Cationized mulberry polysaccharide nanoparticle gene vectors and manufacturing method thereof

Abstract

The invention discloses cationized mulberry polysaccharide nanoparticle gene vectors, which are gene vectors combining mulberry polysaccharides modified by an amine compound and DNA plasmids, wherein the molecular weight distribution of the mulberry polysaccharides is 3 to 3.5*10<4>Da; and the mass ratio of the cationized mulberry polysaccharidesto the DNA plasmids is (0.5-100):1; the particle size of the plasmids is 50 to 150 nanometers; and the amine compounds is spermine, ethylenediamine or polyethyleneimine with a number-average molecular weight of 600Da to 2,000Da. The three kinds of cationized mulberry polysaccharides used in the invention all have good DNA plasmid bonding actions and can form biodegradable, high-efficiency and low-toxicity novel non-viral gene vector materials. And the preparation processes of the cationized mulberry polysaccharides are simple and the prices of the cationized mulberry polysaccharides are low. The invention discloses a manufacturing method of the cationized mulberry polysaccharide nanoparticle gene vectors.

Description

technical field

[0001] The invention relates to traditional Chinese medicine polysaccharides and gene carriers, in particular to a cationized mulberry leaf polysaccharide nanoparticle gene carrier. Background technique

[0002] In recent years, with the continuous development of biotechnology, gene therapy has become an effective means of clinical treatment of many major diseases such as cancer, cardiovascular and cerebrovascular diseases, and diabetes. With the deepening of gene therapy research, it has been found that the key to gene therapy is to select a suitable and effective gene carrier, that is, an appropriate introduction method, so that the target gene can be introduced in a targeted manner and expressed stably and effectively without causing toxic side effects to the body. Currently, there are two types of vectors used in gene therapy: viral vectors and non-viral vectors. Although viral vectors are widely used in the current clinical trials of gene therapy, many ...

Examples

Embodiment 1

[0032] Example 1. Preparation of Refined Mulberry Leaf Polysaccharide

[0033] Weigh 120g of dried mulberry leaves, crush them into powder, and pass through a 60-mesh sieve; extract according to the following process: solid-liquid ratio: 1:12, extraction temperature: 85°C, extraction time 2h / time, extraction times: 2 times; combine two The second extraction solution was centrifuged at 5000rpm for 10min, and the supernatant was taken; then the supernatant was concentrated by rotary evaporation to 1 / 6 of the original volume to obtain a concentrated solution; the concentrated solution was precipitated with 95% medical alcohol until the alcohol concentration was 75%, stand at 4°C for 12 hours; collect the precipitate, wash twice with absolute ethanol, acetone, and ether in turn; dry in vacuum to obtain the crude polysaccharide of mulberry leaves.

[0034] Weigh 3g of mulberry leaf crude polysaccharide, dissolve it completely in 40ml of double-distilled water, add 20% trichloroa...

Embodiment 2

[0037] Example 2. Preparation of Oxidized Mulberry Leaf Polysaccharide

[0038] Weigh 0.5g refined mulberry leaf polysaccharide, dissolve in 30ml double distilled water, add 0.8g KIO 4 , quickly placed in a dark room, stirred magnetically, and reacted at room temperature for 72 hours; adding 12ml ethylene glycol to the reaction solution to terminate the reaction, and continued the reaction for 30 minutes according to the aforementioned conditions; put the reaction solution into a dialysis bag, and dialyzed in double distilled water for 48 hours; Obtain oxidized mulberry leaf polysaccharide.

Embodiment 3

[0039] Example 3. Preparation of cationized mulberry leaf polysaccharide modified by ethylenediamine

[0040] Take 0.3g of oxidized mulberry leaf polysaccharide and dissolve it in 20ml of double distilled water; take 0.1ml of ethylenediamine and dissolve it in 5ml of borate buffer solution (pH=9); Add it into the mulberry leaf polysaccharide solution, and carry out magnetic stirring at the same time; after the addition, magnetically stir, and react at room temperature for 24 hours; after that, add 0.4g sodium borohydride to the reaction solution, and continue the reaction for 48 hours under the same conditions; then add to the reaction solution 0.4 g of sodium borohydride, and continue to react for 24 hours under the same conditions; put the reaction solution into a dialysis bag, and dialyze in double distilled water for 48 hours; freeze-dry the dialysate to obtain cationized mulberry polysaccharide modified with ethylenediamine.

technical field

[0001] The invention relates to traditional Chinese medicine polysaccharides and gene carriers, in particular to a cationized mulberry leaf polysaccharide nanoparticle gene carrier. Background technique

[0002] In recent years, with the continuous development of biotechnology, gene therapy has become an effective means of clinical treatment of many major diseases such as cancer, cardiovascular and cerebrovascular diseases, and diabetes. With the deepening of gene therapy research, it has been found that the key to gene therapy is to select a suitable and effective gene carrier, that is, an appropriate introduction method, so that the target gene can be introduced in a targeted manner and expressed stably and effectively without causing toxic side effects to the body. Currently, there are two types of vectors used in gene therapy: viral vectors and non-viral vectors. Although viral vectors are widely used in the current clinical trials of gene therapy, many ...

Examples

Embodiment 1

[0032] Example 1. Preparation of Refined Mulberry Leaf Polysaccharide

[0033] Weigh 120g of dried mulberry leaves, crush them into powder, and pass through a 60-mesh sieve; extract according to the following process: solid-liquid ratio: 1:12, extraction temperature: 85°C, extraction time 2h / time, extraction times: 2 times; combine two The second extraction solution was centrifuged at 5000rpm for 10min, and the supernatant was taken; then the supernatant was concentrated by rotary evaporation to 1 / 6 of the original volume to obtain a concentrated solution; the concentrated solution was precipitated with 95% medical alcohol until the alcohol concentration was 75%, stand at 4°C for 12 hours; collect the precipitate, wash twice with absolute ethanol, acetone, and ether in turn; dry in vacuum to obtain the crude polysaccharide of mulberry leaves.

[0034] Weigh 3g of mulberry leaf crude polysaccharide, dissolve it completely in 40ml of double-distilled water, add 20% trichloroa...

Embodiment 2

[0037] Example 2. Preparation of Oxidized Mulberry Leaf Polysaccharide

[0038] Weigh 0.5g refined mulberry leaf polysaccharide, dissolve in 30ml double distilled water, add 0.8g KIO 4 , quickly placed in a dark room, stirred magnetically, and reacted at room temperature for 72 hours; adding 12ml ethylene glycol to the reaction solution to terminate the reaction, and continued the reaction for 30 minutes according to the aforementioned conditions; put the reaction solution into a dialysis bag, and dialyzed in double distilled water for 48 hours; Obtain oxidized mulberry leaf polysaccharide.

Embodiment 3

[0039] Example 3. Preparation of cationized mulberry leaf polysaccharide modified by ethylenediamine

[0040] Take 0.3g of oxidized mulberry leaf polysaccharide and dissolve it in 20ml of double distilled water; take 0.1ml of ethylenediamine and dissolve it in 5ml of borate buffer solution (pH=9); Add it into the mulberry leaf polysaccharide solution, and carry out magnetic stirring at the same time; after the addition, magnetically stir, and react at room temperature for 24 hours; after that, add 0.4g sodium borohydride to the reaction solution, and continue the reaction for 48 hours under the same conditions; then add to the reaction solution 0.4 g of sodium borohydride, and continue to react for 24 hours under the same conditions; put the reaction solution into a dialysis bag, and dialyze in double distilled water for 48 hours; freeze-dry the dialysate to obtain cationized mulberry polysaccharide modified with ethylenediamine.

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