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Ferritin-loaded antitumor drug and preparation method thereof

An anti-tumor drug and ferritin technology, applied in anti-tumor drugs, drug combinations, pharmaceutical formulations, etc., can solve the problems of safety risks, large loss of doxorubicin, high production costs, etc., to improve production efficiency, increase loading ratio, The effect of reducing production costs

Inactive Publication Date: 2016-11-16
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although this method has a certain increase in protein yield, reaching 55%, and a loading ratio of 28:1, the operation process is cumbersome, and there are still losses of ferritin and doxorubicin
This process of drug loading by adjusting the pH results in low protein recovery and high loss of doxorubicin, Kim, M et al. (pH-Dependent Structures of Ferritin and Apoferritin in Solution: Disassembly and Reassembly, Biomacromolecules, 2011, 12(5) :1629-1640) believed that the process of opening and restoring the globular structure of ferritin by adjusting the pH would cause structural damage to the ferritin subunits, resulting in low protein recovery
In order to solve the problem of low protein yield during pH adjustment, researcher Minmin Liang et al. ) and Lei, Y et al. (Targeted tumordelivery and controlled release of neuronal drugs with ferritin nanoparticles to regulate pancreatic cancer progression, J Control Release, 2016,232:131-142) further tried to denature ferritin with 8M urea to partially open the spherical structure, Then dialysis is used to refold the ferritin in the process of restoring the spherical structure to realize drug loading. The final loading ratio can reach 33.1:1 and 32.5:1 respectively, and the recovery rate of ferritin is also high, reaching 65%. The process is complicated and cumbersome, there is residual urea in the sample, the dialysis process causes a lot of waste of doxorubicin, and the production cost is too high
Due to the excessive protein loss in the recovery process after opening the globular structure of ferritin, some researchers tried to load drugs without opening the globular structure of ferritin. Zhen, Z et al. (RGD-Modified Apoferritin Nanoparticles for Efficient Drug Delivery toTumors, Acs Nano, 2013,7(6):4830-4837) Although the method of loading small molecule drugs into ferritin mediated by copper ions can increase the loading ratio to a certain extent, reaching 37:1, the protein is still lost Larger, only 33% yield, and there is a certain safety risk in the residual heavy metal ions in the sample

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  • Ferritin-loaded antitumor drug and preparation method thereof
  • Ferritin-loaded antitumor drug and preparation method thereof
  • Ferritin-loaded antitumor drug and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] In this example, the anti-tumor drug with ferritin as the carrier was prepared by the following method (the overall schematic diagram is as follows: figure 1 shown), specifically include the following steps:

[0068] (1) Preparation of ferritin: engineering bacteria expressing ferritin (the construction of engineering bacteria uses PET-28a as a carrier, the ferritin gene is cloned into the carrier to construct a recombinant plasmid, and then the recombinant plasmid is transferred to Escherichia coli BL21 for iron Protein prokaryotic expression) was crushed and the supernatant was collected. The supernatant was heat-treated at 75°C for 10 minutes, and then ice-bathed for 15 minutes to obtain a crude sample. The crude sample was centrifuged at 12,000 rpm for 30 minutes to remove the precipitate. The supernatant was taken and passed through a step of Superdex 200 Gel filtration chromatography column purification to obtain ferritin;

[0069] (2) Add ferritin and antineopla...

Embodiment 2

[0079] In this example, the anti-tumor drug with ferritin as the carrier was prepared by the following method (the overall schematic diagram is as follows: figure 1 shown), specifically include the following steps:

[0080] (1) Preparation of ferritin: crush the engineering bacteria expressing ferritin (the construction of the engineering bacteria is the same as in Example 1) and collect the supernatant. The supernatant is heat-treated at 75° C. for 10 minutes, and then ice-bathed for 15 minutes to obtain crude For the sample, centrifuge the crude sample at 12000rpm for 30min to remove the precipitate, take the supernatant, and purify it with a Superdex 200 gel filtration chromatography column to obtain ferritin;

[0081] (2) Add ferritin and antineoplastic drug DOX with a mass ratio of 1.5:1 to a 20mM acetic acid-sodium acetate buffer solution with a pH value of 5.5, mix well, add the additive arginine to it to obtain a mixed solution, arginine The molar concentration of the...

Embodiment 3

[0089] In this example, the anti-tumor drug with ferritin as the carrier was prepared by the following method (the overall schematic diagram is as follows: figure 1 shown), specifically include the following steps:

[0090] (1) Preparation of ferritin: crush the engineering bacteria expressing ferritin (the construction of the engineering bacteria is the same as in Example 1) and collect the supernatant. The supernatant is heat-treated at 70° C. for 15 minutes, and then ice-bathed for 10 minutes to obtain crude For the sample, centrifuge the crude sample at 8000rpm for 40min to remove the precipitate, take the supernatant, and purify it with a Superdex 200 gel filtration chromatography column to obtain ferritin;

[0091] (2) Add ferritin and antineoplastic drug DOX with a mass ratio of 1:1 to 20mM acetic acid-sodium acetate buffer solution at pH 4.5, mix well, add additive arginine to it to obtain a mixed solution, arginine The molar concentration in the mixed solution is 20m...

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Abstract

The present invention provides an antineoplastic drug with ferritin as a carrier and a preparation method thereof, the method comprising the following steps: preparing ferritin; performing high pressure treatment on a mixed solution of ferritin and antineoplastic drug at a pressure of 200-800MPa6 ‑20h; the product after high pressure treatment is separated and purified to obtain an anti-tumor drug with ferritin as a carrier. The method of the present invention can not only increase the loading ratio of drugs, the yield of drug-loaded ferritin is close to 100%, but also can completely recover free unloaded anti-tumor drugs, improve production efficiency, reduce production costs, and is suitable for large-scale industrial production .

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, and relates to an antitumor drug with ferritin as a carrier and a preparation method thereof. Background technique [0002] Common small-molecule anti-tumor drugs such as doxorubicin, 5-fluorouracil, paclitaxel, and cisplatin have good effects on tumor treatment, among which doxorubicin has particularly obvious anti-tumor activity, but on the other hand, they do not have tumor-targeting properties. Due to the characteristics of cells, single administration is easy to kill normal cells while inhibiting tumor cells, and has certain cytotoxicity and obvious side effects. [0003] Due to its stable spherical structure and its ability to actively target ferritin receptor 1 on the surface of tumor cells, ferritin has been used by researchers to load small molecule anti-tumor drugs, so as to achieve targeted delivery of tumor drugs. The use of anti-tumor drugs with ferritin as a carrier can not only e...

Claims

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Application Information

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IPC IPC(8): A61K47/48A61K47/42A61K45/00A61P35/00
CPCA61K47/42A61K45/00
Inventor 刘永东王祺苏志国张纯
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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