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CN110812491B - Small-particle-size quercetin nano-drug targeting alpha v beta 3 integrin receptor high-expression tumor cells - Google Patents

Small-particle-size quercetin nano-drug targeting alpha v beta 3 integrin receptor high-expression tumor cells Download PDF

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CN110812491B
CN110812491B CN201911126392.5A CN201911126392A CN110812491B CN 110812491 B CN110812491 B CN 110812491B CN 201911126392 A CN201911126392 A CN 201911126392A CN 110812491 B CN110812491 B CN 110812491B
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quercetin
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integrin receptor
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徐鹏程
贾海涛
王海生
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Inner Mongolia Medical University
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Abstract

The invention discloses a quercetin small-particle-size nano-drug targeting an alpha v beta 3 integrin receptor high-expression tumor cell and a preparation method thereof, wherein the drug comprises a carrier and bulk drugs, the bulk drugs are quercetin, and the mass ratio of the bulk drugs to the carrier is 1:10-1:100, wherein the carrier comprises an amphiphilic polymer and a targeting compound, wherein the molar ratio of the amphiphilic polymer to the targeting compound is 1: 100-1: the invention realizes that the medicament has active targeting specific tumor cells and can be quickly and effectively taken up by the cells, the medicament can exert the anti-tumor effect to a greater extent, and the distribution of the medicament in the whole body and the toxic and side effect generated by the anti-tumor effect are reduced.

Description

Small-particle-size quercetin nano-drug targeting alpha v beta 3 integrin receptor high-expression tumor cells
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a quercetin nano-drug with small particle size, which targets alpha v beta 3 integrin receptors to highly express tumor cells.
Background
According to the latest statistical data of the world health organization in 2018, about 1808 ten thousand cancer patients are newly added in the world in 2018, no chemical or natural medicine for curing cancer exists at present, most chemotherapy medicines are not selective, and the medicines can kill tumor cells and normal cells, so that serious side effects can be generated, for example, taxol has an inhibiting effect on bone marrow, adriamycin can generate serious cardiotoxicity, and the like. Therefore, the development of anticancer medicinal preparation with small systemic toxicity and targeting effect has important significance for tumor treatment.
In the field of anti-tumor, some drug-loaded micelles or liposomes are on the market or are undergoing clinical tests, however, most of the products of the micelles or liposomes do not have the active targeting function. Because the targeting micelle can be functionalized by modifying the polypeptide or the antibody on the surface of the targeting micelle by means of a pharmaceutical preparation, the targeting micelle has the capability of targeting specific tissues or cells. Therefore, developing a drug-loaded micelle with an active targeting function becomes a preparation technology with a great clinical application prospect, and particularly, the surface of the micelle can be modified by a peptide chain with high affinity of tumor surface specific protein, so that the micelle has the effect of targeting tumors, and the drug can be accumulated in a tumor area at a higher concentration and taken by tumor cells, thereby realizing better anti-tumor curative effect.
DSPE-PEG2000 is used as a medicinal high molecular material approved by FDA, has good biocompatibility, can be prepared into a nano-medicament by loading a medicament into a glue core formed by DSPE-PEG in self-assembly, and meanwhile, a hydrophilic PEG chain of a micelle shell has good hydration effect, so that the micelle is not easy to be removed by a reticuloendothelial system in vivo after being injected into blood, thereby realizing the long circulation effect of the medicament in blood.
The liposome or nanoparticle capable of targeting the α v β 3 integrin receptor reported in the literature at present has the problem of insufficient targeting function of a carrier modified by targeting peptide due to large particle size or heavy mass, namely, after the carrier is passively reached to a tumor part by a so-called small horse (small horse, namely targeting peptide) cart (large cart, namely liposome or nanoparticle), specifically, after the targeting liposome or nanoparticle passively reaches the tumor part by an EPR effect, the targeting peptide modified on the surface of the targeting liposome or nanoparticle can not enable a drug to enter a tumor cell through receptor-mediated endocytosis due to large mass or volume of the targeting peptide, so that the true targeting peptide-mediated drug entry behavior can not be realized to a great extent, and the anti-tumor effect is not ideal.
At present, a majority of drugs targeting an alphavbeta 3 integrin receptor are pharmacosome or nanoparticles, the preparation method is generally a film hydration method or an anti-solvent method, due to the material properties and the preparation method, the average particle size of the liposome or nanoparticles prepared by the methods is generally 50nm-500nm, and the drug delivery carriers show the phenomenon of insufficient penetration capacity to tumors after passively reaching the tumor parts through an EPR effect.
In addition, many of the carrier materials used by the drug-loaded liposome or nanoparticle capable of targeting the α v β 3 integrin receptor reported at present have not been subjected to effective safety evaluation, and generally do not have the possibility of being marketed.
Disclosure of Invention
Technical problem to be solved
In order to overcome the defects of the prior art, the quercetin small-particle-size nano-drug targeting the alpha v beta 3 integrin receptor and expressing tumor cells at high level and the preparation method thereof are provided, so that the purposes that the drug actively targets specific tumor cells, can be quickly and effectively taken by the cells, can exert the drug effect to a greater extent, and can reduce the distribution of the drug in the whole body and the toxic and side effects generated by the drug are achieved. The small-particle-size quercetin nano-drug targeting the alpha v beta 3 integrin receptor high-expression B16 tumor cells can directly play an anti-tumor role by inhibiting the growth of the tumor cells, the preparation method is simple and easy to implement, and good research and development ideas and application are provided for the development of anti-tumor drugs.
(II) technical scheme
The invention is realized by the following technical scheme: the invention provides a quercetin nano-drug with small particle size for targeting an alpha v beta 3 integrin receptor high-expression tumor cell, which comprises a drug carrier and a raw material drug, wherein the raw material drug is quercetin, the drug carrier is a micelle, and the mass ratio of the raw material drug to the drug carrier is 1:10-1:100, the drug carrier, namely the micelle carrier, comprises an amphiphilic polymer and a targeting compound, wherein the molar ratio of the amphiphilic polymer to the targeting compound is 1: 100-1: 1, the surface of the drug carrier is modified by a ligand with high affinity of an alpha v beta 3 integrin receptor overexpressed on the surface of a tumor cell.
Further, the small-particle-size quercetin nano-drug is formed by self-assembling an amphiphilic copolymer, a targeting compound and quercetin in an aqueous solution.
Further, the amphiphilic copolymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol.
Further, the guide compound is obtained by connecting a ligand to the end of polyethylene glycol of distearoyl phosphatidyl ethanolamine-polyethylene glycol.
Further, the guiding compound is distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDfK.
Further, the molecular composition of the ligand is selected from the group consisting of RGD, iRGD, cRGDfK, cRGDyK, and cyclic polypeptides comprising the RGD sequence.
Further, a preparation method of the small-particle-size quercetin nano-drug targeting the alpha v beta 3 integrin receptor high-expression tumor cells comprises the following steps: the method comprises the following steps: a. weighing the components according to the prescription amount in an eggplant-shaped bottle, adding an organic solvent to completely dissolve the components, and then carrying out reduced pressure rotary evaporation at the temperature of between 25 and 40 ℃ to form a composite film; b. and c, hydrating the composite film obtained in the step a by using a glucose solution or a phosphate buffer solution which is kept in a water bath environment at 25-40 ℃, performing probe ultrasonic treatment or water bath ultrasonic treatment after vortexing until the solution is clear, and filtering the solution by using a 0.22-micron filter.
(III) advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
the invention relates to a quercetin nanometer drug with small particle size and high expression tumor cell of a targeting alpha v beta 3 integrin receptor and a preparation method thereof, which adopts DSPE-PEG2000 amphiphilic block polymer approved by FDA as a carrier material, directly prepares the quercetin nanometer drug with small particle size by a thin film hydration method, greatly reduces the average particle size of the nanometer drug, generally about 15nm, and has better tumor penetrability by reaching the deep part of a tumor through tumor capillaries due to smaller particle size of the nanometer drug, thereby better playing the anti-tumor effect (complex H, Matsumoto Y, Mizuno K, et. Accumulation of 823-100 nm polymeric in porous particulate depends on size. natural nanotechnology.2011; 6(12: 815-), and is very suitable for delivery of drug-coated drugs or drugs due to good biocompatibility of the carrier material, can better exert the anti-tumor curative effect by targeting the alpha v beta 3 integrin receptor on the surface of the tumor cell.
Drawings
FIG. 1 is a graph showing the distribution of the particle size of small particle size quercetin nano-drug PM-QU (left panel A) and cRGDfK-PM-QU (right panel B), both of which have an average particle size of about 15nm and a polydispersity of about 0.16, and the modification of the cRGDfK on micelles has no substantial effect on the particle size.
FIG. 2 is a transmission electron microscope image of small particle size quercetin nano-drug PM-QU (left panel A) and cRGDFK-PM-QU (right panel B), wherein the drug is spherical or quasi-spherical, has complete and uniform appearance, has a particle size of about 15nm, and is consistent with the test result of a particle size analyzer.
FIG. 3 is a diagram showing the physical appearance of small-particle size quercetin nano-drug PM-QU (left panel A) and cRGDFK-PM-QU (right panel B).
FIG. 4 shows that the real-time uptake of cRGDFK-PM and-QU by B16 cells to quercetin nano-drug with small particle size is measured by laser confocal measurement, and the result shows that the rate of the cRGDFK-PM uptake by B16 cells is obviously higher than that of PM after the beginning of the uptake.
FIG. 5 shows the final uptake of cRGDfK-PM-QU and PM-QU by B16 cells on small particle size quercetin nanopharmaceuticals using a FACS (Switzerland cell analyzer) (left panel) and statistical analysis (right panel), showing that B16 cells uptake cRGDfK-PM-QU significantly higher than PM-QU.
Fig. 6 is a living body image of a small-particle-size nano-drug cRGDfK-PM-Dir encapsulated with a fluorescent probe Dir and a passively targeted small-particle-size nano-drug PM-Dir distributed in a B16 tumor-bearing mouse of a tumor cell highly expressing the α v β 3 integrin receptor, and as shown in the figure, because the particle sizes of the two preparations are both small, the two preparations are accumulated at the tumor within 1 hour, however, the accumulation amount of the cRGDfK-PM-Dir in the tumor tissue is obviously enhanced with the lapse of time, which shows that the small-particle-size drug targeting the tumor cell highly expressing the α v β 3 integrin receptor significantly enhances the targeting property and the permeability to the tumor.
FIG. 7 is an in vivo pharmacodynamic evaluation of small-particle size quercetin Nanoparticulate drug cRGDFK-PM-QU targeting α v β 3 integrin receptor-highly expressed tumor cells and passively targeted small-particle size quercetin Nanoparticulate drug PM-QU, as shown (right panel) in the appearance of dissected B16 tumor mass after treatment was over (left panel) and in the re-statistical analysis of dissected B16 tumor mass after treatment was over (description: P <0.01, P < 0.5). The figure shows that the quercetin nano-drug cRGDFK-PM-QU with small particle size targeting the tumor cells with high expression of the alpha v beta 3 integrin receptor can obviously inhibit the tumor growth.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
A small-particle-size quercetin nano-drug targeting an alpha v beta 3 integrin receptor high-expression tumor cell comprises a drug carrier and bulk drugs, wherein the bulk drugs are quercetin, the mass ratio of the bulk drugs to the drug carrier is 1:10-100, the drug carrier comprises an amphiphilic polymer and a guiding compound, and the molar ratio of the amphiphilic polymer to the guiding compound is 1: 100-1: 1, the surface of the drug carrier is modified by a ligand with high affinity of an alpha v beta 3 integrin receptor overexpressed on the surface of a tumor cell.
The bulk drug is quercetin, the targeting compound is distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDfK, the polymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol, and the preparation method comprises the following steps:
a. weighing the components according to the prescription amount in an eggplant-shaped bottle, adding an organic solvent to completely dissolve the components, and then carrying out reduced pressure rotary evaporation at the temperature of between 25 and 40 ℃ to form a composite film;
b. and c, hydrating the composite film obtained in the step a by using a glucose solution or a phosphate buffer solution which is kept in a water bath environment at 25-40 ℃, and performing probe ultrasonic treatment or water bath ultrasonic treatment after vortexing until the solution is clarified.
The organic solvent in step a is preferably: one or more of dichloromethane, acetonitrile, chloroform, ethanol, acetone and methanol, and more preferably methanol.
Preferably, the invention provides a preparation method of quercetin nano-drugs with small particle size for targeting tumor cells highly expressed by an alpha v beta 3 integrin receptor, which comprises the following steps:
first, the nano-drug composed of the targeting compound distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDfK, polymer distearoyl phosphatidyl ethanolamine-polyethylene glycol, Quercetin (Quercetin) is abbreviated as cRGDfK-PM-QU (QU indicates Quercetin, PM indicates polymer micelle, cRGDfK indicates targeting peptide)
1. The molar ratio of distearoyl phosphatidyl ethanolamine-polyethylene glycol/distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDfK is 20: 1
2. According to total lipid: the raw material medicines are 25: 1 (mass ratio), precisely weighing each lipid material and quercetin with the prescription amount, dissolving in an eggplant-shaped bottle by methanol, carrying out reduced pressure rotary evaporation at 37 ℃ to remove organic solvent to form a composite film, adding a proper amount of phosphoric acid buffer solution (pH 7.4) preheated at 37 ℃ into the eggplant-shaped bottle, carrying out vortex until the film falls off, carrying out water ultrasound for 3min to clarify and brighten, passing through a 0.22 mu m filter to form quercetin nano-drug with small particle size, transferring the quercetin nano-drug into an EP tube, centrifuging for 5 min at 10000g to obtain supernatant, removing unencapsulated free quercetin, and measuring the particle size of the micelle by dynamic light scattering, as shown in figures 1 to 3.
Example 1 preparation of Small particle size drug-loaded micelles targeting tumor cells highly expressing α v β 3 integrin receptor
1. Synthesis of the guide Compound
Weighing a certain amount of actively esterified distearoyl phosphatidyl ethanolamine-polyethylene glycol-NHS and cRGDfK peptide powder (the molar ratio is 2: 1-3: 1), and respectively dissolving the powders in anhydrous N, N-dimethylformamide. After the powder is completely dissolved, the polypeptide solution is firstly transferred to an eggplant-shaped bottle, and the distearoyl phosphatidyl ethanolamine-polyethylene glycol-NHS solution is dropwise added into the polypeptide solution under the magnetic stirring. And after uniform mixing, adding a proper amount of triethylamine, adjusting the pH value of the reaction solution to 8.0-9.0, and reacting for 24 hours at room temperature under the protection of dark nitrogen. During the reaction, the progress of the reaction was followed by thin layer chromatography. And (4) dialyzing and freeze-drying at the end of the reaction to obtain the guide compound.
Preparation and characterization of cRGDFK-PM-QU quercetin small-particle-size nano-drug
The formulations of the cRGDFK-PM-QU and PM-QU small-particle-size nano-drugs are distearoyl phosphatidyl ethanolamine-polyethylene glycol and distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDFK (20: 1, molar ratio), respectively, according to the total lipid: quercetin ═ 25: 1 (mass ratio) and distearoylphosphatidylethanolamine-polyethylene glycol: quercetin 25: 1 (mass ratio), precisely weighing each lipid material and bulk drug quercetin according to the prescription amount, dissolving the materials in an eggplant-shaped bottle by methanol, carrying out reduced pressure rotary evaporation at 37 ℃ to remove organic solvent to form a composite film, adding a proper amount of phosphoric acid buffer solution (pH 7.4) preheated at 37 ℃ into the eggplant-shaped bottle, carrying out vortex until the film falls off, carrying out water area ultrasound for 3min to clarify the film to be bright, passing through a 0.22 mu m filter to form the small-particle-size quercetin nano-drug, transferring the small-particle-size quercetin nano-drug into an EP tube, centrifuging for 5 min at 10000g to obtain supernatant, and removing unencapsulated free quercetin.
Example 2 Targeted evaluation of cRGDFK-PM-QU quercetin Small particle size Nanopartic drugs to in vitro B16 tumor cells
1. The uptake of cRGDFK-PM-QU by B16 cells was measured by laser confocal measurements. B16 cells are respectively inoculated in different confocal small dishes, incubated overnight until the cells are completely attached to the wall, the culture solution is discarded, and the cells are washed for 3 times by phosphate buffer solution; 1ml of QU-PM and cRGDFK-PM-QU preparation (the final concentration of quercetin is 10 mu g/ml) are respectively added into different confocal dishes, and the laser confocal observation and the uptake are carried out in real time at 37 ℃, and the fluorescence intensity in cells is recorded by photographing. As shown in FIG. 4, cRGDFK-PM-QU was significantly higher in intake rate and intake amount than QU-PM after the start of intake.
2. The QU-PM and cRGDFK-PM-QU uptake by B16 cells was measured using a FACS.
B16 cells were inoculated in 12-well plates, incubated overnight for 24h after the cells were completely attached, the stock culture was discarded, washed with phosphate buffer, 1ml of QU-PM and cRGDfK-PM-QU preparation (final quercetin concentration of 10. mu.g/ml) were added to each well, incubated at 37 ℃ for 2h, cells were harvested, and B16 cell uptake after QU-PM and cRGDfK-PM-QU incubation was measured using a FACS (flow cytometry), showing that the cRGDfK-PM-QU uptake was significantly higher than that of QU-PM, indicating that the small-particle-size quercetin nanopharmaceuticals targeting tumor cells highly expressing α v β 3 integrin receptor were taken up by B16 tumor cells more than that of the passive targeting preparation, as shown in FIG. 5.
Example 3 evaluation of in vivo targeting and tumor tissue penetration of cRGDfK small-particle size nano-drugs. Establishing a naked mouse model with a lotus B16 tumor, randomly dividing into 2 groups when the tumor volume is 200mm3, and respectively injecting nano-drugs cRGDFK-PM-DiR and PM-DiR encapsulated with fluorescent probes into tail veins. As shown in fig. 6, at 1h and 24h after injection, in vivo distribution of the near-infrared fluorescent probe DiR nano-drug was detected by a living body imaging system, and the accumulation of the fluorescent signal in the tumor tissue of the small-particle-size nano-drug cRGDfK-PM-DiR treated group was significantly increased, and was significantly accumulated at 1h, which indicates that the ability of the small-particle-size nano-drug targeting tumor cells highly expressing α v β 3 integrin receptor was significantly increased. And (3) after living body imaging is finished, dying the nude mouse, taking down tumor of the nude mouse, and continuously observing the fluorescence distribution condition in the tumor of the nude mouse by using a living body imaging system, wherein the cRGDfK-PM remarkably promotes the accumulation and penetration of the nano preparation in the tumor as shown in figure 6.
Example 4 in vivo pharmacodynamic evaluation of cRGDFK-PM-QU
Establishing a naked mouse model of a B16-loaded tumor, wherein the tumor volume is 50mm3 after 10 days of inoculation of B16 cells, and the tumor is randomly divided into 3 groups for administration, wherein 3 groups each comprise:
physiological saline group: the tail vein is given with physiological saline at 300 mu L/mouse in 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 days;
cRGDFK-PM-QU group: cRGDFK-PM-QU was administered in the tail vein on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, at 300 μ L/mouse, at a dose: 50mg/kg (calculated as quercetin);
QU-PM group: QU-PM was administered in the tail vein on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 300 μ L/tube, at doses: 50mg/kg (calculated as quercetin);
after the administration, nude mice were quenched and tumor tissues were removed for tumor mass size comparison and weighing, and the results are shown in FIG. 7. QU-PM micelle group and cRGDFK-PM-QU micelle group both showed tumor-suppressing effect, but cRGDFK-PM-QU micelle group showed better tumor-suppressing effect.
In the above experiments, B16 tumor cells were selected as tumor cells with high expression of α v β 3 integrin receptors for illustration only, and the drugs prepared in some examples were used as experimental drugs, and it should be noted that other small-particle-size nano-drugs targeting tumor cells with high expression of α v β 3 integrin receptors of the present invention can also target tumor cells with high expression of other α v β 3 integrin receptors, and can exert the same or similar tumor suppression effect.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and do not limit the spirit and scope of the present invention. Various modifications and improvements of the technical solutions of the present invention may be made by those skilled in the art without departing from the design concept of the present invention, and the technical contents of the present invention are all described in the claims.

Claims (6)

1. A small-particle-size quercetin nano-drug targeting an alpha v beta 3 integrin receptor high-expression tumor cell comprises a drug carrier and bulk drugs, and is characterized in that: the bulk drug is quercetin, the mass ratio of the bulk drug to a drug carrier is 1:10-1:100, the drug carrier is a micelle carrier and comprises an amphiphilic polymer and a targeting compound, wherein the molar ratio of the amphiphilic polymer to the targeting compound is 1: 100-1: 1, the surface of the drug carrier is modified by a ligand with high affinity of an alpha v beta 3 integrin receptor overexpressed on the surface of a tumor cell, the amphiphilic copolymer is distearoyl phosphatidyl ethanolamine-polyethylene glycol, and the guide compound is distearoyl phosphatidyl ethanolamine-polyethylene glycol-cRGDfK.
2. The quercetin nanometer medicine with small particle size targeting α v β 3 integrin receptor high expression tumor cells as claimed in claim 1, wherein: the small-particle-size quercetin nano-drug has the particle size distribution of 5-25nm, the average particle size of 15nm, the particle sizes are in normal distribution, and the PDI is less than 0.2.
3. The quercetin nanometer medicine with small particle size targeting α v β 3 integrin receptor high expression tumor cells as claimed in claim 1, wherein: the quercetin nano-drug with the small particle size is formed by self-assembling an amphiphilic polymer, a targeting compound and quercetin in an aqueous solution.
4. The quercetin nanometer medicine with small particle size targeting α v β 3 integrin receptor high expression tumor cells as claimed in claim 1, wherein: the drug carrier is a micelle formed by an amphiphilic copolymer and a guide compound.
5. The preparation method of the quercetin nanometer medicine with small particle size for targeting α v β 3 integrin receptor high expression tumor cells as claimed in claim 1, characterized by comprising the following steps: a. weighing the components according to the prescription amount in an eggplant-shaped bottle, adding an organic solvent to completely dissolve the components, and then carrying out reduced pressure rotary evaporation at the temperature of between 25 and 40 ℃ to form a composite film; b. and c, hydrating the composite film obtained in the step a by using a glucose solution or a phosphate buffer solution which is kept in a water bath environment at 25-40 ℃, performing probe ultrasonic treatment or water bath ultrasonic treatment after vortexing until the solution is clear, and filtering the solution by using a 0.22-micron filter.
6. The preparation method of the quercetin nanometer medicine with small particle size targeting the α v β 3 integrin receptor high expression tumor cells as claimed in claim 5, wherein the preparation method comprises the following steps: in the step a, the organic solvent is one or more of dichloromethane, acetonitrile, chloroform, ethanol, acetone and methanol.
CN201911126392.5A 2019-11-18 2019-11-18 Small-particle-size quercetin nano-drug targeting alpha v beta 3 integrin receptor high-expression tumor cells Expired - Fee Related CN110812491B (en)

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