Preparation method for novel red blood cell substitute-artificial red blood cell fluorescent nanoparticles

A nanoparticle and red blood cell technology, which is applied in the direction of extracellular fluid diseases, medical preparations with no active ingredients, medical preparations containing active ingredients, etc., can solve the problems that blood substitutes cannot be directly applied, and achieve low price and source wide range of effects

Inactive Publication Date: 2012-03-14
王革 +2
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Problems solved by technology

Therefore, SFH cannot be directly applied as a blood substitute

Method used

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  • Preparation method for novel red blood cell substitute-artificial red blood cell fluorescent nanoparticles

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Embodiment 1

[0020] Embodiment 1: the preparation of stroma-free hemoglobin (Hb)

[0021] Stroma-free hemoglobin is prepared by lysing bovine red blood cells with low osmosis, extracting with toluene, purifying by high-speed centrifugation, and ion-exchange chromatography. The final solution contained 0.1-0.15 g / mL hemoglobin. In order to reduce the formation of methemoglobin, the operation process was carried out at 4°C under a nitrogen atmosphere, and the pH value of the hemoglobin solution was 7.4.

[0022] (1) Separation of bovine red blood cells

[0023] Take fresh bovine blood and put it into several aseptically treated centrifuge tubes, and centrifuge at 4000xg for 10 minutes. After taking out, the upper layer of plasma and middle layer of white blood cells were sucked out, and the lower layer of red blood cells was mixed with 0.9% normal saline and centrifuged at 4000×g for 10 minutes. The supernatant was discarded, and the red blood cells in the lower layer were washed with nor...

Embodiment 2

[0028] Example 2 Preparation of polyhemoglobin PolyHb-SOD-CAT-CA complex cross-linked with superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA)

[0029] The three enzymes were added to the purified hemoglobin solution at a ratio of 300,000 U catalase: 15,000 U superoxide dismutase: 100 U carbonic anhydrase: 1 g hemoglobin. Before crosslinking, add 1.3M lysine at a molar ratio (lysine:hemoglobin) of 10:1 and shake for 1 hour, then add 0.5M glutaraldehyde at a molar ratio of 17:1 (glutaraldehyde:hemoglobin), and crosslink for 10 -24 hours, the degree of molecular cross-linking was monitored by HPLC, and when the desired degree of cross-linking was reached, 2M lysine solution was added to the solution to terminate the cross-linking reaction. After the Lactate Ringer solution was dialyzed overnight, excess modifiers and other small molecular substances were removed through a Sephadex G-25 chromatographic column. The operation process was carried out at 4°C under...

Embodiment 3

[0030] Example 3 Polylactic acid, polyethylene glycol copolymer (PLA-PEG) nanoparticles embed PolyHb-SOD-CAT-CA complex to prepare artificial red blood cell nanoparticles

[0031] (1), the preparation of PLA-PEG copolymer

[0032] 1.5 g of DL-polylactic acid (molecular weight 16,000) and 0.75 g of methoxypolyethylene glycol (molecular weight 2,000) were dried in vacuo overnight. It was heated to 180° C. for 2 hours under the protection of nitrogen. After adding 10 μL of stannous 2-ethylhexanoate, the mixture was heated to 180 °C for 3 h under nitrogen protection to obtain the final polymer PLA-PEG copolymer, which was soluble in acetone.

[0033] (2), preparation of artificial red blood cell nanoparticles

[0034] Organic phase: 150 mg of PLA-PEG copolymer dissolved in 8 ml of acetone was mixed with 50 mg of hydrogenated soybean lecithin dissolved in 4 ml of ethanol, and 0.05% coumarin-6 was added as a fluorescent marker.

[0035] Water phase: Mix 0.04 ml of Tween 20 with 2...

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Abstract

The present invention discloses a preparation method for a novel red blood cell substitute-artificial red blood cell fluorescent nanoparticles. According to the present invention, a cross-linking technology is adopted to carry out a cross-linking reaction for hemoglobin (Hb), catalase (CAT), superoxide dismutase (SOD) and carbonic anhydrase (CA) to generate a polyHb-SOD-CAT-CA complex body, wherein the cross-linking technology is performed by a bifunctional reagent glutaraldehyde; the polyHb-SOD-CAT-CA complex body is further embedded by polylactic acid and polyethylene glycol copolymer (PLA-PEG) nanoparticles. With the present invention, the antioxidase of the SOD and the CAT in the complex body can eliminate the oxygen free radicals generated during the blood transfusion process, such that the ischemia-reperfusion injury easily generated during the blood replacement process can be avoided; with the CA, the red blood cell substitute of the present invention has oxygen carrying performance and carbon dioxide transportation performance; with the PLA-PEG microcapsule embedding, the half life period of the artificial blood product can be prolonged, the biocompatibility of the artificial blood product can be increased.

Description

technical field [0001] The invention relates to red blood cell substitutes, in particular to embedding bovine hemoglobin, catalase (CAT) and superoxide dismutase through glutaraldehyde crosslinking and polylactic acid, polyethylene glycol copolymer (PLA-PEG) nanoparticles (SOD) and carbonic anhydrase (CA) cross-linking to generate PolyHb-SOD-CAT-CA complex to prepare a method for artificial red blood cell nanoparticles with therapeutic significance. Background technique [0002] In recent years, emergencies such as earthquakes, tsunamis, and typhoons have caused a large number of deaths, and one of the reasons is that blood transfusions cannot be rescued in time. In my country, "blood shortages" have occurred in almost all major cities, and some operations have to wait for the arrival of blood before they can be performed. The transportation and storage of blood require special low temperature conditions, and the storage period is no more than one month at most. The blood t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/48A61K47/42A61K47/34A61K38/42A61P7/08
Inventor 谷劲松王革叶春江
Owner 王革
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