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Multilevel targeted hyaluronic acid nanoparticle loaded with methotrexate and preparing method thereof

A technology loaded with methotrexate and hyaluronic acid, applied in powder delivery, antineoplastic drugs, drug combination, etc., can solve the problems of blood cell destruction, toxic side effects, application limitations, etc., and achieve the goal of promoting deep penetration and reducing toxic and side effects Effect

Inactive Publication Date: 2017-08-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, PAMAM is also greatly limited in its application due to its own shortcomings, mainly because the surface of unmodified PAMAM is enriched with positive charges, which is easy to combine with red blood cells, resulting in the destruction of blood cells, causing serious toxic side effects, and the small particle size PAMAM is more easily recognized and phagocytized by macrophages during systemic circulation

Method used

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  • Multilevel targeted hyaluronic acid nanoparticle loaded with methotrexate and preparing method thereof
  • Multilevel targeted hyaluronic acid nanoparticle loaded with methotrexate and preparing method thereof
  • Multilevel targeted hyaluronic acid nanoparticle loaded with methotrexate and preparing method thereof

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Effect test

specific Embodiment 1

[0024] Step 1: Preparation of fluorescently labeled dendrimers

[0025] PAMAM itself does not have fluorescence. In order to observe its cellular uptake, we designed to link PAMAM with fluorescein isothiocyanate isomer (FITC). Briefly, 6.7 mg of FITC was dissolved in 5 ml of dimethyl sulfoxide (Dimethyl sulfoxide, DMSO), and then slowly added to 5 ml of dimethyl sulfoxide solution containing 50.2 mg of PAMAM. The mixed solution was stirred for 12 h, and then the reaction solution was dialyzed at room temperature for 48 h with deionized water as the dialysis medium in the dark. Finally, the dialysate was freeze-dried to obtain the purified product PAMAM-FITC.

[0026] Step 2: Preparation of Fluorescently Labeled Hierarchical Targeted Hyaluronic Acid Nanoparticles

[0027] 10 mg of hyaluronic acid was weighed and dissolved in an appropriate amount of deionized water, ultrasonically dispersed to dissolve the hyaluronic acid, and 1 mg of PAMAM-FITC solution was added dropwise. ...

specific Embodiment 2

[0029] Step 1: Preparation of drug-containing dendrimers

[0030] Weigh 15 mg of PAMAM, dissolve it in 3 ml of purified water, and ultrasonically mix to obtain a PAMAM aqueous solution. Gradually add 25 mg of MTX powder into the PAMAM aqueous solution, stir at 1000 rpm in the dark for 3 hours at room temperature, filter with a 0.45 μm cellulose acetate membrane to remove unreacted MTX powder, and freeze-dry the obtained filtrate for 72 hours to obtain a yellow powder, which is the drug-loaded PAMAM core, namely PAMAM / MTX.

[0031] Step 2: Preparation of tumor-deeply permeable multistage targeting hyaluronic acid nanoparticles loaded with methotrexate

[0032] Weigh 10 mg of hyaluronic acid, dissolve it in an appropriate amount of purified water, and ultrasonically dissolve the hyaluronic acid. PAMAM / MTX was dissolved into an aqueous solution, and 1 mg of PAMAM / MTX solution was added dropwise. 6.8 ml of acetone solution was added dropwise to the above hyaluronic acid solutio...

specific Embodiment 3

[0034]The uptake of fluorescently labeled multi-stage targeting hyaluronic acid nanoparticles to A549 cells, see the attached figure 2 .

[0035] Take the human lung cancer cell line A549 cells in the logarithmic growth phase, trypsinize and dilute with serum-containing RPMI1640 medium, transfer the cells to a 24-well plate for culture, add about 50,000 cells to each well, and culture for 24 hours Replace the medium, and add PAMAM-FITC, HA / PAMAM-FITC and HA / PAMAM-FITC respectively (add HAase to the medium, 50U / mL), so that the final concentration of PAMAM is 15 μM, in 5% CO 2 , Incubate at 37°C for 6 hours, discard the medium, wash with PBS for 3 times, and observe the cell uptake under a fluorescent inverted microscope.

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Abstract

The invention discloses a multilevel targeted hyaluronic acid nanoparticle loaded with methotrexate and a preparing method thereof, and belongs to the technical field of medicine. The multilevel targeted hyaluronic acid nanoparticle is characterized by being formed by wrapping a polyamido-amine dendrimer with a small particle size (about 5 nm) in a hyaluronic acid nanoparticle with a large particle size (about 200 nm), and methotrexate is used as an anti-tumor drug (encapsulation efficiency and drug loading capacity are 71.62+ / -3.159% and 6.46+ / -0.518% respectively), so that the multilevel targeted hyaluronic acid nanoparticle intravenous injection administration preparation loaded with methotrexate is prepared. After the multilevel targeted hyaluronic acid nanoparticle enters systemic circulation, passive targeting and concentration of the nanoparticle in tumor tissue can be achieved through the hypertonicity effect of tumor tissue blood vessels, meanwhile, the hyaluronic acid nanoparticle can be degraded through high-expression hyaluronidase of tumor tissue, so that the wrapped polyamido-amine dendrimer can be released, and deep permeation of tumor tissue and trans-tumor cell drug delivery can be achieved by means of the small particle size and the positively charged surface of the polyamido-amine dendrimer.

Description

[0001] Technical field: [0002] The invention relates to the technical field of medicine, in particular to a methotrexate-loaded tumor deep penetration multi-stage targeting hyaluronic acid nanoparticle polymer and its preparation. [0003] Background technique: [0004] Polyamide-amine (polyamidoyamine, PAMAM) dendrimer is a new type of highly branched functional material, because of its small particle size, terminal functional group modification, good water solubility, high geometric symmetry and large internal The advantages of hydrophobic cavities have attracted attention in many fields such as materials, analytical chemistry, biology and medicine. Especially in the field of medicine, because PAMAM is positively charged in a physiological environment, it is easy to combine with negatively charged tumor cell membranes, so it has good cell entry ability, and because its PAMAM particle size is 5-10nm, it is beneficial to cross tumor tissue Therefore, it is considered to be o...

Claims

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Application Information

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IPC IPC(8): A61K9/14A61K31/519A61K47/36A61K47/34A61P35/00
Inventor 吴正红霍蒙蒙祁小乐
Owner CHINA PHARM UNIV
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