;
adding the silk fibroin nano particles into a secondary solution to enable the concentration of the silk fibroin nano particles to be 1-4mg/mL, carrying out light-shielding reaction for 8-12h at room temperature, and washing with water to obtain the FA-PX478-DOX-SFNPs.
The silk fibroin nano drug-loading system can be applied to the treatment of breast cancer.
In summary, due to the adoption of the technical scheme, the beneficial effects of the invention are as follows:
1. the silk fibroin nano drug-loading system has good biocompatibility, and is easier to penetrate into tumor tissues and stay for a long time;
2. the method has the advantages that the cytotoxicity of the chemotherapeutic drug to the multidrug resistant tumor cells can be increased, the expression of a multidrug resistant gene (MDR 1) is reduced, so that the multidrug resistant cells can inhibit the drug from being discharged outwards, meanwhile, the targeting molecule FA enables the silk fibroin nano drug-carrying system to have the capability of actively targeting the tumor cells, and finally, when chemotherapy is carried out, the toxic and side effects on normal tissues can be reduced, the retention of the chemotherapeutic drug in the multidrug resistant tumor cells can be obviously increased, the half inhibition concentration of doxorubicin is effectively reduced, and the multidrug resistance problem generated when the doxorubicin is administered is overcome;
3. the multi-drug resistant cells can be inhibited from discharging chemotherapeutic drugs, so that the chemotherapeutic effect is enhanced, and meanwhile, the toxic and side effects of the chemotherapeutic drugs can be reduced, particularly, when doxorubicin is clinically used, a plurality of toxic and side effects such as cardiotoxicity, bone marrow suppression, gastrointestinal tract reaction and the like are shown, when the doxorubicin is pumped out of tumor cells due to drug resistance, the chemotherapeutic effect is reduced, and stronger toxic and side effects are caused, and the silk fibroin nano drug-loading system can effectively inhibit the tumor cells from discharging the chemotherapeutic drugs, so that the toxic and side effects caused by discharging are greatly reduced, and the chemotherapy is safer;
4. cell uptake experiments show that the uptake rate of MCF-7/ADR cells on FA-PX478-DOX-SFNPs after 4 hours of drug addition is up to 84.63 percent, which is about 27.8 times of the uptake rate of free DOX (3.04 percent), and the FA-PX478-DOX-SFNPs can obviously promote the uptake of drug-resistant breast cancer cells on DOX and increase the content of DOX in drug-resistant cells;
5. cytotoxicity experiments showed that FA-PX478-DOX-SFNPs resulted in half-Inhibitory Concentration (IC) of DOX on MCF-7/ADR cells 50 ) The dosage is reduced to 0.403 mug/mL, which is reduced by about 63.4 times compared with free DOX, namely FA-PX478-DOX-SFNPs has better in-vitro anti-tumor activity, and can effectively reverse the multi-drug resistance of breast cancer cells.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a Scanning Electron Microscope (SEM) observation diagram of FA-PX478-DOX-SFNPs obtained in example 1;
FIG. 2 is a flow cytometer analysis histogram (4 h incubation), cell uptake ratio vs. average fluorescence intensity vs. MCF-7/ADR cells versus DOX, PX478-DOX-SFNPs, FA-PX478-DOX-SFNPs. P <0.05, P <0.01, P <0.001, P < 0.0001);
FIG. 3 is a graph showing in vitro comparison of anti-tumor activity of DOX, PX478-DOX-SFNPs, FA-PX478-DOX-SFNPs, i.e., comparison of experimental results of cytotoxicity detection by CCK 8;
FIG. 4 shows the differential gene expression analysis of MCF-7/ADR cells after drug addition.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the particular embodiments described herein are illustrative only and are not intended to limit the invention, i.e., the embodiments described are merely some, but not all, of the embodiments of the invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
It should be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The invention provides a silk fibroin nano drug-loading system which is mainly used for the growth of multi-drug resistant breast cancer cells all the time, and comprises a carrier, a drug and a targeting molecule, wherein the carrier is silk fibroin nano particles, the drug is DOX and hypoxia inducible factor inhibitor which are loaded on silk fibroin, the targeting molecule is FA (Folic acid ), and the FA is grafted on the silk fibroin nano particles loaded with the DOX and the hypoxia inducible factor inhibitor to form the silk fibroin nano drug-loading particles.
Wherein the hypoxia inducible factor inhibitor is one or more of PX478, oltipraz, LW6 and Acriflavine. In addition, the hydrated particle size of the silk fibroin nanoparticle is maintained between 100-300nm so that it better enters tumor cells and stays for a long period of time.
The preparation method of the silk fibroin nano drug-loading system comprises the following steps:
cutting cocoon shells into pieces, degumming to obtain silk fibroin fibers, dissolving the silk fibroin fibers, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
dissolving freeze-dried silk fibroin in water to form a silk fibroin solution, adding DOX to obtain a mixed solution of DOX and silk fibroin, and then dripping the mixed solution into an acetone solution containing an hypoxia inducible factor inhibitor to obtain silk fibroin nano particles loaded with a drug and the hypoxia inducible factor inhibitor;
and carrying out FA targeted modification on the silk fibroin nano particles to obtain silk fibroin nano drug-loaded particles.
When the hypoxia inducible factor inhibitor is PX478, the drug-loaded silk fibroin nanoparticles are PX478-DOX-SFNPs, and the silk fibroin nanoparticle is FA-PX478-DOX-SFNPs.
In the actual preparation process, after silk fibroin fibers are dissolved in a ternary solution, dialysis and freeze-drying treatment are carried out, wherein the ternary solution comprises calcium chloride, ethanol and water in a molar ratio of 1:2:8, and 1g silk fibroin fibers are correspondingly 15-25mL ternary solution.
In addition, the concentration of the freeze-dried silk fibroin after water dissolution is 5-20mg/mL, the concentration of DOX added into silk fibroin solution is 0.1-0.5mg/mL, and the concentration of PX478 mixed with acetone solution is 6.25-50 mug/mL.
The FA grafting to the silk fibroin nanoparticle surface was as follows:
dissolving EDC and NHS in 0.1mol/mL MES solution to make the concentrations of EDC and NHS be 2-15mg/mL, then adding FA to make the concentration of FA be 50-100 mug/mL, and reacting for 1-3h to obtain primary solution;
adding NaOH to regulate the pH value of the primary solution to 7-8 to obtain a secondary solution;
adding the silk fibroin nano particles into a secondary solution to enable the concentration of the silk fibroin nano particles to be 1-4mg/mL, carrying out light-shielding reaction for 8-12h at room temperature, and washing with water to obtain the FA-PX478-DOX-SFNPs.
Since some of the treatment methods of the present invention can be operated by using the existing methods, only the differences from the existing methods are described in the above description, and the existing methods are not particularly limited, so long as the objects of the present invention can be achieved.
The following are examples as references:
step 1) selecting clean silkworm cocoon shells, and shearing the silkworm cocoon shells.
Step 2) the silkworm cocoon shells are dried in 0.5 percent of Na 2 CO 3 Degumming in the solution, wherein the bath ratio is 1:30-50; degumming in a water bath at 95 ℃ for 30min, degumming twice, washing the obtained silk fibroin fiber with pure water, and drying in a 60 ℃ oven.
And 3) dissolving the dried silk fibroin fibers in a calcium chloride-ethanol-water ternary solution (the molar ratio is 1:2:8), dissolving in a water bath at 60-80 ℃, pouring the solution into a dialysis bag after dissolving, dialyzing in pure water for 3 days, changing water once every 3-6 hours, collecting the dialyzed silk fibroin solution, centrifuging for 10-20min at 8000-12000g to remove impurities, and then freeze-drying the silk fibroin solution to obtain the freeze-dried silk fibroin.
Step 4) dissolving freeze-dried silk fibroin in water to obtain 5-20mg/mL silk fibroin solution, adding DOX to obtain silk fibroin mixed solution containing 0.1-0.5mg/mL DOX, then dripping the mixed solution into acetone solution (6.25-50 mug/mL) containing hypoxia inducible factor inhibitor PX478, wherein the volume ratio of the silk fibroin mixed solution to the acetone solution is preferably about 1:5, stirring while dripping, magnetically stirring until acetone is completely volatilized, and obtaining silk fibroin nano particles PX-DOX-SFNPs carrying DOX and PX478 together, and collecting nanoparticles after three times of water washing.
Grafting the surface of the targeting molecule FA value nanoparticle by EDC-NHS method, wherein the concentration of EDC and NHS is 2-15mg/mL, dissolving in 0.1mol/mL MES solution, the pH is 5-6, the concentration of FA is 50-100 mug/mL, and reacting for 2h; then adding 10mol/mL NaOH solution to adjust the pH to 7-8, adding 1-4mg/mL drug-loaded silk fibroin nanoparticles, performing light-proof reaction for 8-12h at room temperature, washing with water for three times, collecting the nanoparticles, and finally performing freeze drying to obtain freeze-dried nanoparticles, drying and storing at 4 ℃, and marking as FA-PX478-DOX-SFNPs.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Examples
(1) Placing 50g of sheared silk cocoon shell in 2L of 0.5% Na 2 CO 3 Degumming treatment is carried out in the solution for 2 times, each time is carried out in a water bath at 95 ℃ for 30min, then the solution is washed three times by pure water, and the solution is placed in a baking oven at 60 ℃ for drying to obtain silk fibroin fibers;
dissolving 10g of silk fibroin fibers in 200mL of ternary solution (calcium chloride-ethanol-water ternary solution) with the molar ratio of 1:2:8, dissolving the silk fibroin fibers in 80 ℃ water bath for 20-30min, then pouring the solution into a dialysis bag, dialyzing for 3 days in pure water, changing water once every 6h, collecting the silk fibroin solution after dialysis, centrifuging for 15min by 12000g, taking supernatant, and freeze-drying to obtain the freeze-dried silk fibroin.
(2) 100mg of the lyophilized silk fibroin was dissolved in 10mL of pure water, 250. Mu.L of 10mg/mL DOX aqueous solution was added, and then mixed well, and added dropwise to 50mL acetone solution containing 2.5mg of PX478 while stirring, and then stirring was continued in a fume hood until the acetone was completely volatilized. Subsequently, the mixture was centrifuged at 13000g for 10min, washed with water three times, and collected to obtain PX478-DOX-SFNPs.
(3) 410mg of EDC and 100mg of NHS are taken and dissolved in 25mL of 0.1mol/mL MES solution, 3mg of FA is added, and the reaction is carried out for 2 hours to obtain 1 time solution; then adding 10mol/mL NaOH solution to adjust the pH to 7.5 to obtain 2 times of solution, adding 50mg of PX478-DOX-SFNPs, carrying out light-shielding reaction for 10 hours at room temperature, and washing with water for three times to obtain FA-PX478-DOX-SFNPs;
freeze-drying FA-PX478-DOX-SFNPs to obtain freeze-dried nano particles, and drying and preserving at 4 ℃.
Test example 1
The FA-PX478-DOX-SFNPs prepared in example 1 were characterized by morphology using a scanning electron microscope: adding a small amount of nano particles into a proper amount of water, fully dispersing the particles in ultrasonic waves for 1min, then dripping the particles on a monocrystalline silicon wafer, naturally drying, and photographing by using a scanning electron microscope, wherein the result is shown in figure 1.
As is apparent from FIG. 1, the FA-PX478-DOX-SFNPs particles are spherical and have a relatively uniform particle size.
The average value of the hydration particle size is 247nm and the polydispersity coefficient is 0.086, and the particle size can realize high permeation long retention effect (Enhanced permeability and retention effect, EPR) in human body, namely, the phenomenon that macromolecular substances with specific size are easier to permeate into tumor tissues and retain for a long time compared with normal tissues after entering the body, thereby realizing passive targeting of tumor cells.
Test example 2
Cell uptake assay of DOX by MCF-7/ADR cells.
Taking MCF-7/ADR cells in logarithmic growth phase, digesting with trypsin, and dividing into 3 groups of 10 each 5 The density of each cell per well was inoculated into a 12-well cell plate and cultured under the same conditions for 24 hours in a culture environment of 37℃and 5% CO 2 A constant temperature cell incubator with saturated humidity.
After 24h of culture, DOX, PX478-DOX-SFNPs and FA-PX478-DOX-SFNPs (the concentration of DOX loaded in the nanoparticles is 2 mug/mL) are dispersed in a serum-free basal medium to replace the original complete medium in an orifice plate, namely, each group is added with one drug to form an independent DOX group, a PX478-DOX-SFNPs group and a FA-PX478-DOX-SFNPs group. After incubation for 1, 4 and 8 hours under the same conditions, the well plate was washed 3 times with PBS, then cells were resuspended in PBS buffer after digestion with pancreatin, and the fluorescence intensity of DOX (PE red fluorescence channel) in each group of cells was analyzed by flow cytometry, as shown in FIG. 2, which is a graph obtained by flow cytometry analysis.
As can be seen from fig. 2:
MCF-7/ADR cells had a weaker uptake of single DOX, with cell uptake increasing from 0.48% to 3.04% over incubation time from 1h to 8 h;
under the action of PX478-DOX-SFNs, the DOX is rapidly accumulated in MCF-7/ADR cells, the cell uptake rate is 8.21% after 1h, and the cell uptake rate is increased to 58.83% after 4 h;
the FA-PX478-DOX-SFNPs with targeting molecules can ensure that the cell uptake rate reaches 80.9% only by incubating for 1h, and reaches 84.63% after 4 h;
wherein, for ease of characterization, the cell extraction rates were each used as average values for each group.
That is, it is apparent that the cell uptake rates of the PX478-DOX-SFNs and the FA-PX478-DOX-SFNPs are significantly improved as compared with the single DOX group, while the cell uptake rates of the PX478-DOX-SFNs and the FA-PX478-DOX-SFNPs are significantly improved as compared with the single DOX group, and the trend of the average fluorescence intensity of each group is the same as that of the cell uptake rate. The results of flow analysis show that the FA-PX478-DOX-SFNPs have obvious targeting effect, can greatly improve the uptake of MCF-7/ADR cells to nanoparticles, and increase the DOX content in drug-resistant cells.
Test example 3
Cytotoxicity detection of MCF-7/ADR cells by FA-PX478-DOX-SFNPs.
After taking MCF-7/ADR cells in logarithmic growth phase and digesting with trypsin, inoculating 5×10 cells in three groups 4 100. Mu.L of cell suspension/mL was applied to 96-well cell plates at 37℃under 5% CO 2 Culturing in a constant temperature cell incubator with saturated humidity.
Dispersing DOX, PX478-DOX-SFNPs and FA-PX478-DOX-SFNPs in serum-free basal medium instead of well plate after culturing for 24 hrThe original complete culture medium is adopted, and the DOX concentration in each group of culture medium is the same, namely, each group is added with one drug to form an independent DOX group, a PX478-DOX-SFNPs group and an FA-PX478-DOX-SFNPs group. After 48h incubation, the well plate was washed 3 times with PBS, then 100. Mu.L of serum-free medium containing 10% CCK8 reagent was added to each well, and after 1h incubation in a cell incubator, the absorbance of each well at 450nm was measured with a microplate reader, and the cell viability of each group and half-maximal Inhibitory Concentration (IC) on MCF-7/ADR cells were calculated 50 ) The results are shown in FIG. 3 and Table 1 below.
TABLE 1DOX and nanoparticle to MCF-7/ADR cell IC 50 Value of
As can be seen from FIG. 3 and Table 1, MCF-7/ADR cells have strong resistance to DOX, IC 50 25.56 μg/mL was achieved, while PX478-DOX-SFNPs reduced DOX IC to MCF-7/ADR cells 50 To 1.075 μg/mL. After grafting targeting molecule FA, FA-PX478-DOX-SFNPs make DOX IC on MCF-7/ADR cells 50 Reduced to 0.403 μg/mL, about 63.4 fold reduced compared to free DOX, and greater killing of drug resistant tumor cells compared to PX478-DOX-SFNs without targeting molecule. The result shows that the FA-PX478-DOX-SFNPs have better in-vitro anti-tumor activity and can effectively reverse the multi-drug resistance of breast cancer cells.
Test example 4
MCF-7/ADR cells were grouped into 4 groups at 2X 10 5 Is inoculated into a 12-well cell plate, and is cultured in a constant temperature cell incubator at 37 ℃ under 5% CO2 and saturated humidity for 24 hours.
Then, three groups of the original culture mediums are replaced by basal culture mediums containing DOX-SFNPs, PX478-DOX-SFNPs and FA-PX478-DOX-SFNPs, wherein the DOX content in 3 drug-loaded nano particles is 2.5 mug/mL, which are respectively marked as a DOX-SFNPs group, a PX478-DOX-SFNPs group and an FA-PX478-DOX-SFNPs group, and the last group is marked as a Control group by taking the basal culture mediums as a Control.
After 24h incubation, the medium was aspirated, the well plate was washed 3 times with PBS and 800. Mu.L of lysate was added to each well for total RNA extraction. Reverse transcription of RNA into cDNA, real-time fluorescent quantitative PCR detection, and detection of HIF-1 alpha, MDR1, VEGF and GLUT1 genes in different groups by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene.
HIF-1 can regulate and control MDR1 gene encoding multi-drug resistance protein P-gp (P-glycoprotein), pumping chemotherapeutic drugs out of tumor cells, which is one of the main mechanisms of tumorigenic multi-drug resistance. Meanwhile, HIF-1 (Hypoxia-inducible factor 1) also regulates the expression of VEGF, GLUT-1, wherein Vascular Endothelial Growth Factor (VEGF) is related to angiogenesis, tumor growth and metastasis; glucose transporter (GLUT-1) can regulate tumor growth, and the proteins can mediate the drug resistance of tumors to chemotherapeutic drugs such as doxorubicin and the like.
As shown in FIG. 4, after DOX-SFNPs were added to the MCF-7/ADR cells, HIF-1α and MDR1 genes were significantly up-regulated due to DOX stimulation of the MCF-7/ADR cells. However, PX478-DOX-SFNPs and FA-PX478-DOX-SFNPs treated groups were significantly lower in HIF-1α expression than the DOX-SFNPs groups due to the presence of PX478; since VEGF, GLUT1 are also regulated by HIF-1α, they show the same trend as HIF-1α expression in each group. In addition, since FA-PX478-DOX-SFNPs have targeting effect, the inhibition effect on the whole of HIF-1 alpha and the downstream gene of HIF-1 alpha is superior to that of untargeted group PX478-DOX-SFNPs. In general, FA-PX478-DOX-SFNPs overcome tumor multidrug resistance by down-regulating HIF-1. Alpha. To achieve inhibition of drug resistance-associated genes MDR1, VEGF, GLUT 1.
Wherein, the PX478-DOX-SFNPs and the FA-PX478-DOX-SFNPs used in the above test examples are prepared by the method of the present invention, and the DOX-SFNPs are prepared by the method of the present invention without adding PX 478.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (8)
1. The silk fibroin nano drug-loading system is characterized by comprising a carrier, a drug and a targeting molecule, wherein the carrier is silk fibroin nano particles, the drug is DOX and hypoxia inducible factor inhibitor which are co-carried on silk fibroin, the targeting molecule is FA, and the FA is grafted on the silk fibroin nano particles loaded with the DOX and the hypoxia inducible factor inhibitor to form the silk fibroin nano drug-loading particles, and the steps are as follows:
cutting cocoon shells into pieces, degumming to obtain silk fibroin fibers, dissolving the silk fibroin fibers, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
dissolving freeze-dried silk fibroin in water to form a silk fibroin solution, adding DOX to obtain a mixed solution of DOX and silk fibroin, and then dripping the mixed solution into an acetone solution containing an hypoxia inducible factor inhibitor to obtain silk fibroin nano particles loaded with a drug;
carrying out FA targeting modification on the silk fibroin nano particles to obtain silk fibroin nano drug-loaded particles;
the hypoxia inducible factor inhibitor is PX478;
the concentration of the freeze-dried silk fibroin after water dissolution is 5-20mg/mL, the concentration of DOX added into silk fibroin solution is 0.1-0.5mg/mL, and the concentration of PX478 mixed with acetone solution is 6.25-50 mug/mL.
2. The silk fibroin nanoscopic drug delivery system of claim 1 wherein the silk fibroin nanoscopic drug delivery particles have a hydrated particle size of 100-300nm.
3. A method for preparing the silk fibroin nano-drug delivery system according to any one of claims 1-2, comprising the steps of:
cutting cocoon shells into pieces, degumming to obtain silk fibroin fibers, dissolving the silk fibroin fibers, dialyzing and freeze-drying to obtain freeze-dried silk fibroin;
dissolving freeze-dried silk fibroin in water to form a silk fibroin solution, adding DOX to obtain a mixed solution of DOX and silk fibroin, and then dripping the mixed solution into an acetone solution containing an hypoxia inducible factor inhibitor to obtain silk fibroin nano particles loaded with a drug;
and carrying out FA targeting modification on the silk fibroin nano particles to obtain silk fibroin nano drug-loaded particles.
4. The method of claim 3, wherein the hypoxia inducible factor inhibitor is PX478, the drug-loaded silk fibroin nanoparticles are PX478-DOX-SFNPs, and the silk fibroin nanoparticle is FA-PX478-DOX-SFNPs.
5. The method according to claim 3, wherein the silk fibroin fibers are dissolved in a ternary solution, and then subjected to dialysis and freeze-drying treatment, wherein the ternary solution comprises calcium chloride, ethanol and water in a molar ratio of 1:2:8, and 1g silk fibroin fibers are 15-25mL of the ternary solution.
6. The method according to claim 3, wherein the concentration of the freeze-dried silk fibroin after dissolution in water is 5-20mg/mL, the concentration of DOX after addition of silk fibroin solution is 0.1-0.5mg/mL, and the concentration of PX478 after mixing with acetone solution is 6.25-50. Mu.g/mL.
7. A method of preparation according to claim 3, wherein the FA is grafted to the surface of the silk fibroin nanoparticle as follows:
dissolving EDC and NHS in 0.1mol/mL MES solution to make the concentrations of EDC and NHS be 2-15mg/mL, then adding FA to make the concentration of FA be 50-100 mug/mL, and reacting for 1-3h to obtain primary solution;
adding NaOH to regulate the pH value of the primary solution to 7-8 to obtain a secondary solution;
adding the silk fibroin nano particles into a secondary solution to enable the concentration of the silk fibroin nano particles to be 1-4mg/mL, carrying out light-shielding reaction for 8-12h at room temperature, and washing with water to obtain the FA-PX478-DOX-SFNPs.
8. Use of the silk fibroin nano-drug delivery system of claim 1 in the preparation of a medicament for treating breast cancer.
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| Bano Subia等.Folate conjugated silk fibroin nanocarriers for targeted drug delivery.Integrative Biology.2014,第6卷第203-204页. * |
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